HK1257593A1 - New drug for treating diabetes and use thereof - Google Patents

Abstract

The invention relates to a novel method for treating diabetes. The novel method comprises dosing a diabetic subject with an effective amount of plasminogen. The invention further relates to a drug used to treat diabetes.

Description

New medicine for treating diabetes and its use

Technical Field

The present invention relates to a novel method for the treatment of diabetes comprising administering to a diabetic subject an effective amount of plasminogen, as well as to a medicament for the treatment of diabetes.

Background

Diabetes mellitus (diabetes)mellitis, DM) is a common inherited predisposition to abnormal glucose metabolism and endocrine disturbance caused by absolute or relative insulin hyposecretion. In 2015, 4.15 hundred million diabetics exist all over the world, and the number of diabetics reaches 6.42 hundred million by 2040 years^[1]. Diabetes is one of the major diseases seriously harming human health.

Diabetes is mainly manifested by abnormal carbohydrate metabolism and metabolic disorders of substances such as fat, protein and the like, and long-term hyperglycemia can cause serious diabetic complications including microvascular complications, diabetic nephropathy, diabetic cardiomyopathy, diabetic nervous system lesions, diabetic skin lesions, diabetic complicated infection and the like. The diabetic nephropathy and diabetic nervous system lesions have great influence on the life quality of patients and serious harm.

Clinically common diabetes can be divided into four types: type 1diabetes (T1 DM), type 2diabetes (T2 diabetes, T2DM), gestational diabetes, special type diabetes. Among them, T1DM and T2DM are the most common, and gestational diabetes and special diabetes are relatively rare.

T1DM is thought to be associated with genetic factors, environmental factors (e.g., viral infection, diabetogenic chemicals, dietary factors) and autoimmune factors, studies have shown that at least 17 loci of genes associated with T1DM are located on different chromosomes environmental factors, environmental factors contributing to the onset of T1DM include viral infection, diabetogenic chemicals and dietary factors, of which viral factors are most important, mumps, rubella virus, cytomegalovirus, etc. have been found to be involved in the onset of T1DM, the mechanism is that the virus can directly destroy islet β cells and provoke an autoimmune response to further damage islet β cells after the virus damages islet β cells, diabetogenic chemicals such as alloxan, Streptozotocin (STZ), pentamidine, etc. act on islet β cells, resulting in the destruction of islet β cells.

T2DM is a polygenic hereditary disease, and its occurrence is generally considered to be polygenic, wherein the environmental factors and the genetic factors act together to cause insulin resistance, and it is shown that the insulin at the same level concentration cannot play a normal role due to the body's resistance, and the body will excessively secrete insulin to relieve the ' low efficiency ' state of insulin use in order to reach the normal blood sugar level, and the previous requirement on the insulin β cell is higher and higher, and finally the insulin β cell is damaged by itself due to "overworking", and the absolute lack of insulin is developed.

Pathogenesis of DM

The pathogenesis of DM is complex and mainly related to familial genetic predisposition, ethnic heterogeneity, insulin receptor deficiency, insulin receptor substrate damage, protein tyrosine phosphatase related gene up-regulation, excessive immune inflammatory response, lipotoxicity, oxidative stress, mitochondrial damage and the like^[2-3]。

1. Free fatty acids

The research shows that the long-term high-fat diet can cause dysfunction of islet β cells, because the high-fat diet can cause the increase of abdominal fat content and the reduction of lipolysis inhibition ability of insulin besides peripheral insulin resistance, thereby promoting the increase of free fatty acid content, further inhibiting the phosphorylation of tyrosine sites of insulin receptors and IRS-l and 1RS-2 substrates thereof, inhibiting the activity of P13K and causing the formation of insulin resistance due to the obstruction of an insulin signal transduction pathway.

2. Inflammatory reaction

1) Inflammation and insulin resistance

T2DM is a mild non-specific inflammatory disease. Recent researches show that the main mechanism of insulin resistance caused by inflammation is the crossing of signal transduction of inflammatory factors and insulin receptor substrates, on one hand, the inflammatory factors generated by nonspecific inflammation have the effect of inhibiting IRS/PI3K signal paths, on the other hand, a series of kinases activated by the inflammatory factors can induce the phosphorylation of IRS silk and threonine sites to block the normal tyrosine phosphorylation, and finally, the insulin resistance is induced by the reduction of the insulin signal transduction capability^[2-3]。

In target cells, insulin can activate receptors by binding with the receptors, then intracellular signal transduction pathways generate a series of intracellular transduction molecules, and enzymatic cascade reactions are carried out to finish the step-by-step transmission and amplification of signals in cells, and the signals are finally transmitted to target organs to generate a series of biological effects. There are two major signaling pathways, one is the IRS-1-PI3K-PKB/AKT pathway, and the other is the mitogen-activated protein kinase (Shc/Raf/MAPK) pathway. In the first pathway, insulin binding to its receptor occurs first upon stimulation by exogenous insulin and/or glucose, thereby activating the receptor's endogenous tyrosine kinase. The activated tyrosine kinase induces tyrosine site phosphorylation of the insulin receptor substrate IRS while achieving its own phosphorylation. Activated IRS migrates to the cell membrane, phosphotyrosine is anchored to IRS tyrosine kinase by a phosphotyrosine binding domain (PTB), and tyrosine phosphorylated IRS is recruited via its SH2 domain to the regulatory subunit P85 of PI 3K. P85 binds to the inositol phosphate 3-phosphate molecule, converting phosphatidylinositol-monophosphate (PIP) to phosphatidylinositol diphosphate (PIP)2) And phosphatidylinositol triphosphate (PIP3), which is a second messenger for insulin and other growth factors, and is an anchor site for a subset of the downstream signaling molecules phosphoinositide-dependent protein kinase-1 (PDK1) and/or protein kinase c (pkc). PDK1 activates protein kinase B (PKB, also known as Akt) and some atypical PKC subtypes. The activated PKB inactivates glycogen synthesis kinase-3 (GSK3) through serine/threonine phosphorylation, and activates mammalian target of rapamycin (mTOR) protein kinase, thereby inducing phosphorylation activation of 70ku-S6 kinase (p70S6K) downstream thereof. mTOR protein kinase acts as an "ATP receptor" activating p70s6K without the need for Ca transport₂ ⁺cAMP, effects the synthesis of control proteins, enhances transcription of genes, promotes hypertrophy of pancreatic islet β cells and other biological effects PKB can directly induce the phosphorylation of certain transcription factors serine/threonine to promote mitosis of cells^[4-5]. In the second pathway, Ras activation can be achieved through two pathways. 1) The activated insulin receptor activates IRS-2 protein, and IRS-2 protein can transmit signal to the aptamer growth factor receptor binding protein 2(Grb2), and then interact with the signal protein GDP/GTP exchange factor (mSOS) to activate Ras-GT converted from inactivated Ras-GDP, thereby activating Ras. Direct action of the insulin receptor phosphorylates tyrosine of the signal protein Shc, which then binds to Grb2 to activate Ras via the mSOS pathway. Activated Ras-GTP recruits Raf serine kinase, in turn phosphorylating MAPK kinase, MAPK. The activated MAPK can activate other protein kinases to participate in the processes of inducing gene transcription, regulating and controlling cell apoptosis and the like^[6]。

It has been demonstrated that IRS-1 serine residues can be phosphorylated by a variety of inflammatory kinases, such as c-Jun amino terminal kinase (JNK), I.kappa.B kinase β (I.kappa.K β), and Protein Kinase C (PKC) -theta. radioimmunoassay showed that serine 307 site is the major site for JNK phosphorylation of IRS-1, and its mutation abolishes JNK-induced IRS-1 phosphorylation and the inhibitory effect of TNF on insulin-induced IRS-1 tyrosine phosphorylation. JNK reduces insulin receptor substrate phosphorylation tyrosine by phosphorylating IRS-1 serine 307, inhibits transduction of insulin signals^[7]. The discovery of Hiorsumi et alThe JNK activity in the liver, muscle and fat tissues of diet obese mice and ob/ob mice is obviously increased. The gene knockout (JNK1-/-) can weaken the insulin resistance phenomenon of diet-induced obese mice and relieve obesity, hyperglycemia and hyperinsulinemia of ob/ob mice. The phosphorylation level of IRS-1 serine 307 site in the liver tissue of obese mice is higher than that of lean mice, but the phosphorylation level is not increased in obese mice with gene knockout (JNK1-/-), so that IRS-1 serine 307 site is a target point of JNK action in vivo^[,8]The research shows that in a model of inducing hepatocyte insulin resistance by TNF α stimulation, a JNK inhibitor can completely block phosphorylation of serine 307, and Ikappa K β can influence insulin signaling through at least two ways, can directly induce phosphorylation of Ser307 site of IRS-1, and can also indirectly trigger insulin resistance by stimulating expression of various inflammatory factors through phosphorylation of Ikappa B to further activate NF-kappa B.

Inflammatory responses are defensive responses of the human immune system to infection, tissue damage and stress responses against these injuries, and are also causative or pathogenic for diabetes, cardiovascular diseases and tumors.

As early as 1993, Hotmaissligill et al^[9]Animal experiments show that the fat tissue of obese rats with insulin resistance has high levels of proinflammatory cytokines and TNF- α, since then, a plurality of researchers began to explore the relationship between inflammation and obesity and insulin resistance and explore the molecular pathogenesis of the obesity and insulin resistance, Hotmahissligill 2006^[10]The first time a new medical definition of metabolic inflammation (metabolic inflammation) was proposed, emphasizing that this low-grade, chronic systemic inflammation is mainly caused by excess nutrients and metabolites. Metabolic inflammation may present molecular and signaling pathways similar to typical inflammation, unlike typical inflammation as we have recognized previously, metabolic inflammation does not present the symptoms of redness, swelling, heat, pain, and dysfunction. Normally, the environment in the body is at a steady state level, and inflammation and metabolism are each and every other in a state of dynamic equilibrium. When the organism is in metabolic disorder, the balance state of the organism is broken, and the immune system is causedThe imbalance of the cells stimulates an inflammatory signal conduction pathway, promotes an organism to release a series of inflammatory factors, and some inflammatory factors even amplify the inflammatory reaction to form an inflammatory waterfall effect, so that the organism generates insulin resistance, thereby causing the occurrence of metabolic syndrome.

The research proves that TNF- α is closely related to metabolic syndrome, TNF is called cachectin and is mainly generated by activated macrophages, Natural Killer (NK) cells and T lymphocytes, TNF secreted by the macrophages is called TNF- α, lymphotoxin secreted by the T lymphocytes is called TNF- β, the biological activity of TNF- α accounts for 70% -95% of the total activity of the TNF, so that the TNF frequently involved at present is called TNF- α, through years of research and discussion, TNF- α is clearly related to various diseases such as insulin resistance, autoimmune diseases, tumors, chronic hepatitis B and the like, TNF- α plays a vital role in the generation and development process of the insulin resistance, Swaroop and the like^[11]The level of TNF- α of a T2DM patient is increased by detecting the serum TNF- α level of 50T 2DM patients, and is obviously related to BMI, fasting insulin level and steady-state model insulin resistance index (HOMA-IR), which indicates that TNF- α plays an important role in the pathogenesis of T2DM^[12]。

2) Inflammation and apoptosis of islet β cells

The chronic low-grade inflammatory response is closely related to the dysfunction of the pancreatic island β, the dysfunction of the pancreatic island β cells caused by the reduction of the number of β cells is another important reason for the onset of T2DM, and the apoptosis of β cells is the most important reason for the reduction of the number of β cells, because of heredity or diet, a T2DM patient is easy to generate insulin resistance, the blood sugar of the patient is increased, the hyperglycemic state can promote the production of IL-6, IL-6 can not only reduce the expression of GLUT4, reduce the transportation of glucose by fat cells and block the synthesis of glycogen,reducing insulin sensitivity, promoting insulin cell to secrete IL-6 to cause vicious circle, inducing large amount of IL-1 β by hyperglycemia, inducing islet cell apoptosis by activating NF-kB, MAPK, Fas, NO and other channels, promoting multiple inflammation channels to cross with each other, promoting islet cell apoptosis, and finally causing islet function failure^[13]In addition, IL-1 β can mediate the interaction between white blood cells, and interact with other cytokines such as IFN-gamma, TNF- α, etc. to restrict, playing an important role in β cell injury process, dyslipidemia of T2DM can cause the increase of hormone substances such as leptin and IL-6 level, leptin can increase the release of IL-1 β to induce β apoptosis, and can negatively regulate the secretion of insulin^[14]ROS, in addition to causing insulin resistance, also have effects on the damage of the β cells of the islets of langerhans, under oxidative stress conditions, the expression of insulin gene transcription factors and insulin binding sites are significantly reduced, thereby affecting the production and secretion of insulin, other adipocyte factors such as TNF- α and leptin can also reduce β cell function^[15]In addition, part of the inflammatory factors can also act on the key position of the insulin receptor substrate 2 to ensure that serine/threonine is phosphorylated, so that the degradation of the insulin receptor substrate 2 is accelerated, and the apoptosis of the insulin β cells is promoted.

3. Oxidative stress

Research has shown that oxidative stress is an important factor in the development and progression of T2 DM. Oxidative stress refers to imbalance between the generation of Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS) and the elimination of antioxidant defense system in the body, resulting in excessive production of ROS and RNS and damage to biological macromolecules such as body tissue cells, proteins and nucleic acids^[13]. Hyperglycemia, which is the major cause of oxidative stress, is produced through the mitochondrial electron transport chain^[14]Pathways for glucose autooxidation and polyol pathway^[15]Increasing ROS and RNS content in the organism, wherein the mitochondrial electron transport chain is the main pathway for generating ROS. The mitochondrial electron transport chain is mainlyEnzyme complexes I to IV, cytochrome c and coenzyme Q are involved, small amounts of superoxide products including superoxide anion, hydrogen peroxide and hydroxyl radical are continuously produced in the enzyme complexes I and III, and superoxide dismutase, catalase and glutathione peroxidase catalytically convert the superoxide products into oxygen and water. However, under obese or hyperglycemic conditions, superoxide production is greatly increased, and oxidative stress is produced when the rate of production of superoxide exceeds the rate of its removal.

Multiple studies^[16-18]The ROS can directly damage β cells, particularly destroy the mitochondrial structure of the cells, promote β apoptosis, indirectly inhibit β cell functions by influencing an insulin signal transduction pathway, such as activating a nuclear transcription factor kappa B (NF-kappa B) signal pathway to cause β cell inflammatory response, inhibiting nuclear translocation of pancreatic and duodenal homeobox factor 1 (PDX-1), inhibiting mitochondrial energy metabolism, reducing insulin synthesis and secretion and the like, inhibiting β cell damage NF-kappa B to be a dimer consisting of p50 and RelA through the NF-kappa B pathway, combining with the inhibitor Ikappa B in resting cells, existing in cytoplasm in an inactive trimer form, and mainly participating in the response of cells to the stimulation of stress, cytokines, free radicals, bacteria and viruses and the like, and instantaneously regulating gene expression and the like^[19]Studies have shown that hyperglycemia-induced ROS can activate NF- κ B by disrupting intracellular signal transduction, inducing β cell damage^[20]. Mariappan et al^[21]The pyrrolidine dithiocarbamic acid (PDTC) is used for inhibiting the expression of NF-kappa B in obese db/db mice, and the damage degree of oxidative stress on mouse β cell mitochondria is found to be obviously reduced, Hofmann and the like^[22]The antioxidant α -lipoic acid is used for treating diabetic patients, and the results show that the NF-kB activity in the patients is obviously reduced, the patient's condition is improved, Eldor and the like^[23]The transgenic technology is utilized to specifically inhibit the expression of mouse NF-kB, and the incidence rate of diabetes of the mouse after STZ induction is obviously reduced.

NF-kB as a multi-nuclear transcription factor, activated metanchoRegulation of various genes related to cell proliferation, apoptosis, inflammation and immunity^[24]In diabetic organisms, NF-. kappa.B causes an increase in pancreatic islet leukocytes by regulating the gene expression of cytokines and chemokines, such as IL-1(interleukin-1) and MCP-1 (monoclonal/macroporous chemoattractant protein-1) factors, etc., resulting in β cell damage^[25]In addition, many gene products regulated by NF-kB, such as tumor necrosis factor α (tumor necrosis factor α - α) and the like, can further activate NF-kB and aggravate β cell damage^[26]。

Mahadev et al^[27]Research shows that ROS has a regulatory effect on insulin signaling, and that the effect is multifaceted. Under the stimulation of insulin, the body can rapidly generate trace ROS through a nox (NADPH oxidase) dependent mechanism, the ROS is used as a second messenger and can promote insulin cascade reaction mainly through inhibiting the activity of PTP1B through oxidation^[28]And after the NOx is inhibited by DPI (diphenyleneiodonium), the phosphorylation of insulin-stimulated insulin receptor (InsR) and Insulin Receptor Substrate (IRS) is reduced by 48 percent^[29]. Loh et al^[30]The studies of (a) show that physiological ROS can promote the sensitivity of the body to insulin. Although the micro amount of ROS produced by insulin stimulation under physiological conditions promotes the action of insulin, long-term hyperglycemia causes the body to produce a large amount of ROS through the mitochondrial pathway^[31]Causing insulin resistance.

InsR and IRS are important signaling elements in the insulin signaling pathway: the former is the initiating element of insulin signaling, while the IRS is the bridge of the connection of the former to the downstream elements of the pathway. Numerous studies have shown that oxidative stress can interfere with the phosphorylation reactions of InsR and IRS through multiple pathways, impeding insulin signaling. IKK is activator of NF-kB inhibitory subunit IkB, and can be used as serine/threonine phosphorylation kinase of InsR and IRS under ROS stimulation to promote serine phosphorylation of InsR and IRS, inhibit normal tyrosine phosphorylation, and block insulin signaling^[32]。 Brownlee^[33]Research shows that IKK can directly phosphorylate IRS 307 filamentsThe amino acid residue, which results in a decrease in the normal tyrosine phosphorylation of the IRS, blocks the binding of InsR to the IRS, thereby causing insulin resistance.

In addition to IKK, multiple members of the MAPK family also have an effect on instr and IRS. JNK, extracellular regulated protein kinases (ERKs) and p38 mitogen-activated protein kinases (p38MAPK) are members of the MAPK family, have serine/threonine protein kinase activity, and can be activated under the action of oxidative stress, cytokines, G-protein coupled receptor agonists and the like. Several studies have shown that activation of JNK, ERK and p38MAPK aggravates the serine/threonine phosphorylation of InsR and IRS, reducing the protein binding capacity between InsR and IRS and the ability of IRS to activate SH-2 domain-containing signaling molecules downstream of the IRS^[34-36]。

Oxidative stress due to the hyperglycemic state of diabetes is one of the key causes of the development of various chronic complications and is also an important factor inducing DNA damage^[37]. When diabetes occurs, extracellular fluid is seen to be continuously high in sugar. Under the condition, electrons generated by a mitochondrial electron transfer chain are increased obviously, excessive ROS is generated, and the intracellular environment and biomacromolecules such as lipid, protein, DNA and the like are damaged. Active oxygen generated by the organism in the aerobic metabolic pathway is used as a mutation inducer to oxidize guanine on a DNA chain into 8-hydroxyguanine (8-hydroxy-2' -deoxyguanosine, 8-OhdG). During DNA replication, 8-OHdG is prone to mismatch with adenine, resulting in a G: C to T: A transversion mutation, resulting in DNA damage. In addition, ROS can cause other forms of DNA damage, including DNA strand breaks, DNA site mutations, DNA double strand aberrations, and mutations in proto-oncogenes and tumor suppressor genes. Also, DNA damage may exacerbate ROS and oxidative stress processes, e.g., DNA damage may induce ROS production through the H2 AX-reduced coenzyme ii oxidase 1(Nox1)/Rac1 pathway. ROS further promote large amounts of Ca₂ ⁺Enter mitochondria to cause cell necrosis and apoptosis, or directly damage mitochondria to cause mitochondrial dysfunction, further damage islet β cells, and aggravate pathological process of diabetes^[38]。

ROS can cause insulin resistance, also has effect on damage of islet β cells, under oxidative stress state, expression of insulin gene transcription factor and insulin binding site are obviously reduced, thereby influencing generation and secretion of insulin, other adipocyte factors such as TNF- α can also reduce function of β cells^[15]In addition, part of the inflammatory factors can also act on the key position of the insulin receptor substrate 2 to ensure that serine/threonine is phosphorylated, so that the degradation of the insulin receptor substrate 2 is accelerated, and the apoptosis of the insulin β cells is promoted.

ROS can be used as a signal molecule to activate some stress sensitive channels besides directly damaging islet β cells, regulate the expression of related factors, cause apoptosis or necrosis of β cells, inhibit insulin secretion, induce insulin resistance, and finally initiate or aggravate diabetes.

Treatment of DM

Diabetes is usually treated by medicines, and traditional medicines comprise insulin medicines and oral hypoglycemic medicines.

Insulin is mainly extracted from pancreas of animals such as pig and cattle in early stage, and obvious anaphylactic reaction can occur after the insulin is applied to a human body. The insulin is more and more mature in 90 years in the 20 th century, so that the insulin analogue is gradually applied, the pharmacokinetics of the traditional insulin can be obviously changed, and the insulin has the advantages of low incidence rate of hypoglycemia, quick response, lasting effect and the like. At present, with the continuous and deep exploration of insulin preparations, some oral insulin preparations are already in the experimental stage, but no effective oral preparations are yet used clinically because of the technical difficulty.

The most of traditional oral hypoglycemic drugs are (1) biguanides such as metformin, which have good cardiovascular protection effect and good hypoglycemic effect, and are currently used as first-line drugs for treating T2DM (2) sulfonylureas which belong to insulin secretagogues and stimulate pancreatic islet β cells to secrete insulin so as to achieve the effect of improving blood sugar level in a plurality of countries, however, the insulin allowed to be on the market in China mainly comprises glimepiride, glibenclamide, glipizide, gliclazide, gliquidone and the like, but some researches show that if the drugs are taken for a long time, the hypoglycemic effect fails, complications such as hypoglycemia and increase of body mass easily occur, (3) Thiazolidinediones (TZD) are frequently used, namely the drugs are used for approving the glitazone and the pioglitazone in T2DM in 1999, the risk of heart disease of the former people may be aggravated, and then are used as second-line drugs, and the drugs are also used for inhibiting the clinical release of glipizide and the glipizide, and the glucose absorption of the glipizide is even prohibited by the enteric glucose receptor for the epithelial cell inhibitors, so that the drugs are used for relieving the clinical blood sugar level of the glimepiride, and the glipizeigosaccharin, and the enteric glucose release of the glimepiridoid receptor thereof, and the clinical release of the glimepiridoid receptor thereof, and the glimepiridoid thereof, which are indicated by the clinical examination of the glimepiridoid receptor for the clinical trial drug for the clinical trial.

With the intensive research on the basic theory of diabetes, in order to avoid the side effect of the traditional hypoglycemic drugs and bring protection to pancreatic islet β cells, new diabetes treatment targets are actively searched, and targets related to the pathogenesis of diabetes are found at present and mainly comprise glucagon-like peptide-1 (glucagon-like peptide-1, GLP-1), dipeptidyl peptidase-4 (dipeptidyl peptidase-4, DPP-4), sodium-glucose cotransporter-2 (sodium-glucose transporter-2, SGLT-2), glycogen synthase kinase-3 (glycogen synthase kinase-3, DPP-3-tyrosine kinase), glucose-glucose co-transporter-2 (glucagon-kinase-4, DPP-kinase-3, glucagon-kinase-4, glucagon-like kinase-1-kinase-2, glucagon-kinase-2 (glucagon-kinase-4), and so on, and the like, and the development of glucagon-like receptor kinase (glucagon-kinase-like kinase-4, DPP-3, glucagon-glucose-kinase-4, DPP-glucose cotransporter-2 (glucagon-kinase-like) can be effectively maintained by glucagon-like, and DPP-4, and the like.

With the deeper and comprehensive understanding of the pathogenesis of diabetes, the research on the diabetes treatment drugs is also transited from the drug research on the traditional mechanism to the drug research on new targets and new action mechanisms, some of the drugs are on the market, such as GLP-1 receptor agonists, DPP-4 inhibitors, SGLT-2 inhibitors and the like, and some drugs are in clinical or preclinical research stages, such as GPR119 receptor agonists, 11 β -HSD1 inhibitors, PTP1B inhibitors, GK agonists and the like, the curative effect and the safety of the drugs are still to be clinically verified.

The plasminogen is found to be a brand new medicine which is expected to be comprehensively directed at multiple aspects of the pathogenesis of diabetes and can reduce the damage of the pancreatic tissue of a diabetes experimental mouse, control inflammation, reduce the apoptosis of the pancreatic β cells, repair the pancreatic tissue, recover the secretion function of the pancreatic β cells and reduce blood sugar.

Brief description of the invention

The invention includes the following:

1. a method of lowering blood glucose in a diabetic subject comprising administering to the subject an effective amount of plasminogen.

2. The method of item 1, wherein the blood glucose is selected from one or more of: serum glucose levels, serum fructosamine levels, serum glycated hemoglobin levels.

3. The method of item 2, wherein the blood glucose is serum glucose level.

4. The method of any one of items 1-3, wherein the diabetes is T1DM or T2 DM.

5. A method of increasing glucose tolerance in a diabetic subject comprising administering to the subject an effective amount of plasminogen.

6. The method of item 5, wherein the diabetes is T2 DM.

7. A method of promoting postprandial blood glucose lowering in a diabetic subject comprising administering to the subject an effective amount of plasminogen.

8. The method of item 7, wherein the plasminogen is administered 30 minutes to 1.5 hours before the subject meals.

9. The method of item 8, wherein the plasminogen is administered 30 minutes to 1 hour before the subject meals.

10. A method of promoting glucose utilization in a diabetic subject comprising administering to the subject an effective amount of plasminogen.

11. A method of promoting insulin secretion in a diabetic subject comprising administering to the subject an effective amount of plasminogen.

12. The method of clause 11, wherein said plasminogen further promotes insulin expression in a diabetic subject.

13. The method of clause 11 or 12, wherein the diabetes is T1DM or T2 DM.

14. The method of any of claims 10-13, wherein the plasminogen promotes insulin secretion in the diabetic subject after eating.

15. The method of any of claims 10-13, wherein the plasminogen promotes insulin secretion in the fasting state in a diabetic subject.

16. The method of any of claims 10-15, wherein the plasminogen promotes insulin secretion in the diabetic subject in response to a blood glucose elevation stimulus, returning blood glucose to normal or near normal levels.

17. The method of any of claims 11-16, wherein said plasminogen reduces expression and/or secretion of glucagon in a subject while promoting expression and/or secretion of said insulin.

18. The method of item 17, wherein said plasminogen effects a return of the subject's blood glucose to normal or near normal levels by promoting expression and/or secretion of said insulin while reducing expression and/or secretion of glucagon in the subject.

19. A method of reducing glucagon secretion in a diabetic subject comprising administering to the subject an effective amount of plasminogen.

20. The method of item 19, wherein said plasminogen further reduces glucagon expression in a diabetic subject.

21. The method of item 19 or 20, wherein the diabetes is T1DM or T2 DM.

22. The method of any of claims 19-21, wherein said plasminogen reduces glucagon secretion in a diabetic subject after eating.

23. The method of any of claims 19-22, wherein said plasminogen reduces glucagon secretion in the fasting state in a diabetic subject.

24. The method of any of claims 19-23, wherein said plasminogen reduces glucagon secretion in a diabetic subject at elevated blood glucose levels to return blood glucose to normal or near normal levels.

25. The method of any of claims 19-24, wherein said plasminogen promotes expression and/or secretion of said insulin while reducing expression and/or secretion of glucagon in the subject.

26. The method of item 25, wherein said plasminogen effects a return of the subject's blood glucose to normal or near normal levels by promoting said insulin expression and/or secretion in conjunction with reducing the expression and/or secretion of glucagon in the subject.

27. The method of any of items 11-26, wherein the plasminogen promotes expression of insulin receptor substrate 2 (IRS-2).

28. A method of promoting islet cell damage repair in a diabetic subject, comprising administering to the subject an effective amount of plasminogen.

29. The method of item 28, wherein said plasminogen promotes expression of insulin receptor substrate 2 (IRS-2).

30. The method of clause 28 or 29, wherein said plasminogen promotes expression of the cytokine TNF- α.

31. The method of any of claims 28-30, wherein the plasminogen promotes expression of the subject's multiple nuclear transcription factor NF- κ B.

32. The method of any one of items 28-31, wherein the islet cell injury is an impairment of the function of islet β cells in synthesizing and secreting insulin.

33. The method of any one of claims 28-32, wherein the islet cell damage is an islet tissue structure damage.

34. The method of any one of items 28-33, wherein the islet cell damage is islet collagen deposition.

35. The method of any one of claims 28-34, wherein the islet cell damage is fibrosis of the islets.

36. The method of any one of claims 28-35, wherein the islet cell injury is islet cell apoptosis.

37. The method of any one of claims 28-36, wherein the islet cell injury is a disturbance in the balance of glucagon and insulin secretion from the islets.

38. The method of any one of items 28-37, wherein the islet cell injury is an inability of the islets to secrete glucagon and insulin at levels compatible with the subject's blood glucose level.

39. The method of any of claims 28-38, wherein said plasminogen causes a decrease in glucagon secretion and an increase in insulin secretion in said diabetic subject.

40. The method of item 39, wherein said normal balance of islet glucagon and insulin secretion is restored.

41. A method of promoting islet inflammation repair in a diabetic subject, comprising administering to the subject an effective amount of plasminogen.

42. The method of clause 41, wherein said plasminogen promotes expression of the cytokine TNF- α.

43. The method of clause 41 or 42, wherein the plasminogen promotes expression of the nuclear multiple transcription factor NF- κ B in the subject.

44. The method of any of claims 41-43, wherein said plasminogen reduces islet collagen deposition.

45. The method of clause 44, wherein the plasminogen reduces fibrosis of the pancreatic islets.

46. The method of any of claims 41-45, wherein said plasminogen inhibits islet cell apoptosis.

47. The method of items 41-46, wherein the diabetic patient is T1DM or T2 DM.

48. The method of clause 47, wherein the T1DM subject is a subject with normal PLG activity or impaired PLG activity.

49. The method of any one of items 1-48, wherein said plasminogen can be used in combination with one or more other drugs or therapies.

50. The method of clause 49, wherein the plasminogen can be combined with one or more drugs selected from the group consisting of: antidiabetic, cardiovascular and cerebrovascular disease resistant, antithrombotic, antihypertensive, antilipemic, anticoagulant, antiinfective.

51. The method of any one of items 1-50, wherein the plasminogen has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to sequence 2, 6, 8, 10 or 12 and still possesses plasminogen activity.

52. The method of any one of items 1 to 51, wherein the plasminogen is a protein which has 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 1 amino acid and still has plasminogen activity, and is added, deleted and/or substituted on the basis of the sequence 2, 6, 8, 10 or 12.

53. The method of any one of items 1 to 52, said plasminogen is a protein comprising a plasminogen active fragment and still having plasminogen activity.

54. The method of any one of items 1 to 53, wherein the plasminogen is selected from Glu-plasminogen, Lys-plasminogen, miniplasminogen, microplasminogen, delta-plasminogen or a variant thereof retaining plasminogen activity.

55. The method of any one of items 1 to 54, wherein the plasminogen is native or synthetic human plasminogen, or a variant or fragment thereof which still retains plasminogen activity.

56. The method of any one of items 1 to 54, wherein the plasminogen is a human plasminogen ortholog from a primate or rodent, or a variant or fragment thereof that still retains plasminogen activity.

57. The method of any one of items 1 to 56, wherein the amino acid sequence of plasminogen is as shown in SEQ ID No.2, 6, 8, 10 or 12.

58. The method of any one of items 1-57, wherein the plasminogen is human native plasminogen.

59. The method of any one of items 1-58, wherein the subject is a human.

60. The method of any one of claims 1-59, wherein the subject lacks or lacks plasminogen.

61. The method of any one of items 1-60, wherein the deficiency or deletion is congenital, secondary, and/or local.

62. A plasminogen for use in a method according to any of claims 1-61.

63. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and plasminogen for use in the method of any one of items 1-61.

64. A prophylactic or therapeutic kit comprising: (i) plasminogen for use in a method according to any of claims 1-61 and (ii) means for delivering said plasminogen to said subject.

65. The kit of item 64, wherein the component is a syringe or a vial.

66. The kit of item 64 or 65, further comprising a label or instructions for administering the plasminogen to the subject for performing the method of any of items 1-61.

67. An article of manufacture, comprising:

a container containing a label; and

comprising (i) plasminogen or a pharmaceutical composition comprising plasminogen for use in a method according to any of claims 1 to 61, wherein the label indicates that the plasminogen or composition is to be administered to the subject for carrying out a method according to any of claims 1 to 61.

68. The kit of any one of items 64-66 or the article of manufacture of item 67, further comprising one or more additional components or containers comprising an additional pharmaceutical.

69. The kit or article of manufacture of item 68, wherein the additional agent is selected from the group consisting of: antidiabetic, cardiovascular and cerebrovascular disease resistant, antithrombotic, antihypertensive, antilipemic, anticoagulant, antiinfective.

In one aspect, the invention relates to a method for preventing and treating diabetes comprising administering to a subject an effective amount of plasminogen or plasmin.

In another aspect, the invention relates to a method of lowering blood glucose in a diabetic subject comprising administering to the subject an effective amount of plasminogen. The invention also relates to the use of plasminogen for lowering blood glucose in a diabetic subject. The invention also relates to the use of plasminogen for the preparation of a medicament for lowering blood glucose in a diabetic subject. Furthermore, the present invention relates to plasminogen for use in lowering blood glucose in a diabetic subject. In some embodiments, the blood glucose is selected from one or more of: serum glucose levels, serum fructosamine levels, serum glycated hemoglobin levels. In other embodiments, the blood glucose is serum glucose level. In the above embodiments, the diabetes is T1DM or T2 DM.

In another aspect, the invention relates to a method of increasing glucose tolerance in a diabetic subject comprising administering to the subject an effective amount of plasminogen. The invention also relates to the use of plasminogen for increasing glucose tolerance in a diabetic subject. The invention also relates to the use of plasminogen for the preparation of a medicament for increasing glucose tolerance in a diabetic subject. Furthermore, the present invention relates to plasminogen for use in increasing glucose tolerance in a diabetic subject. In some embodiments, the diabetes is T2 DM.

In one aspect, the invention relates to a method of promoting postprandial blood glucose lowering in a diabetic subject comprising administering to the subject an effective amount of plasminogen. The invention also relates to the use of plasminogen for promoting postprandial blood glucose lowering in a diabetic subject. The invention also relates to the use of plasminogen for the manufacture of a medicament for promoting postprandial blood glucose lowering in a diabetic subject. In addition, the invention relates to plasminogen for promoting postprandial blood glucose lowering in diabetic subjects. In some embodiments, the plasminogen is administered 30 minutes to 1.5 hours before the subject meals. In other embodiments, the plasminogen is administered 30 minutes to 1 hour before the subject's meal.

In one aspect, the invention relates to a method of promoting glucose utilization in a diabetic subject comprising administering to the subject an effective amount of plasminogen. The invention also relates to the use of plasminogen for promoting glucose utilisation in a diabetic subject. The invention also relates to the use of plasminogen for the preparation of a medicament for promoting glucose utilisation by a diabetic subject. Furthermore, the present invention relates to plasminogen for use in promoting glucose utilisation by a diabetic subject. In another aspect, the invention relates to a method of promoting insulin secretion in a diabetic subject comprising administering to the subject an effective amount of plasminogen. In some embodiments, the plasminogen also promotes insulin expression in diabetic subjects. In the above embodiments, the diabetes is T1DM or T2 DM. In some embodiments, the plasminogen promotes insulin secretion in a diabetic subject after eating. In other embodiments, the plasminogen promotes insulin secretion in the fasting state in a diabetic subject. In some embodiments, the plasminogen promotes insulin secretion in diabetic subjects in response to a blood glucose elevation stimulus, returning blood glucose to normal or near normal levels. In other embodiments, the plasminogen reduces the expression and/or secretion of glucagon in the subject while promoting expression and/or secretion of the insulin, in particular, the plasminogen effects a return of the subject's blood glucose to normal or near normal levels by reducing the expression and/or secretion of glucagon in the subject while promoting expression and/or secretion of the insulin.

In one aspect, the invention relates to a method of reducing glucagon secretion in a diabetic subject comprising administering to the subject an effective amount of plasminogen. The present invention also relates to the use of plasminogen for reducing glucagon secretion in a diabetic subject. The invention also relates to the use of plasminogen for the preparation of a medicament for reducing glucagon secretion in a diabetic subject. In addition, the invention relates to plasminogen for use in reducing glucagon secretion in a diabetic subject. In some embodiments, the plasminogen also reduces glucagon expression in a diabetic subject. In the above embodiments, the diabetes is T1DM or T2 DM. In some embodiments, the plasminogen reduces glucagon secretion in a diabetic subject after eating. In other embodiments, the plasminogen reduces glucagon secretion in a diabetic subject in the fasted state. In some embodiments, the plasminogen reduces glucagon secretion in a diabetic subject at elevated blood glucose levels, returning blood glucose to normal or near normal levels. In some embodiments, the plasminogen reduces glucagon secretion in a diabetic subject at elevated blood glucose levels, returning blood glucose to normal or near normal levels. In other embodiments, the plasminogen promotes expression and/or secretion of insulin while reducing expression and/or secretion of glucagon in the subject, in particular, the plasminogen promotes expression and/or secretion of insulin while reducing expression and/or secretion of glucagon in the subject to achieve a return of blood glucose in the subject to normal or near normal levels. In the above embodiments, the plasminogen promotes expression of insulin receptor substrate 2 (IRS-2).

In one aspect, the present invention relates to a method of promoting repair of islet cell damage in a diabetic subject, comprising administering to the subject an effective amount of plasminogen, the present invention also relates to the use of plasminogen for promoting repair of islet cell damage in a diabetic subject, the present invention also relates to the use of plasminogen for the manufacture of a medicament for promoting repair of islet cell damage in a diabetic subject, the present invention further relates to plasminogen for promoting repair of islet cell damage in a diabetic subject, in some embodiments, the plasminogen promotes expression of insulin receptor substrate 2(IRS-2), in other embodiments, the plasminogen promotes expression of the cytokine TNF- α, in other embodiments, the plasminogen promotes expression of the multidirectional transcription factor NF- κ B in the subject.

In another aspect, the present invention relates to a method of protecting pancreatic islets in a subject comprising administering to the subject an effective amount of plasminogen, the present invention also relates to the use of plasminogen for protecting pancreatic islets in a subject, the present invention also relates to plasminogen for protecting pancreatic islets in a subject, in some embodiments, the plasminogen reduces pancreatic islet collagen deposition, in other embodiments, the plasminogen reduces fibrosis of pancreatic islets, in other embodiments, the plasminogen reduces islet cell apoptosis, in other embodiments, the plasminogen promotes expression of insulin receptor substrate 2(IRS-2), in some embodiments, the plasminogen promotes repair of islet inflammation, in other embodiments, the plasminogen promotes expression of the cytokine TNF- α, in other embodiments, the plasminogen promotes expression of nuclear transcription factor NF- κ B in a subject, in the above embodiments, the subject is diabetic, in particular, the diabetic is T1DM or T2dm, in some embodiments, the plasminogen is normal activity of the subject PLG DM.

In another aspect, the present invention relates to a method of promoting repair of inflammation of pancreatic islets in a diabetic subject comprising administering to the subject an effective amount of plasminogen, the present invention also relates to the use of plasminogen for promoting repair of inflammation of pancreatic islets in a diabetic subject, the present invention also relates to plasminogen that promotes repair of inflammation of pancreatic islets in a diabetic subject, in some embodiments, the plasminogen promotes expression of the cytokine TNF- α, in other embodiments, the plasminogen promotes expression of the subject's multiple nuclear transcription factor NF- κ B, in other embodiments, the plasminogen reduces islet collagen deposition, in other embodiments, the plasminogen reduces fibrosis of pancreatic islets, in other embodiments, the plasminogen inhibits apoptosis of pancreatic islets in the above embodiments, the diabetic is T1DM or T2DM, in particular, the T1DM subject is a subject with normal or impaired PLG activity.

In one aspect, the invention relates to a method for promoting expression of the cytokine TNF- α in a diabetic subject, comprising administering to the subject an effective amount of plasminogen, the invention also relates to the use of plasminogen for promoting expression of the cytokine TNF- α in a diabetic subject, the invention also relates to the use of plasminogen for the manufacture of a medicament for promoting expression of the cytokine TNF- α in a diabetic subject, and furthermore, the invention also relates to plasminogen for promoting expression of the cytokine TNF- α in a diabetic subject.

In another aspect, the invention relates to a method of promoting expression of the multiple nuclear transcription factor NF- κ B in a diabetic subject, comprising administering to the subject an effective amount of plasminogen. The invention also relates to the use of plasminogen for promoting expression of the multiple nuclear transcription factor NF-kB in diabetic subjects. The invention also relates to the use of plasminogen for the preparation of a medicament for promoting the expression of the multiple nuclear transcription factor NF-kB in a diabetic subject.

In another aspect, the invention relates to a method of promoting expression of insulin receptor substrate 2(IRS-2) comprising administering to a subject an effective amount of plasminogen. The invention also relates to the use of plasminogen for promoting insulin receptor substrate 2(IRS-2) expression. The invention also relates to the use of plasminogen for the preparation of a medicament for promoting the expression of insulin receptor substrate 2 (IRS-2). In addition, the present invention relates to plasminogen for use in promoting expression of insulin receptor substrate 2 (IRS-2).

In another aspect, the invention relates to a method of promoting insulin secretion in a diabetic subject comprising administering to the subject an effective amount of plasminogen to promote expression of insulin receptor substrate 2 (IRS-2). The invention also relates to the use of plasminogen for promoting insulin secretion in a diabetic subject. The invention also relates to the use of plasminogen for the preparation of a medicament for promoting insulin secretion in a diabetic subject. Furthermore, the present invention relates to plasminogen for use in promoting insulin secretion in a diabetic subject.

In another aspect, the invention relates to a method of promoting an increase in the number of pancreatic islets β in a diabetic subject comprising administering to the subject an effective amount of plasminogen the invention also relates to the use of plasminogen for promoting an increase in the number of pancreatic islets β in a diabetic subject the invention also relates to the use of plasminogen for the manufacture of a medicament for promoting an increase in the number of pancreatic islets β in a diabetic subject the invention also relates to plasminogen for promoting an increase in the number of pancreatic islets β in a diabetic subject in some embodiments, the plasminogen promotes insulin receptor substrate 2(IRS-2) expression.

In another aspect, the present invention relates to a method of reducing apoptosis in pancreatic islets β comprising administering to a subject an effective amount of plasminogen, the present invention also relates to the use of plasminogen for reducing apoptosis in pancreatic islets β the present invention also relates to the use of plasminogen for the manufacture of a medicament for reducing apoptosis in pancreatic islets β the present invention also relates to plasminogen for reducing apoptosis in pancreatic islets β.

In another aspect, the invention relates to a method of promoting islet β cell injury repair comprising administering to a subject an effective amount of plasminogen, the invention also relates to the use of plasminogen for promoting islet β cell injury repair, the invention also relates to the use of plasminogen for the manufacture of a medicament for promoting islet β cell injury repair, the invention also relates to plasminogen for promoting islet β cell injury repair.

In another aspect, the invention relates to a method of promoting the recovery of cellular function of pancreatic islets β comprising administering to a subject an effective amount of plasminogen the invention also relates to the use of plasminogen for promoting the recovery of cellular function of pancreatic islets β the invention also relates to the use of plasminogen for the manufacture of a medicament for promoting the recovery of cellular function of pancreatic islets β furthermore the invention also relates to plasminogen for promoting the recovery of cellular function of pancreatic islets β in some embodiments said plasminogen promotes the expression of insulin receptor substrate 2 (IRS-2).

In the above embodiments, the plasminogen can be used in combination with one or more other drugs or therapies. In particular, the plasminogen may be used in combination with one or more drugs selected from the group consisting of: antidiabetic, cardiovascular and cerebrovascular disease resistant, antithrombotic, antihypertensive, antilipemic, anticoagulant, antiinfective.

In the above embodiments, the plasminogen has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to sequence 2, 6, 8, 10 or 12 and still possesses plasminogen activity.

In the above embodiments, the amino acid of said plasminogen is as shown in sequence 2, 6, 8, 10 or 12. In some embodiments, the plasminogen is a protein that adds, deletes and/or replaces 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2, 1 amino acid on the basis of sequence 2, 6, 8, 10 or 12 and still has plasminogen activity.

In the above embodiments, the plasminogen is a protein comprising a plasminogen active fragment and still having plasminogen activity. In particular, the plasminogen is selected from Glu-plasminogen, Lys-plasminogen, miniplasminogen, microplasminogen, delta-plasminogen or variants thereof retaining plasminogen activity.

In the above embodiments, the plasminogen is native or synthetic human plasminogen, or a variant or fragment thereof which still retains plasminogen activity. In some embodiments, the plasminogen is a human plasminogen ortholog from a primate or rodent, or a variant or fragment thereof that still retains plasminogen activity. For example, plasminogen orthologs from primates or rodents, such as plasminogen orthologs from gorilla, rhesus, mouse, cow, horse, dog. Most preferably, the amino acid sequence of the plasminogen of the present invention is as shown in seq id No.2, 6, 8, 10 or 12.

In the above embodiments, the subject is a human. In some embodiments, wherein the subject lacks or lacks plasminogen. In particular, the deficiency or deletion is congenital, secondary and/or local.

In one embodiment, the plasminogen is administered systemically or locally, preferably by the following route: topically, intravenously, intramuscularly, subcutaneously, inhalationally, intraspinally, topically, intraarticularly, or rectally. In one embodiment, the topical administration is directly to the area of osteoporosis, for example by means of a dressing, catheter or the like.

In one embodiment, the plasminogen is administered in combination with a suitable polypeptide carrier or stabilizer. In one embodiment, the plasminogen is present in an amount of 0.0001-2000mg/kg, 0.001-800 mg/kg, 0.01-600mg/kg, 0.1-400mg/kg, 1-200mg/kg, 1-100mg/kg, 10-100 mg/kg (calculated per kg body weight) or 0.0001-2000mg/cm per day²、0.001-800 mg/cm²、0.01-600mg/cm²、0.1-400mg/cm²、1-200mg/cm²、1-100 mg/cm²、10-100mg/cm²The dose (calculated per square centimeter of body surface area) is preferably administered at least once, preferably at least daily. In the case of topical administration, the above dosages may be further adjusted as appropriate. In one aspect, the invention relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and plasminogen for use in the methods of the invention.

In another aspect, the invention relates to a prophylactic or therapeutic kit comprising: (i) plasminogen for use in the method of the invention and (ii) means (means) for delivering said plasminogen to said subject, in particular said means is a syringe or vial. In some embodiments, the kit further comprises a label or instructions for administering the plasminogen to the subject for performing the methods of the invention.

In another aspect, the present invention is also directed to an article comprising: a container containing a label; and (i) plasminogen or a pharmaceutical composition comprising plasminogen for use in the method of the invention, wherein the label indicates that the plasminogen or composition is to be administered to the subject in order to carry out the method of the invention.

In the above embodiments, the kit or article of manufacture further comprises one or more additional components or containers containing other drugs. In some embodiments, the additional agent is selected from the group consisting of: antidiabetic, cardiovascular and cerebrovascular disease resistant, antithrombotic, antihypertensive, antilipemic, anticoagulant, antiinfective.

Detailed Description

Diabetes mellitus is a series of metabolic disorder syndromes of sugar, protein, fat, water, electrolyte and the like caused by hypofunction of pancreatic islets, insulin resistance and the like due to the action of various pathogenic factors such as genetic factors, immune dysfunction, microbial infection and toxins thereof, free radical toxins, mental factors and the like on organisms, and is clinically characterized by hyperglycemia.

"diabetic complications" are damage or dysfunction of other organs or tissues of the body caused by poor blood glucose control during diabetes, including damage or dysfunction of the liver, kidney, heart, retina, nervous system, etc. According to the statistics of the world health organization, the diabetes complications are more than 100, and are the most known diseases at present.

"insulin resistance" refers to the decrease in the efficiency of insulin in promoting glucose uptake and utilization due to various causes, and the compensatory hypersecretion of insulin in the body to produce hyperinsulinemia to maintain the stability of blood glucose.

"plasmin" is a very important enzyme present in the blood which is capable of degrading fibrin polymers.

"plasminogen (plg)" is a zymogen form of plasmin, and is a glycoprotein consisting of 810 amino acids, having a molecular weight of about 90kD, synthesized mainly in the liver and capable of circulating in the blood, calculated from the sequence in swiss prot according to the natural human plasminogen amino acid sequence (SEQ ID NO: 4) containing a signal peptide, and the cDNA sequence encoding this amino acid sequence is shown in SEQ ID NO: 3. Full-length PLG comprises seven domains: a serine protease domain at the C-terminus, a Pan Apple (PAP) domain at the N-terminus, and 5 Kringle domains (Kringle 1-5). Referring to the sequence in swiss prot, the signal peptide includes residues Met1-Gly19, PAP includes residues Glu20-Val98, Kringle1 includes residues Cys103-Cys181, Kringle2 includes residues Glu184-Cys262, Kringle3 includes residues Cys275-Cys352, Kringle4 includes residues Cys377-Cys454, and Kringle5 includes residues Cys481-Cys 560. According to NCBI data, the serine protease domain includes residues Val581-Arg 804.

Glu-plasminogen is native full-length plasminogen, and consists of 791 amino acids (without 19 amino acid signal peptide), and the cDNA sequence encoding this sequence is shown in SEQ ID No. 1The amino acid sequence is shown as sequence 2. In vivo, there is also Lys-plasminogen which is formed by hydrolysis from amino acids 76-77 of Glu-plasminogen as shown in SEQ ID No. 6, and cDNA sequence encoding the amino acid sequence as shown in SEQ ID No. 5. Delta-plasminogen is a fragment of full-length plasminogen lacking the structure of Kringle2-Kringle5, and contains only Kringle1 and the serine protease domain^[39,40]The delta-plasminogen amino acid sequence (SEQ ID NO: 8) has been reported^[40]The cDNA sequence encoding the amino acid sequence is shown as sequence 7. Mini-plasminogen, which consists of Kringle5 and the serine protease domain, has been reported to include residues Val443-Asn791 (starting with the Glu residue of the Glu-plg sequence which does not contain a signal peptide)^[41]The amino acid sequence is shown as sequence 10, and the cDNA sequence for coding the amino acid sequence is shown as sequence 9. While Micro-plasminogen contains only serine protease domain, its amino acid sequence has been reported to include residues Ala 543-Asn 791 (starting with Glu residue of Glu-plg sequence not containing signal peptide)^[42]In addition, the patent CN102154253A reports that the sequence includes residues Lys531-Asn791 (Glu residues of Glu-plg sequence not containing signal peptide are used as initial amino acids), the patent sequence refers to patent CN102154253A, the amino acid sequence is shown as sequence 12, and the cDNA sequence coding the amino acid sequence is shown as sequence 11.

The plasmin and the plasmin can be used interchangeably and have the same meaning; "plasminogen" is used interchangeably with "plasminogen" and is synonymous.

In the present application, the plasminogen "deficient" means that the plasminogen content or activity in a subject is lower than in a normal human, low enough to affect the normal physiological function of the subject; the plasminogen is "deficient" in the sense that the content or activity of plasminogen in a subject is significantly lower than that of a normal human, even the activity or expression is minimal, and normal physiological function can be maintained only by external supply.

It will be appreciated by those skilled in the art that all of the solutions for plasminogen of the present invention are applicable to plasmin, and thus the solutions described herein encompass both plasminogen and plasmin.

In embodiments of the invention, "aging" and "premature aging" may be used interchangeably and are intended to have the same meaning.

During circulation, plasminogen adopts a closed inactive conformation, but when bound to a thrombus or cell surface, it is converted to active PLM in an open conformation, mediated by a PLG Activator (PA). The active PLM can further hydrolyze fibrin clots into fibrin degradation products and D-dimers, thereby dissolving the thrombus. Wherein the PAp domain of PLG comprises an important determinant for maintaining plasminogen in an inactive closed conformation, and the KR domain is capable of binding to lysine residues present on the receptor and substrate. A variety of enzymes are known that can act as PLG activators, including: tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), kallikrein, and coagulation factor XII (Hageman factor), and the like.

"plasminogen active fragment" refers to an active fragment of a plasminogen protein that is capable of binding to a target sequence in a substrate and performing proteolytic functions. The present invention relates to the technical scheme of plasminogen, and the technical scheme of replacing plasminogen by plasminogen active fragments is covered. The plasminogen active fragment is a protein containing serine protease domain of plasminogen, preferably, the plasminogen active fragment contains sequence 14 and a protein with amino acid sequence which has at least 80%, 90%, 95%, 96%, 97%, 98% and 99% homology with the sequence 14. Thus, the plasminogen of the present invention includes proteins that contain the plasminogen active fragment and still retain the plasminogen activity.

Currently, methods for determining plasminogen and its activity in blood include: the assay for tissue plasminogen activator activity (t-PAA), the assay for plasma tissue plasminogen activator antigen (t-PAAg), the assay for plasma tissue plasminogen activity (plgA), the assay for plasma tissue plasminogen antigen (plgAg), the assay for plasma tissue plasminogen activator inhibitor activity (PlGA), the assay for plasma tissue plasminogen activator inhibitor antigen (pPAP), the assay for plasma plasmin-antiplasmin complex (PAP). Among the most commonly used detection methods are chromogenic substrate methods: streptokinase (SK) and chromogenic substrate are added to the detected plasma, the PLG in the detected plasma is converted into PLM under the action of SK, the PLM acts on the chromogenic substrate, and then the absorbance is increased in proportion to the activity of plasminogen by measuring with a spectrophotometer. In addition, the plasminogen activity in blood can also be measured by immunochemistry, gel electrophoresis, immunoturbidimetry, radioimmunodiffusion, and the like.

"orthologues or orthologs" refers to homologues between different species, including both protein homologues and DNA homologues, also referred to as orthologs, orthologs. It specifically refers to proteins or genes evolved from the same ancestral gene in different species. The plasminogen of the invention includes native human plasminogen, as well as plasminogen orthologs or orthologs derived from different species having plasminogen activity.

"conservative substitution variants" refer to variants in which a given amino acid residue is changed without changing the overall conformation or function of the protein or enzyme, and include, but are not limited to, substitution of amino acids in the amino acid sequence of a parent protein with amino acids of similar characteristics (e.g., acidic, basic, hydrophobic, etc.). Amino acids with similar properties are well known. For example, arginine, histidine and lysine are hydrophilic basic amino acids and may be interchanged. Likewise, isoleucine is a hydrophobic amino acid and may be replaced by leucine, methionine or valine. Thus, the similarity of two proteins or amino acid sequences of similar function may differ. For example, 70% to 99% similarity (identity) based on the MEGALIGN algorithm. "conservatively substituted variants" also includes polypeptides or enzymes having greater than 60% amino acid identity, preferably greater than 75%, more preferably greater than 85%, and even greater than 90% as determined by the BLAST or FASTA algorithms, and having the same or substantially similar properties or functions as the native or parent protein or enzyme.

By "isolated" plasminogen is meant plasminogen protein that is isolated and/or recovered from its natural environment. In some embodiments, the plasminogen is purified (1) to a purity (by weight) of greater than 90%, greater than 95%, or greater than 98%, as determined by the Lowry method, e.g., greater than 99% (by weight), (2) to an extent sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using a spinning cup sequencer, or (3) to homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using coomassie blue or silver stain under reducing or non-reducing conditions. Isolated plasminogen also includes plasminogen that has been prepared from recombinant cells by bioengineering techniques and isolated by at least one purification step.

The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymeric forms of amino acids of any length, which may include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including but not limited to fusion proteins having heterologous amino acid sequences, fusions with heterologous and homologous leader sequences (with or without an N-terminal methionine residue); and so on.

"percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with amino acid residues in the reference polypeptide sequence, after introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be accomplished in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art will be able to determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared. However, for the purposes of the present invention, percent amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2.

In the case where ALIGN-2 is used to compare amino acid sequences, the% amino acid sequence identity of a given amino acid sequence a relative to a given amino acid sequence B (or a given amino acid sequence a that can be expressed as having or comprising some% amino acid sequence identity relative to, with, or for a given amino acid sequence B) is calculated as follows:

fractional X/Y times 100

Wherein X is the number of amino acid residues scored as identical matches in the A and B alignments of the sequence alignment program by the program ALIGN-2, and wherein Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence a is not equal to the length of amino acid sequence B, the% amino acid sequence identity of a relative to B will not be equal to the% amino acid sequence identity of B relative to a. Unless otherwise specifically indicated, all% amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

As used herein, the terms "treatment" and "preventing" refer to obtaining a desired pharmacological and/or physiological effect. The effect may be a complete or partial prevention of the disease or symptoms thereof, and/or a partial or complete cure of the disease and/or symptoms thereof, and includes: (a) preventing the occurrence of a disease in a subject, which may have a predisposition to the disease but has not yet been diagnosed as having the disease; (b) inhibiting the disease, i.e. blocking its formation; and (c) alleviating the disease and/or symptoms thereof, i.e., causing regression of the disease and/or symptoms thereof.

The terms "individual," "subject," and "patient" are used interchangeably herein to refer to a mammal, including, but not limited to, a mouse (rat, mouse), a non-human primate, a human, a dog, a cat, an ungulate (e.g., horse, cow, sheep, pig, goat), and the like.

"therapeutically effective amount" or "effective amount" refers to an amount of plasminogen that is sufficient to effect such prevention and/or treatment of a disease when administered to a mammal or other subject to treat the disease. The "therapeutically effective amount" will vary depending on the plasminogen used, the severity of the disease and/or its symptoms in the subject to be treated, as well as the age, weight, etc.

2. Preparation of plasminogen in accordance with the invention

Plasminogen can be isolated from nature and purified for further therapeutic use, and can also be synthesized by standard chemical peptide synthesis techniques. When the polypeptide is synthesized chemically, the synthesis may be carried out via a liquid phase or a solid phase. Solid Phase Polypeptide Synthesis (SPPS), in which the C-terminal amino acid of the sequence is attached to an insoluble support, followed by sequential addition of the remaining amino acids in the sequence, is a suitable method for plasminogen chemical synthesis. Various forms of SPPS, such as Fmoc and Boc, can be used to synthesize plasminogen. Techniques for Solid Phase Synthesis are described in Barany and Solid-Phase Peptide Synthesis; pages 3-284 from The Peptides: Analysis, Synthesis, biology, Vol.2: special Methods in peptide Synthesis, Part A., Merrifield, et al J.am.chem.Soc.,85: 2149-; stewart et al, Solid Phase Peptide Synthesis,2nd ed.Pierce chem.Co., Rockford, Ill. (1984); and Ganesan A.2006Mini Rev. Med chem.6:3-10 and Camarero JA et al 2005Protein PeptLett.12: 723-8. Briefly, small insoluble porous beads are treated with a functional unit on which peptide chains are constructed. After repeated cycles of coupling/deprotection, the attached solid phase free N-terminal amine is coupled to a single N-protected amino acid unit. This unit is then deprotected, revealing a new N-terminal amine that can be attached to another amino acid. The peptide remains immobilized on the solid phase, after which it is cleaved off.

The plasminogen of the present invention can be produced using standard recombinant methods. For example, a nucleic acid encoding plasminogen is inserted into an expression vector, operably linked to regulatory sequences in the expression vector. Expression control sequences include, but are not limited to, promoters (e.g., naturally associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences. Expression control may be a eukaryotic promoter system in a vector capable of transforming or transfecting a eukaryotic host cell (e.g., a COS or CHO cell). Once the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high level expression of the nucleotide sequence and collection and purification of plasminogen.

Suitable expression vectors are typically replicated in the host organism as episomes or as an integral part of the host chromosomal DNA. Typically, expression vectors contain selectable markers (e.g., ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance) to facilitate detection of those cells that have been exogenously transformed with the desired DNA sequence.

Coli (Escherichia coli) is an example of a prokaryotic host cell that may be used to clone the subject antibody-encoding polynucleotides. Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis and other Enterobacteriaceae (Enterobacteriaceae), such as Salmonella (Salmonella), Serratia (Serratia), and various Pseudomonas species. In these prokaryotic hosts, expression vectors may also be produced, which will typically contain expression control sequences (e.g., origins of replication) that are compatible with the host cell. In addition, there will be many well known promoters such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or a promoter system from bacteriophage lambda. Promoters will generally control expression, optionally in the case of operator sequences, and have ribosome binding site sequences and the like to initiate and complete transcription and translation.

Other microorganisms, such as yeast, may also be used for expression. Yeast (e.g., saccharomyces cerevisiae (s. cerevisiae)) and Pichia (Pichia) are examples of suitable yeast host cells, wherein suitable vectors have expression control sequences (e.g., promoters), origins of replication, termination sequences, and the like, as desired. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeasts start from promoters which include, inter alia, those from alcohol dehydrogenases, isocytochrome C, and enzymes responsible for maltose and galactose utilization.

In addition to microorganisms, mammalian cells (e.g., mammalian cells cultured in vitro cell cultures) can also be used to express plasminogen of the present invention. See Winnacker, From Genes to Clones, VCH Publishers, n.y., n.y. (1987). Suitable mammalian host cells include CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, and transformed B cells or hybridomas. Expression vectors for these cells may contain expression control sequences such as origins of replication, promoters and enhancers (Queen et al, Immunol. Rev.89:49(1986)), as well as necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. Examples of suitable expression control sequences are promoters derived from the immunoglobulin genes, SV40, adenovirus, bovine papilloma virus, cytomegalovirus, and the like. See Co et al, J. Immunol.148:1149 (1992).

Once synthesized (chemically or recombinantly), the plasminogen described herein can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, High Performance Liquid Chromatography (HPLC), gel electrophoresis, and the like. The plasminogen is substantially pure, e.g., at least about 80% to 85% pure, at least about 85% to 90% pure, at least about 90% to 95% pure, or 98% to 99% pure or more pure, e.g., free of contaminants, such as cellular debris, macromolecules other than the subject antibody, and the like.

3. Pharmaceutical formulations

Therapeutic formulations can be prepared by mixing plasminogen having the desired purity with an optional Pharmaceutical carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences,16 th edition, Osol, a. ed. (1980)) to form a lyophilized formulation or an aqueous solution. Acceptable carriers, excipients, stabilizers, are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants include ascorbic acid and methionine; preservatives (for example octadecyl dimethyl benzyl ammonium chloride; hexane diamine chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol); low molecular weight polypeptides (less than about 10 residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, fucose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc-protein complexes); and/or a non-ionic surfactant, such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG). A preferred lyophilized anti-VEGF antibody formulation is described in WO 97/04801, which is incorporated herein by reference.

The formulations of the invention may also contain more than one active compound as required for the particular condition to be treated, preferably those with complementary activities and without side effects on each other. For example, antihypertensive drugs, antiarrhythmic drugs, drugs for treating diabetes, etc.

The plasminogen of the present invention can be encapsulated in microcapsules prepared by techniques such as coacervation or interfacial polymerization, for example, hydroxymethylcellulose or gel-microcapsules and poly- (methylmethacylate) microcapsules that can be placed in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. These techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980).

The plasminogen of the present invention for in vivo administration must be sterile. This can be readily achieved by filtration through sterile filtration membranes before or after lyophilization and reconstitution.

The plasminogen of the invention can be prepared into sustained release preparation. Suitable examples of sustained release formulations include shaped, glycoprotein-containing, solid hydrophobic polymer semi-permeable matrices, such as membranes or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (such as poly (2-hydroxyethyl-methacrylate) (Langer et al, J. biomed. Mater. Res.,15:167-277 (1981); Langer, chem. Tech.,12: 98-105 (1982)) or poly (vinyl alcohol), polylactide (U.S. Pat. No. 3773919, EP 58,481), copolymers of L-glutamic acid and □ ethyl-L-glutamic acid (Sidman, et al, Biopolymers 22:547(1983)), non-degradable ethylene-vinyl acetate (Langer, et al, supra), or lactic acid-glycolic acid copolymers such as Lupron DepotTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly D- (-) -3-hydroxybutyric acid polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid, are capable of sustained release of the molecule for more than 100 days, while some hydrogels release proteins for a shorter period of time. Rational strategies for protein stabilization can be designed based on the relevant mechanisms. For example, if the mechanism of aggregation is found to be intermolecular S — S bond formation through thio-disulfide interchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling humidity, employing appropriate additives, and developing specific polymer matrix compositions.

4. Administration and dosage

Administration of the pharmaceutical compositions of the invention can be achieved by different means, e.g., by intravenous, intraperitoneal, subcutaneous, intracranial, intrathecal, intraarterial (e.g., via the carotid artery), intramuscular, intranasal, topical or intradermal administration or spinal cord or brain delivery. Aerosol formulations such as nasal spray formulations comprise a purified aqueous or other solution of the active agent together with a preservative and an isotonicity agent. Such formulations are adjusted to a pH and isotonic state compatible with the nasal mucosa.

In some cases, the plasminogen pharmaceutical composition of the invention can be modified or formulated in such a way as to provide its ability to cross the blood-brain barrier.

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, ringer's dextrose, dextrose and sodium chloride, or fixed oils. Intravenous vehicles include liquid and nutritional supplements, electrolyte supplements, and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.

In some embodiments, the plasminogen of the present invention is formulated with an agent that facilitates transport across the blood-brain barrier. In some cases, the plasminogen of the present invention is fused, directly or via a linker, to a carrier molecule, peptide or protein that facilitates crossing the blood brain barrier. In some embodiments, the plasminogen of the present invention is fused to a polypeptide that binds to an endogenous Blood Brain Barrier (BBB) receptor. Joining plasminogen to polypeptides that bind endogenous BBB receptors facilitates passage across the BBB. Suitable polypeptides that bind to an endogenous BBB receptor include antibodies, such as monoclonal antibodies, or antigen-binding fragments thereof, that specifically bind to an endogenous BBB receptor. Suitable endogenous BBB receptors include, but are not limited to, insulin receptors in some cases, the antibodies are encapsulated in liposomes. See, for example, U.S. patent publication No. 2009/0156498.

Medical personnel will determine dosage regimens based on various clinical factors. As is well known in the medical arts, the dosage for any one patient depends on a variety of factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, number and route of administration, general health, and other drugs being administered concurrently. The dosage range of the plasminogen-containing pharmaceutical composition of the invention may be, for example, about 0.0001 to 2000mg/kg, or about 0.001 to 500mg/kg (e.g., 0.02mg/kg, 0.25 mg/kg, 0.5mg/kg, 0.75mg/kg, 10mg/kg, 50mg/kg, etc.) of the subject's body weight per day. For example, the dose may be 1mg/kg body weight or 50mg/kg body weight or in the range 1-50mg/kg, or at least 1 mg/kg. Doses above or below this exemplary range are also contemplated, particularly in view of the above factors. Intermediate doses within the above ranges are also included within the scope of the present invention. The subject may administer such doses daily, every other day, weekly, or according to any other schedule determined by empirical analysis. An exemplary dosage schedule includes 1-10mg/kg for several consecutive days. Real-time evaluation and regular evaluation of the therapeutic effect and safety of thrombi and thrombus-related diseases are required during the administration of the drug of the present invention.

5. Therapeutic efficacy and therapeutic safety

One embodiment of the invention relates to the assessment of therapeutic efficacy and therapeutic safety following treatment of a subject with plasminogen. The monitoring and evaluation contents of the commonly used osteoporosis treatment effect include follow-up (adverse reaction, standard medicine taking, basic measures, fracture risk factor reevaluation and the like), new fracture evaluation (clinical fracture, height reduction and imaging examination), bone density (BMD) measurement and bone transition biochemical marker (BTM) detection, comprehensive reevaluation based on the data and the like. Among them, BMD is currently the most widely used method for monitoring and evaluating therapeutic effects. BMD can be measured, for example, by dual energy X-ray bone Densitometry (DXA), Quantitative CT (QCT), single photon absorption assay (SPA), or ultrasonography. BMD can be measured 1 time per year after treatment initiation and can be monitored at appropriately extended intervals, e.g., 1 time in 2 years, after BMD has stabilized. For BTM, the most commonly used bone formation index among the serological indexes at present is procollagen type 1N-terminal peptide (PINP) of serum type 1, and the bone resorption index is procollagen C-terminal peptide (S-CTX) of serum type 1. More reasonable detection indexes can be timely adjusted according to research progress. A baseline value is detected before treatment, and detection is performed 3 months after treatment with the formation promoting drug and 3-6 months after treatment with the absorption inhibiting drug. BTMs can provide dynamic skeletal information, are independent of BMD in function and function, and become complementary monitoring means with BMD, and have higher clinical value when combined. In general, a therapeutic response is considered to be good if there is an expected change in BTMs if BMD rises or stabilizes after treatment, while no fractures occur during treatment. In addition, the present invention relates to the use of plasminogen and variants thereof for the determination of the safety of a subject during and after treatment, including but not limited to the statistics of the serum half-life of the drug in the subject, the treatment half-life, the median toxic dose (TD50), the median lethal dose (LD50), or the observation of various adverse events, such as sensitization, that occur during or after treatment.

6. Articles of manufacture or kits

One embodiment of the present invention relates to a preparation or kit comprising the plasminogen of the present invention. The article preferably comprises a container, label or package insert. Suitable containers are bottles, vials, syringes, etc. The container may be made of various materials such as glass or plastic. The container contains a composition that is effective in treating the disease or condition of the invention and has a sterile access port (e.g., the container may be an intravenous solution bag or vial containing a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is plasminogen. The label on or attached to the container indicates that the composition is for use in treating the aging or aging-related disorder of the present invention. The article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as phosphate buffered saline, ringer's solution, and dextrose solution. It may further comprise other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes. In addition, the article of manufacture comprises a package insert with instructions for use, including, for example, instructing a user of the composition to administer the plasminogen composition to a patient, as well as other drugs to treat the accompanying disease.

Brief Description of Drawings

FIG. 124-25 weeks old diabetic mice blood glucose test results after 10, 31 days administration of plasminogen. The results showed that mice given plasminogen were significantly lower in blood glucose than the vehicle-given PBS control group, and the statistical difference was significant (P <0.05, P < 0.01). In addition, the blood sugar of mice in the vehicle-PBS control group tends to increase along with the prolonging of the administration time, and the blood sugar of mice in the plasminogen group gradually decreases. Indicating that the plasminogen has the function of reducing blood sugar.

FIG. 2 Effect of plasminogen administration on serum fructosamine concentration in diabetic mice. The results of the assay showed a significant decrease in serum fructosamine concentration following administration of plasminogen with a statistically significant difference compared to pre-administration (P < 0.01). Thus showing that the plasminogen can obviously reduce the blood sugar of the diabetic mouse.

FIG. 326 results of plasma glycated hemoglobin assay 35 days after plasminogen administration in week-old diabetic mice. The results showed that the OD values of glycated hemoglobin in mice given plasminogen were significantly lower than those of PBS control group given vehicle, and the statistical difference was very significant (P < 0.01). Indicating that the plasminogen has the function of reducing the blood sugar of the diabetic mice.

FIG. 426 week-old diabetic mice IPGTT assay results 10 days after plasminogen administration. The results showed that after intraperitoneal injection of glucose, the blood glucose level of mice given plasminogen was lower than that of the vehicle-given PBS control group, and the glucose tolerance curve of the plasminogen was closer to that of the normal mice group than that of the vehicle-given PBS control group. Thus showing that the plasminogen can obviously improve the glucose tolerance of diabetic mice.

FIG. 5T 1DM model PLG Activity Normal mice were administered plasminogen for 10 days, followed by fasting blood glucose measurements. The results showed that mice in the vehicle-given PBS control group had significantly higher blood glucose than the plasminogen-given group, and the statistical difference was very significant (P < 0.001). Indicating that the plasminogen can obviously reduce the blood sugar level of the mice with normal PLG activity in the T1DM model.

FIG. 6T 1DM model PLG Activity Normal mice were given plasminogen for 28 days before IPGTT assay results. The results show that the blood sugar concentration of the mice in the vehicle PBS control group after glucose injection is obviously higher than that of the mice in the plasminogen group, and compared with the mice in the vehicle PBS control group, the glucose tolerance curve of the plasminogen group is closer to that of normal mice. Indicating that the plasminogen can improve the sugar tolerance of the SPLG activity normal mice in the T1DM model.

FIG. 726 week old diabetic mice serum insulin test results 35 days after plasminogen administration. The results showed that serum insulin levels were significantly higher in the plasminogen-administered group than in the vehicle-administered PBS control group, and the statistical difference was significant (. about.p < 0.05). Indicating that the plasminogen can effectively promote the secretion of insulin.

FIG. 824-25 weeks old diabetic mice HE stained image of pancreas and islet area ratio after administration of plasminogen for 31 days. A. B is a vehicle-giving PBS control group, C, D is a plasminogen group, and E is a islet area quantitative analysis result. The results show that most of the islets in the vehicle PBS control group are atrophied, the atrophied islet cells are replaced by acini (↓ mark), and the acini at the edge of the islets are proliferated, so that the demarcation between the islets and the acini is unclear; most of the islets of the plasminogen group have larger area than that of the control group, acinus hyperplasia does not exist in the islets, only a few acinus remain in the islets, and the boundary between the islets and the acinus is clear. Comparing the area ratio of pancreatic islets in the plasminogen-administered group and the control group, it was found that the administered group was almost one-fold larger than the control group. The plasminogen can promote the repair of the islet injury of 24-25 weeks old diabetic mice, thereby treating diabetes by repairing the damaged islet.

FIG. 924-25 weeks old diabetic mice were observed by Langerhans' Langerhans red staining 31 days after administration of plasminogen. A is given vehicle PBS control group, B is given plasminogen group, C is quantitative analysis result. The results showed that islet collagen deposition (arrow) was significantly less in mice given plasminogen than in the vehicle-given PBS control group, and the statistical difference was significant (. + -. denotes P < 0.05). Indicating that the plasminogen can improve the fibrosis of the islet of the diabetic animal.

FIG. 1024-25 weeks old diabetic mice showed immunohistochemical staining of islet Caspase-3 after 31 days administration of plasminogen. A is given vehicle PBS control group, B is given plasminogen group. The results showed that Caspase-3 expression (indicated by arrows) was significantly lower in the plasminogen group than in the vehicle-administered PBS control group. Indicating that the plasminogen can reduce the apoptosis of islet cells and protect the pancreas tissue of the diabetic mouse.

FIG. 1118 week-old diabetic mice insulin immunohistochemical staining results 35 days after administration of plasminogen. A is given vehicle PBS control group, B is given plasminogen group, C is quantitative analysis result. The results show that the expression of insulin (indicated by arrows) was significantly higher in the plasminogen group than in the vehicle-administered PBS control group, and the statistical difference was close to significant (P ═ 0.15). Indicating that the plasminogen can promote the functional repair of the pancreatic islet and promote the production and secretion of insulin.

FIG. 1224-25 weeks old diabetic mice insulin immunohistochemical staining observations of islets 35 days after administration of plasminogen. A is given vehicle PBS control group, B is given plasminogen group, C is quantitative analysis result. The results showed that the expression of insulin (arrow) was significantly higher in the plasminogen group than in the vehicle-administered PBS control group, and the statistical difference was significant (. about.p < 0.05). Indicating that the plasminogen can promote the restoration of the pancreatic islet function and promote the generation and the secretion of insulin.

FIG. 1326 shows the results of insulin immunohistochemical staining of pancreatic islets 35 days after administration of plasminogen to diabetic mice of week-old age. A is given vehicle PBS control group, B is given plasminogen group, C is quantitative analysis result. The results showed that the expression of insulin (arrow) was significantly higher in the plasminogen group than in the vehicle-administered PBS control group, and the statistical difference was very significant (. + -. denotes P < 0.01). Thus showing that the plasminogen can effectively promote the functional repair of the pancreatic islet and promote the generation and the secretion of the insulin.

FIG. 1424-25 weeks old diabetic mice were observed by NF- κ B immunohistochemical staining of pancreatic tissue 31 days after administration of plasminogen. A is a normal control group, B is a vehicle-giving PBS control group, C is a plasminogen-giving group, and D is a quantitative analysis result. The results showed that the expression of NF- κ B (arrow) was significantly higher for the plasminogen group than for the vehicle PBS control group, and the statistical difference was significant (toindicate P < 0.05). The plasminogen can promote the expression of the multi-nuclear transcription factor NF-kB, thereby promoting the repair of the islet inflammation of the diabetic mouse with the age of 24-25 weeks.

FIG. 1518 shows the observation of islet glucagon immunohistochemistry in diabetic mice after 35 days of plasminogen administration, A is the normal control group, B is the vehicle-administered PBS control group, C is the plasminogen group, and D is the quantitative analysis result, showing that glucagon is expressed in the peripheral α cell region of islets in the normal control mice, compared to the vehicle-administered PBS control group, glucagon positive cells (indicated by arrows) are significantly increased in the vehicle-administered PBS control group, infiltrated into the central region of islets, and the statistical difference of the mean optical density quantitative analysis result is significant (P <0.01), glucagon positive cells in the plasmin-administered PBS control group are scattered around the islets, and the islet morphology is closer to that of the normal mice compared to the PBS group, indicating that plasminogen can significantly inhibit proliferation of islet α cells and secretion of glucagon, and correct islet α cell distribution disorder, thereby promoting repair of islet injury.

FIG. 1624-25 weeks old diabetic mice were treated with plasminogen for 35 days, and then the results of islet glucagon immunohistochemistry observed showed that glucagon was expressed in α cell areas around islets in the normal control mice, as compared with the plasminogen-fed group, glucagon-positive cells (indicated by arrows) were significantly increased in the vehicle-fed PBS control group, and infiltrated into the central region of islets, and glucagon-positive cells were scattered around islets in the plasminogen group, and the islet morphology was closer to that of the normal mice in the plasminogen-fed group than in the PBS group, indicating that plasminogen could significantly inhibit proliferation of islet α cells and secretion of glucagon, and correct the islet α cell distribution disorder, thereby promoting repair of islet injury.

FIG. 1726 shows the results of 35 days post-administration of plasminogen to diabetic mice aged at week 1726, showing that glucagon is expressed in α cell areas around islets in normal control mice, showing that glucagon-positive cells (marked by arrows) are significantly increased in vehicle-administered PBS control group, infiltrated into the central region of islets, and the results of quantitative analysis of average optical density show statistical differences (P <0.05), compared with plasminogen-administered group, that glucagon-positive cells are scattered around islets in plasminogen group, and that plasminogen is closer to normal mice in form, showing that plasminogen can significantly inhibit proliferation of islet α cells and secretion of glucagon, correct islet α cell distribution disorder, and thus promote repair of islet injury.

FIG. 18 shows that the glucagon positive expression of the vehicle PBS control group is obviously more than that of the plasmin-feeding group, and the statistical difference of the quantitative analysis result of the average optical density (P <0.05) shows that the plasminogen can obviously reduce the glucagon secretion of the diabetes mouse islet α cells and promote the repair of islet injury.

FIG. 1918 shows that 35 days after administration of plasminogen, the observation results of islet IRS-2 immunohistochemistry of diabetic mice of week-old mice show that islet IRS-2 positive expression (marked by an arrow) is significantly less than that of the mice of the normal control group, and the statistical difference is very significant (P <0.01), when the mice of the vehicle PBS control group are administered with plasminogen, the expression level of the islet IRS-2 is closer to that of the mice of the normal control group than that of the vehicle PBS group, which indicates that the plasminogen can effectively increase the expression of islet cell IRS-2, improve insulin signal transduction, and reduce islet β cell damage of the diabetic mice.

FIG. 2024-25 weeks old diabetic mice were administered plasminogen for 31 days, and then islet IRS-2 immunohistochemical observations were made, A was a normal control group, B was a vehicle-administered PBS control group, C was a plasminogen group, and D was a quantitative analysis result, showing that islet IRS-2 positive expression (indicated by an arrow) was significantly less than that of the plasminogen group in the vehicle-administered PBS control group, and that the statistical difference was significant (. beta. -indicates that P <0.05), and showing that the expression level of islet IRS-2 in the plasminogen group was closer to that of the normal control group than that of the vehicle-administered PBS group, plasminogen was able to effectively increase the expression of islet cell IRS-2, improve insulin signal transduction, and reduce islet β cell damage in diabetic mice.

FIG. 2126 shows that the level of IRS-2 positive expression (arrow) in islet of mice in vehicle-administered PBS control group is significantly lower than that in plasmin-administered group, and the level of IRS-2 expression in plasmin-administered group is closer to that in normal control group than that in vehicle-administered PBS group, indicating that plasminogen can effectively increase the expression of IRS-2 in islet cells, improve insulin signal transduction, and reduce injury of β cells in islet of diabetic mice.

FIG. 22 shows the observation results of immunohistochemistry of pancreatic islet IRS-2 in mice with normal PLG activity T1DM after 28 days of plasminogen administration, wherein A is a normal control group, B is a vehicle-administered PBS control group, and C is a vehicle-administered plasminogen group, and the results show that the positive expression (marked by an arrow) of pancreatic islet IRS-2 in mice with the vehicle-administered PBS control group is obviously less than that in mice with the vehicle-administered plasminogen group, and the expression level of the pancreatic islet IRS-2 in the plasminogen group is closer to that in the normal control group than that in the vehicle-administered PBS group, which shows that the plasminogen can effectively increase the expression of pancreatic islet cell IRS-2, improve insulin signal transduction, and reduce the damage of pancreatic islet β cells in mice with normal PLG activity T1 DM.

FIG. 2326 Observation of islet neutrophil immunohistochemistry 35 days after plasminogen administration in week-old diabetic mice. A is a normal control group, B is a vehicle-giving PBS control group, and C is a plasminogen-giving group. The results showed that the cells positively expressed to the plasminogen group (indicated by arrows) were less in the vehicle-administered PBS control group, and the vehicle-administered PBS group was closer to the normal control group than the vehicle-administered PBS group. Indicating that plasminogen can reduce neutrophil infiltration.

FIG. 24 Observation of islet neutrophil immunohistochemistry 28 days after plasminogen administration in mice with impaired PLG activity in the T1DM model. A is blank control group, B is vehicle-giving PBS control group, and C is plasminogen-giving group. The results showed that the cells positively expressed to the plasminogen group (indicated by arrows) were less in the vehicle-administered PBS control group, and the vehicle-administered PBS group was closer to the blank control group than the vehicle-administered PBS group. Indicating that plasminogen could reduce islet neutrophil infiltration in the T1DM model in mice with impaired PLG activity.

FIG. 25 observation of islet neutrophil immunohistochemistry in mice with normal PLG activity 28 days after plasminogen administration in the T1DM model. A is blank control group, B is vehicle-giving PBS control group, and C is plasminogen-giving group. The results showed that the cells positively expressed to the plasminogen group (indicated by arrows) were less in the vehicle-administered PBS control group, and the vehicle-administered PBS group was closer to the blank control group than the vehicle-administered PBS group. Indicating that the plasminogen can promote the infiltration of islet neutrophils in a T1DM model of a mouse with normal PLG activity.

FIG. 26 Observation of insulin immunohistochemistry 28 days after plasminogen administration in mice with impaired PLG activity in the T1DM model. A is blank control group, B is vehicle-giving PBS control group, and C is plasminogen-giving group. Immunohistochemistry results showed that the positive expression of insulin (arrow) was significantly greater for the plasminogen group than for the vehicle-PBS control group, and that the plasminogen group was closer to the blank control group than for the vehicle-PBS group. Indicating that plasminogen can promote synthesis and secretion of insulin in mice with impaired PLG activity in the T1DM model.

FIG. 27 observation of insulin immunohistochemistry of mice with normal PLG activity 28 days after plasminogen administration in T1DM model. A is blank control group, B is vehicle-giving PBS control group, and C is plasminogen-giving group. Immunohistochemistry results showed that the positive expression of insulin (arrow) was significantly greater for the plasminogen group than for the vehicle-PBS control group, and that the plasminogen group was closer to the blank control group than for the vehicle-PBS group. Indicating that the plasminogen promotes synthesis and expression of insulin of mice with normal PLG activity in the T1DM model.

FIG. 28 Observation of islet NF- κ B immunohistochemistry in mice with impaired PLG activity 28 days after plasminogen administration in the T1DM model. A is blank control group, B is vehicle-giving PBS control group, and C is plasminogen-giving group. The results showed that NF-. kappa.B expression (indicated by an arrow) was significantly higher in the plasminogen-administered group than in the vehicle-administered PBS control group. The plasminogen can promote the expression of the inflammatory repair factor NF-kB, thereby promoting the repair of the islet inflammation.

FIG. 2918 Observation of islet NF- κ B immunohistochemistry after 35 days of plasminogen administration in week-old diabetic mice. A is given vehicle PBS control group, B is given plasminogen group. The experimental results show that the expression (marked by an arrow) of NF-kB given to the plasminogen group is obviously higher than that given to the vehicle PBS control group. Indicating that the plasminogen can promote the expression of the multi-nuclear transcription factor NF-kB, thereby promoting the repair of the islet inflammation of relatively young (18 weeks old) diabetic mice.

FIG. 3026 Observation of islet NF- κ B immunohistochemistry 35 days after plasminogen administration in week-old diabetic mice. A is a normal control group, B is a vehicle-giving PBS control group, and C is a plasminogen-giving group. The experimental result shows that the expression (marked by an arrow) of the NF-kB of the plasminogen group is obviously higher than that of the vehicle PBS control group. The plasminogen can promote the expression of a multi-nuclear transcription factor NF-kB, thereby promoting the repair of the islet inflammation of relatively old (26 weeks old) diabetic mice.

FIG. 3124-25 weeks old diabetic mice were subjected to 31 days plasminogen administration, and then islet TNF- α immunohistochemical observations were made, A was the normal control group, B was the vehicle-administered PBS control group, and C was the vehicle-administered plasminogen group.

FIG. 3226 week-old diabetic mice showed 31 days after administration of plasminogen, islet TNF- α immunohistochemical observation, in which A was the normal control group, B was the vehicle-administered PBS control group, and C was the vehicle-administered plasminogen group.

FIG. 33 shows the observation of islet TNF- α immunohistochemistry after 28 days of plasminogen administration in the PLG activity-impaired mouse T1DM model, A is the vehicle-administered PBS control group, B is the vehicle-administered plasminogen group, the results of the study show that the positive expression (marked by an arrow) of TNF- α in the vehicle-administered PBS control group is significantly higher than that in the vehicle-administered PBS control group, indicating that plasminogen can promote the expression of TNF- α, thereby promoting islet injury repair in the PLG activity-impaired mouse T1DM model.

FIG. 34 shows the observation of islet IgM immunohistochemistry after 28 days of plasminogen administration in T1DM model mice with impaired PLG activity. A is blank control group, B is vehicle-giving PBS control group, and C is plasminogen-giving group. The research result of the experiment shows that the positive expression (marked by an arrow) of the IgM in the plasminogen group is obviously lower than that in the vehicle-feeding PBS control group, and the plasminogen group is closer to the normal control group than the vehicle-feeding PBS group. Indicating that plasminogen can reduce IgM expression and thus reduce islet damage in mice with impaired PLG activity in the T1DM model.

FIG. 3524-25 weeks old diabetic mice were observed by TUNEL staining of pancreatic islets 31 days after administration of plasminogen. A is a normal control group, B is a vehicle-giving PBS control group, and C is a plasminogen-giving group. The results of this experiment show that the number of positive cells (indicated by the arrow) given to the plasminogen group is significantly less than that given to the vehicle PBS control group. TUNEL positive staining was very low in the normal control group. The apoptosis rate of the normal control group is about 8%, the apoptosis rate of the vehicle-giving PBS group is about 93%, and the apoptosis rate of the plasminogen group is about 16%. Thus showing that the plasminogen group can obviously reduce the apoptosis of the islet cells of the diabetic mice.

FIG. 3626 week-old diabetic mice serum fructosamine assay results 35 days after administration of plasminogen. The detection result shows that the concentration of fructosamine in serum given to the plasminogen group is obviously lower than that of the vehicle-given PBS control group, and the statistical difference is close to significance (P is 0.06). Indicating that the plasminogen can obviously reduce the blood sugar level of diabetic mice.

FIG. 37 shows the results of blood glucose measurements 20 days after plasminogen administration in T1DM model mice. The results showed that the blood glucose was significantly higher in the vehicle-administered PBS control group than in the plasminogen group, and the statistical difference was significant (P ═ 0.04). Therefore, the plasminogen can promote the glucose decomposition capability of the T1DM mouse, thereby reducing the blood sugar.

FIG. 38 shows the results of serum insulin measurements 20 days after plasminogen administration in T1DM model mice. The results show that the serum insulin concentration of the mice in the vehicle-given PBS control group is obviously lower than that of the mice in the plasminogen-given group, and the statistical difference is close to significant (P ═ 0.08). Indicating that the plasminogen can promote the secretion of the insulin of the T1DM mouse.

Examples

Example 1 plasminogen reduction of blood glucose in diabetic mice

24-25 week old db/db male mice were randomly divided into two groups, 5 for plasminogen and 3 for vehicle PBS control. The day of experiment start was recorded as day 0 and weighed into groups, day 1 on which plasminogen or PBS was administered, the tail vein of plasminogen group was injected with 2mg/0.2 ml/l/day of human plasminogen, and the tail vein of vehicle PBS control group was injected with the same volume of PBS for 31 consecutive days. After fasting for 16 hours on days 10, 31, blood glucose measurements were performed using blood glucose test strips (Roche, Mannheim, Germany).

The results showed that mice given plasminogen were significantly lower in blood glucose than the vehicle-given PBS control group, and the statistical difference was significant (P <0.05, P < 0.01). In addition, there was a tendency for blood glucose to rise in mice given vehicle PBS control group and to gradually decrease in blood glucose in plasminogen group with the time of administration (fig. 1). Indicating that the plasminogen has the function of reducing the blood sugar of diabetic animals.

Example 2 plasminogen reduction of fructosamine levels in diabetic mice

24-25 weeks old db/db male mice were treated with 50. mu.l blood from the venous plexus of the eyeball of each mouse the day before administration to measure the serum fructosamine concentration and recorded as day 0, plasminogen administration was started on the first day and administration was continued for 31 days. On day 32, blood was collected from the eye and the concentration of fructosamine in the serum was measured. The fructosamine concentration was detected using fructosamine detection kit (Nanjing institute of technology, A037-2).

Fructosamine concentration reflects the mean level of blood glucose over 1-3 weeks. The results show a significant decrease in serum fructosamine concentration following plasminogen administration, with a statistically significant difference compared to pre-administration (fig. 2). Thus showing that the plasminogen can effectively reduce the blood sugar of diabetic animals.

Example 3 plasminogen reduction of glycated hemoglobin levels in diabetic mice

9 db/db male mice 26 weeks old were randomly divided into two groups according to body weight after the day notes were weighed at the beginning of the experiment, 4 for plasminogen group and 5 for vehicle PBS control group. Plasminogen or PBS was administered at day 1, human plasminogen 2mg/0.2 ml/day was administered to tail vein of plasminogen group, and PBS of the same volume was administered to tail vein of vehicle PBS control group for 35 days. Mice were fasted for 16 hours on day 35 and blood was collected from the eyeball on day 36 to measure plasma glycated hemoglobin concentration.

The content of the glycosylated hemoglobin can generally reflect the blood sugar control condition of a patient for about 8-12 weeks. The results showed that the concentration of glycated hemoglobin in mice given plasminogen was significantly lower than that of the vehicle-given PBS control group, and the statistical difference was significant (fig. 3). Thus showing that the plasminogen can effectively reduce the blood sugar level of diabetic animals.

Example 4 plasminogen improves glucose tolerance in diabetic mice

26 weeks old db/db male mice 9 and db/m mice 3. On the day of the start of the experiment, db/db mice were weighed and randomly divided into two groups based on body weight, 4 for the plasminogen group and 5 for the vehicle PBS control group, and db/m mice were used as normal control groups. Plasminogen or PBS was administered starting on day 1, human plasminogen 2mg/0.2 ml/day was administered to tail vein of plasminogen group, and PBS of the same volume was administered to tail vein of vehicle PBS control group for 10 days. After fasting the mice for 16 hours on day 11, each mouse was intraperitoneally injected with 5% glucose solution at 5g/kg body weight, and blood glucose concentration was measured using blood glucose paper (Roche, Mannheim, Germany) at 0, 30, 60, 90, 120, 180 minutes.

The glucose tolerance test (IPGTT) can detect the glucose tolerance of the body. It is known in the prior art that diabetic patients have impaired glucose tolerance.

The results of the experiment show that the blood sugar level of mice given plasminogen group after intraperitoneal injection of glucose is lower than that of the mice given the vehicle PBS control group, and the glucose tolerance curve of the mice given the plasminogen group is closer to that of the mice given the normal group compared with the mice given the vehicle PBS control group (figure 4). Thus showing that the plasminogen can obviously improve the glucose tolerance of diabetic mice.

Example 5 plasminogen reduction of PLG activity normal mice blood glucose levels in the T1DM model

10 PLG active normal male mice 9-10 weeks old were randomly divided into two groups, 5 mice each, vehicle PBS control group and plasminogen group. T1DM was induced by a single intraperitoneal injection of 200mg/kg Streptozotocin (STZ) (sigma S0130) after 4 hours fasting in two groups of mice^[43]. Administration was initiated 12 days after STZ injection and recordedOn day 1 of administration, 1mg/0.1 ml/d of human-derived plasmin was injected into the tail vein of the plasminogen group, and the same volume of PBS was injected into the tail vein of the vehicle PBS control group for 10 consecutive days. After fasting for 6 hours on day 11, the blood glucose was measured using blood glucose test strips (Roche, Mannheim, Germany).

The results showed that mice given vehicle PBS control group had significantly higher blood glucose than mice given plasminogen, and the statistical difference was very significant (fig. 5). Indicating that the plasminogen can obviously reduce the blood sugar level of a T1DM model of a mouse with normal PLG activity.

Example 6 plasminogen improves glucose tolerance levels in T1DM model mice

15 PLG-active normal male mice 9-10 weeks old were randomly divided into three groups, a blank control group, a vehicle-administered PBS control group, and a plasminogen group, each of which was 5 mice. T1DM was induced by a single intraperitoneal injection of 200mg/kg STZ (sigma S0130) after fasting for 4 hours in vehicle PBS control group and plasminogen group mice^[43]The blank control group was not treated. The administration was started 12 days after STZ injection and recorded as day 1 of administration, 1mg/0.1 ml/day of human-derived plasmin was administered to the tail vein of the plasminogen group, and the same volume of PBS was administered to the tail vein of the vehicle PBS control group for 28 consecutive days. After fasting for 6 hours on day 28, mice were intraperitoneally injected with 5% glucose solution at 5g/kg body weight, and blood glucose concentration was measured with blood glucose test paper (Roche, Mannheim, Germany) at 0, 15, 30, 60, and 90 minutes after injection.

The glucose tolerance test (IPGTT) can detect the glucose tolerance of the body. It is known in the prior art that diabetic patients have impaired glucose tolerance.

The results showed that the blood glucose concentration after glucose injection was significantly higher in the vehicle PBS control group than in the plasminogen group, and that the glucose tolerance curve was closer to that of the normal mice than in the vehicle PBS control group (fig. 6). Indicating that the plasminogen can improve the glucose tolerance of a mouse T1DM model with normal PLG activity.

Example 7 plasminogen function in promoting insulin secretion in diabetic mice

9 db/db male mice 26 weeks old, scored day 0 at the start of the experiment, weighed and randomized into two groups based on body weight, 4 for plasminogen and 5 for vehicle PBS control. Plasminogen or PBS was administered at day 1, human plasminogen 2mg/0.2 ml/day was administered to tail vein of plasminogen group, and PBS of the same volume was administered to tail vein of vehicle PBS control group for 35 days. After fasting the mice for 16 hours on day 35, the eyes were bled on day 36, the supernatants were centrifuged and the serum insulin levels were measured using an insulin detection kit (Mercodia AB) according to the instructions.

The detection result shows that the serum insulin level of the plasminogen group is obviously higher than that of the vehicle-fed PBS control group, and the statistical difference is significant (figure 7). Thus showing that the plasminogen can obviously improve the insulin secretion of the diabetic mice.

Example 8 protective Effect of plasminogen on pancreas in diabetic mice

7 db/db male mice 24-25 weeks old, scored day 0 on the day of start of the experiment and weighed, randomly divided into two groups according to body weight, 4 for the plasminogen group and 3 for the vehicle PBS control group. On day 1, 2mg/0.2 ml/day of human plasminogen was administered to tail vein of plasminogen group, and the same volume of PBS was administered to tail vein of vehicle PBS control group for 31 consecutive days. Mice were sacrificed on day 32 and pancreata fixed in 4% paraformaldehyde. The fixed pancreas tissue is embedded in paraffin after alcohol gradient dehydration and xylene transparence. The tissue sections were 3 μm thick, the sections were dewaxed and rehydrated and stained with hematoxylin and eosin (HE stain), differentiated with 1% hydrochloric acid alcohol, rewetted with ammonia water, and alcohol-gradient dehydrated and mounted, and the sections were observed under 200 and 400-fold optical microscope.

The results showed that most of the islets were atrophied, the atrophied islet cells were replaced by acini (arrows) and the acini at the islet margins were hyperplastic, resulting in unclear demarcation between islets and acini, given to vehicle PBS control group (fig. 8A, 8B); most of the islets of Langerhans in the plasminogen group (FIGS. 8C and 8D) had larger area than the control group, and had no acinar hyperplasia in the islets, but a small number of acinus remained in the islets, and the boundary between the islets and acinus was clear. Comparing the area ratio of pancreatic islets in the pancreas between the administered group and the control group, it was found that the administered group was almost one-fold larger than the control group (fig. 8E). The plasminogen can promote the repair of the islet injury of the diabetic mouse, and the plasminogen is suggested to be possible to cure the diabetes radically by promoting the repair of the islet injury.

Example 9 plasminogen reduces islet collagen deposition in diabetic mice

16 db/db male mice 24-25 weeks old, scored day 0 on the day of start of the experiment and weighed, randomly divided into two groups according to body weight, 10 for the plasminogen group and 6 for the vehicle PBS control group. Plasminogen or PBS was administered from day 1, human plasminogen 2mg/0.2 ml/day was administered to tail vein of plasminogen group, and PBS of the same volume was administered to tail vein of vehicle PBS control group for 31 days. Mice were sacrificed on day 32 and pancreata fixed in 4% paraformaldehyde. The fixed pancreas tissue is embedded in paraffin after alcohol gradient dehydration and xylene transparence. The thickness of the tissue section is 3 μm, the section is washed with water 1 time after being dewaxed to water, and after being stained with 0.1% sirius red for 60 minutes, the section is washed with running water, stained with hematoxylin for 1 minute, washed with running water, differentiated by 1% hydrochloric acid alcohol and ammonia water and turned to blue, washed with running water, dried and then sealed, and the section is observed under an optical microscope of 200 times.

The sirius red staining can lead the collagen to be stained durably, and as a special staining method for pathological sections, the sirius red staining can specifically display the collagen tissues.

Staining results showed that islet collagen deposition (arrow) was significantly lower in mice given plasminogen (fig. 9B) than in vehicle-PBS control (fig. 9A) and the statistical difference was significant (fig. 9C). Indicating that the plasminogen can reduce the fibrosis of the islet of the diabetic animal.

Example 10 plasminogen reduction of islet cell apoptosis in diabetic mice

6 male mice 24-25 weeks old db/db, recorded as day 0 on the day of start of the experiment and weighed, were randomly divided into two groups based on body weight, 4 for the plasminogen group and 2 for the vehicle PBS control group. Plasminogen or PBS was administered from day 1, human plasminogen 2mg/0.2 ml/day was administered to tail vein of plasminogen group, and PBS of the same volume was administered to tail vein of vehicle PBS control group for 31 days. Mice were sacrificed on day 32 and pancreata fixed in 4% paraformaldehyde. The fixed pancreas tissue is embedded in paraffin after alcohol gradient dehydration and xylene transparence. The thickness of the tissue slice is 3 μm, and the slice is washed with water 1 time after dewaxing and rehydrating. Incubate with 3% hydrogen peroxide for 15 minutes, wash with water for 2 times, each time for 5 minutes. 5% normal sheep blood serum (vectorlaborators, inc., USA) was blocked for 1 hour; afterwards, the sheep serum was discarded and the tissue was circled with a PAP pen. Rabbit anti-mouse Caspase-3(Abcam) was incubated overnight at 4 ℃ and washed 2 times with PBS for 5 minutes each. Goat anti-rabbit igg (hrp) antibody (Abcam) secondary antibody was incubated for 1 hour at room temperature and washed 2 times with PBS for 5 minutes each. Color was developed according to DAB kit (Vector laboratories, Inc., USA), 3 washes were followed by hematoxylin counterstaining for 30 seconds and 5 minutes running water wash. The sections were observed under a 200-fold optical microscope.

Caspase-3 is the most important terminal cleavage enzyme in the process of apoptosis, and the more it expresses, the more cells in the apoptotic state^[44]。

The experimental results of the present invention showed that Caspase-3 expression (indicated by arrows) was significantly lower in the plasminogen group (FIG. 10B) than in the vehicle-administered PBS control group (FIG. 10A). Indicating that plasminogen can reduce apoptosis in islet cells.

Example 11 plasminogen promotes the expression and secretion of insulin in 18 week old diabetic mice

8 male mice, 18 weeks old db/db, were scored as day 0 and weighed on the day of the start of the experiment, and were randomly divided into two groups based on body weight, 4 each for the plasminogen group and vehicle-PBS control group. Plasminogen or PBS was administered from day 1, human plasminogen 2mg/0.2 ml/day was administered to tail vein of plasminogen group, and PBS of the same volume was administered to tail vein of vehicle PBS control group for 31 days. Mice were sacrificed on day 36 and pancreases were fixed in 4% paraformaldehyde. The fixed pancreas tissue is embedded in paraffin after alcohol gradient dehydration and xylene transparence. The thickness of the tissue slice is 3 μm, and the slice is washed with water 1 time after dewaxing and rehydrating. Incubate with 3% hydrogen peroxide for 15 minutes, wash with water for 2 times, each time for 5 minutes. 5% normal sheep blood serum (vectorlaborators, inc., USA) was blocked for 1 hour; afterwards, the sheep serum was discarded and the tissue was circled with a PAP pen. Rabbit anti-mouse insulin antibody (Abcam) was incubated overnight at 4 ℃ and washed 2 times with PBS for 5 minutes each. Goat anti-rabbit igg (hrp) antibody (Abcam) secondary antibody was incubated for 1 hour at room temperature and washed 2 times with PBS for 5 minutes each. Color was developed according to DAB kit (Vector laboratories, Inc., USA), 3 washes were followed by hematoxylin counterstaining for 30 seconds and 5 minutes running water wash. The sections were examined under a microscope at 200 x.

The results show that the expression of insulin (arrow) was significantly higher for the plasminogen group (fig. 11A) than for the vehicle PBS control group (fig. 11A) and the statistical difference was close to significant (P ═ 0.15) (fig. 11C). Indicating that the plasminogen can promote the functional repair of the pancreatic islet and promote the expression and secretion of insulin.

Example 12 plasminogen promotes the expression and secretion of insulin in 24-25 week old diabetic mice

24-25 weeks old db/db male mice were scored as day 0 and weighed on the day of the start of the experiment, and randomly divided into two groups based on body weight, 5 for the plasminogen group and 3 for the vehicle PBS control group. Plasminogen or PBS was administered from day 1, human plasminogen 2mg/0.2 ml/day was administered to tail vein of plasminogen group, and PBS of the same volume was administered to tail vein of vehicle PBS control group for 31 days. Mice were sacrificed on day 32 and pancreata fixed in 4% paraformaldehyde. The fixed pancreas tissue is embedded in paraffin after alcohol gradient dehydration and xylene transparence. The thickness of the tissue slice is 3 μm, and the slice is washed with water 1 time after dewaxing and rehydrating. Incubate with 3% hydrogen peroxide for 15 minutes, wash with water for 2 times, each time for 5 minutes. 5% normal sheep blood serum (vectorlaborators, inc., USA) was blocked for 1 hour; afterwards, the sheep serum was discarded and the tissue was circled with a PAP pen. Rabbit anti-mouse insulin antibody (Abcam) was incubated overnight at 4 ℃ and washed 2 times with PBS for 5 minutes each. Goat anti-rabbit igg (hrp) antibody (Abcam) secondary antibody was incubated for 1 hour at room temperature and washed 2 times with PBS for 5 minutes each. Color was developed according to DAB kit (Vector laboratories, Inc., USA), 3 washes were followed by hematoxylin counterstaining for 30 seconds and 5 minutes running water wash. The sections were examined under a microscope at 200 x.

The results showed that the expression of insulin given to the plasminogen group (arrow) was significantly higher than that given to the vehicle PBS control group, and the statistical difference was significant (P ═ 0.02) (fig. 12). Thus indicating that the plasminogen can effectively repair the function of the pancreatic islet and promote the expression and the secretion of the insulin.

Example 13 repair of plasminogen promoting insulin Synthesis and secretion in diabetic mice

9 db/db male mice 26 weeks old, scored day 0 on the day of start of the experiment and weighed, randomly divided into two groups based on body weight, 4 plasminogen groups and 5 vehicle PBS control groups. Plasminogen or PBS was administered at day 1, human plasminogen 2mg/0.2 ml/day was administered to tail vein of plasminogen group, and PBS of the same volume was administered to tail vein of vehicle PBS control group for 35 days. After fasting for 16 hours on day 35, mice were sacrificed on day 36 and pancreases were fixed in 4% paraformaldehyde. The fixed pancreas tissue is embedded in paraffin after alcohol gradient dehydration and xylene transparence. The thickness of the tissue slice is 3 μm, and the slice is washed with water 1 time after dewaxing and rehydrating. Incubate with 3% hydrogen peroxide for 15 minutes, wash with water for 2 times, each time for 5 minutes. 5% normal sheep blood serum (Vector laboratories, inc., USA) was blocked for 1 hour; afterwards, the sheep serum was discarded and the tissue was circled with a PAP pen. Rabbit anti-mouse insulin antibody (Abcam) was incubated overnight at 4 ℃ and washed 2 times with PBS for 5 minutes each. Goat anti-rabbit igg (hrp) antibody (Abcam) secondary antibody was incubated for 1 hour at room temperature and washed 2 times with PBS for 5 minutes each. Color was developed according to DAB kit (Vector laboratories, Inc., USA), 3 washes were followed by hematoxylin counterstaining for 30 seconds and 5 minutes running water wash. The sections were examined under a microscope at 200 x.

The results showed that the expression of insulin given to the plasminogen group (arrow) was significantly higher than that given to the vehicle PBS control group, and the statistical difference was very significant (P ═ 0.005) (fig. 13). Thus indicating that the plasminogen can effectively repair the functions of the diabetes mouse islet and improve the expression and secretion of insulin.

Example 14 plasminogen promotes the expression of the islet multidirection nuclear transcription factor NF- κ B in 24-25 week old diabetic mice

10 db/db male mice 24-25 weeks old, recording as day 0 on the day of the start of the experiment, weighing, randomly dividing into two groups according to body weight, giving 4 plasminogen groups and 6 vehicle PBS control groups, taking 4 db/m as a normal control group, and not processing the normal control group. Plasminogen or PBS was administered from day 1, human plasminogen 2mg/0.2 ml/day was administered to tail vein of plasminogen group, and PBS of the same volume was administered to tail vein of vehicle PBS control group for 31 days. Mice were sacrificed on day 32 and pancreata fixed in 4% paraformaldehyde. The fixed pancreas tissue is embedded in paraffin after alcohol gradient dehydration and xylene transparence. The thickness of the tissue slice is 3 μm, and the slice is washed with water 1 time after dewaxing and rehydrating. Incubate with 3% hydrogen peroxide for 15 minutes, wash with water for 2 times, each time for 5 minutes. 5% normal sheep blood serum (Vector laboratories, inc., USA) was blocked for 1 hour; afterwards, the sheep serum was discarded and the tissue was circled with a PAP pen. Rabbit anti-mouse NF-. kappa.B (Abcam) was incubated overnight at 4 ℃ and washed 2 times with PBS for 5 minutes each. Goat anti-rabbit igg (hrp) antibody (Abcam) secondary antibody was incubated for 1 hour at room temperature and washed 2 times with PBS for 5 minutes each. Color was developed according to DAB kit (Vector laboratories, Inc., USA), 3 washes were followed by hematoxylin counterstaining for 30 seconds and 5 minutes running water wash. The sections were examined under a microscope at 200 x.

NF-kB is a member of transcription factor protein family and plays an important role in the inflammatory repair process^[45]。

The experimental results of the invention show that the expression (marked by an arrow) of NF-kB given to the plasminogen group is obviously higher than that of a vehicle-given PBS control group, and the statistical difference is significant (FIG. 14). Indicating that the plasminogen can promote the expression of the multi-nuclear transcription factor NF-kB.

Example 15 plasminogen reduces proliferation of islet α cells in 18-week-old diabetic mice, restores normal islet α cell distribution and reduces glucagon secretion

8 db/db male mice 18 weeks old and 3 db/m male mice were scored as day 0 and weighed on the day of the start of the experiment, and the db/db mice were randomly divided into two groups according to body weight, 4 mice each were given to the plasminogen group and the vehicle-PBS control group, and the db/m mice were used as normal control groups. Plasminogen or PBS was given starting on day 1. 2mg/0.2 ml/day of human plasminogen is injected into tail vein of plasminogen group, and PBS with the same volume is injected into tail vein of vehicle PBS control group for continuous administration for 35 days. Mice were sacrificed on day 36 and pancreases were fixed in 4% paraformaldehyde. The fixed pancreas tissue is embedded in paraffin after alcohol gradient dehydration and xylene transparence. The thickness of the tissue slice is 3 μm, and the slice is washed with water 1 time after dewaxing and rehydrating. Tissues were circled with PAP pens, incubated with 3% hydrogen peroxide for 15 minutes, washed 2 times with 0.01MPBS, 5 minutes each. 5% normal sheep blood serum (vectorlaborators, inc., USA) was blocked for 30 minutes; at the end of the time, the sheep serum was discarded, and rabbit anti-mouse glucagon antibody (Abcam) was added dropwise and incubated overnight at 4 ℃ and washed 2 times with 0.01M PBS for 5 minutes each. Goat anti-rabbit igg (hrp) antibody (Abcam) secondary antibody was incubated for 1 hour at room temperature and washed 2 times with 0.01M PBS for 5 minutes each. Color was developed according to DAB kit (Vectorlab, Inc., USA), 3 washes were followed by hematoxylin counter staining for 30 seconds and 5 minutes in running water. Gradient alcohol dehydration, xylene clarity and neutral gum mounting, and sections were viewed under a 200-fold optical microscope.

The pancreatic islets α synthesize and secrete glucagon, which is mainly scattered in the peripheral regions of the islets.

The results show that compared with the plasminogen group (figure 15C), the glucagon positive cells (marked by arrows) are obviously increased in the vehicle PBS control group (figure 15B), the positive cells infiltrate into the central region of the pancreatic islets, and the result of the average optical density quantitative analysis has statistical difference (P <0.01) (figure 15D), the glucagon positive cells are dispersedly distributed at the periphery of the pancreatic islets in the plasminogen group, and the islet morphology of the plasminogen group is closer to the normal control group (15A) than that of the vehicle PBS group, which indicates that the plasminogen can obviously inhibit the proliferation of the pancreatic islets α cells and the secretion of the glucagon in 18-week-old diabetic mice, corrects the islet α cell distribution disorder, and prompts that the plasminogen promotes the repair of the pancreatic islet injury.

Example 16 plasminogen reduces proliferation of islet α cells in 24-25 week old diabetic mice, restores normal islet α cell distribution and reduces glucagon secretion

11 db/db male mice 24-25 weeks old and 5 db/m male mice, which were recorded as day 0 and weighed on the day of the start of the experiment, were randomly divided into two groups after weighing db/db mice, 5 plasminogen groups were given, 6 vehicle PBS control groups were given, and db/m mice were used as normal control groups. Plasminogen or PBS was given starting on day 1. 2mg/0.2 ml/day of human plasminogen is injected into tail vein of plasminogen group, and PBS with the same volume or without any liquid is injected into tail vein of vehicle PBS control group for 31 days. Mice were sacrificed on day 32 and pancreata fixed in 4% paraformaldehyde. The fixed pancreas tissue is embedded in paraffin after alcohol gradient dehydration and xylene transparence. The thickness of the tissue slice is 3 μm, and the slice is washed with water 1 time after dewaxing and rehydrating. Tissues were circled with PAP pens, incubated with 3% hydrogen peroxide for 15 minutes, and washed 2 times with 0.01MPBS for 5 minutes each. 5% normal sheep blood serum (Vector laboratories, inc., USA) was blocked for 30 minutes; at the end of the time, the sheep serum was discarded, and rabbit anti-mouse glucagon antibody (Abcam) was added dropwise and incubated overnight at 4 ℃ and washed 2 times with 0.01M PBS for 5 minutes each. Goat anti-rabbit igg (hrp) antibody (Abcam) secondary antibody was incubated for 1 hour at room temperature and washed 2 times with 0.01M PBS for 5 minutes each. Color was developed according to DAB kit (Vectorlab, Inc., USA), 3 washes were followed by hematoxylin counter staining for 30 seconds and 5 minutes in running water. Gradient alcohol dehydration, xylene clarity and neutral gum mounting, and sections were viewed under a 200-fold optical microscope.

The pancreatic islets α synthesize and secrete glucagon, which is mainly scattered in the peripheral regions of the islets.

The results show that compared with a plasminogen group (figure 16C), glucagon positive cells (marked by arrows) are obviously increased in a vehicle PBS control group (figure 16B), the positive cells infiltrate into the central region of pancreatic islets, the glucagon positive cells in the plasminogen group are scattered and distributed around the pancreatic islets, the pancreatic islet morphology in the plasminogen group is closer to that in a normal control group (16A) than that in the vehicle PBS group, which indicates that plasminogen can obviously inhibit proliferation of pancreatic islet α cells and secretion of glucagon in 24-25-week-old diabetic mice, corrects pancreatic islet α cell distribution disorder, and prompts that plasminogen can promote repair of pancreatic islet injury.

Example 17 plasminogen inhibits proliferation of islet α cells, restores normal distribution of islet α cells and reduces glucagon secretion in 26-week-old diabetic mice

9 db/db male mice and 3 db/m male mice of 26 weeks old were scored as day 0 and weighed on the day of the start of the experiment, and the db/db mice were weighed and then randomly divided into two groups, 4 plasmin group was given, 5 vehicle PBS control group was given, and db/m mice were used as normal control group. Plasminogen or PBS was given starting on day 1. 2mg/0.2 ml/day of human plasminogen is injected into tail vein of plasminogen group, and PBS with the same volume is injected into tail vein of vehicle PBS control group for continuous administration for 35 days. Mice were sacrificed on day 36 and pancreases were fixed in 4% paraformaldehyde. The fixed pancreas tissue is embedded in paraffin after alcohol gradient dehydration and xylene transparence. The thickness of the tissue slice is 3 μm, and the slice is washed with water 1 time after dewaxing and rehydrating. Tissues were circled with PAP pens, incubated with 3% hydrogen peroxide for 15 minutes, and washed 2 times with 0.01MPBS for 5 minutes each. 5% normal sheep blood serum (Vector laboratories, inc., USA) was blocked for 30 minutes; at the end of the time, the sheep serum was discarded, and rabbit anti-mouse glucagon antibody (Abcam) was added dropwise and incubated overnight at 4 ℃ and washed 2 times with 0.01M PBS for 5 minutes each. Goat anti-rabbit igg (hrp) antibody (Abcam) secondary antibody was incubated for 1 hour at room temperature and washed 2 times with 0.01M PBS for 5 minutes each. Color was developed according to DAB kit (Vector laboratories, Inc., USA), 3 washes were followed by hematoxylin counterstaining for 30 seconds and 5 minutes running water wash. Gradient alcohol dehydration, xylene clarity and neutral gum mounting, and sections were viewed under a 200-fold optical microscope.

The pancreatic islets α synthesize and secrete glucagon, which is mainly scattered in the peripheral regions of the islets.

The results show that compared with the plasminogen group (figure 17C), the glucagon positive cells (marked by arrows) are obviously increased in the vehicle PBS control group (figure 17B), the positive cells infiltrate into the central region of the pancreatic islets, and the result of the quantitative analysis of the average optical density has statistical difference (P <0.01) (figure 17D), the glucagon positive cells in the plasminogen group are scattered and distributed at the periphery of the pancreatic islets, and the islet morphology of the plasminogen group is closer to the normal control group (17A) than the vehicle PBS group, which indicates that the plasminogen can obviously inhibit the proliferation of the pancreatic islets α cells and the secretion of the glucagon of the diabetic mice with the age of 26 weeks, corrects the islet α cell distribution disorder, and prompts that the plasminogen can promote the repair of the pancreatic islet injury.

Example 18 secretion of glucagon in a mouse T1DM model with reduced PLG Activity of plasminogen

15 PLG-active normal male mice 9-10 weeks old were randomly divided into three groups, a blank control group, a vehicle-administered PBS control group, and a plasminogen group, each of which was 5 mice. Vehicle-administered PBS control group and plasminogen-administered groupMice were fasted for 4 hours and then induced by a single intraperitoneal injection of 200mg/kg STZ (Sigma, cat # S0130) into the T1DM model^[43]The blank control group was not treated. The administration is started 12 days after the injection and is set as the 1 st day, 1mg/0.1 ml/day of human-derived plasmin is injected into tail vein of plasminogen group, and PBS with the same volume is injected into tail vein of vehicle PBS control group for 28 days continuously. Mice were sacrificed on day 29 and pancreata fixed in 4% paraformaldehyde. The fixed pancreas tissue is embedded in paraffin after alcohol gradient dehydration and xylene transparence. The thickness of the tissue slice is 3 μm, and the slice is washed with water 1 time after dewaxing and rehydrating. Tissues were circled with PAP pens, incubated with 3% hydrogen peroxide for 15 minutes, and washed 2 times with 0.01MPBS for 5 minutes each. 5% normal sheep blood serum (Vector laboratories, inc., USA) was blocked for 30 minutes; at the end of the time, the sheep serum was discarded, and rabbit anti-mouse glucagon antibody (Abcam) was added dropwise and incubated overnight at 4 ℃ and washed 2 times with 0.01M PBS for 5 minutes each. Goat anti-rabbit igg (hrp) antibody (Abcam) secondary antibody was incubated for 1 hour at room temperature, washed 2 times with 0.01MPBS, each for 5 minutes. Color was developed according to DAB kit (Vector laboratories, Inc., USA), 3 washes were followed by hematoxylin counterstaining for 30 seconds and 5 minutes running water wash. Gradient alcohol dehydration, xylene clarity and neutral gum mounting, and sections were viewed under a 200-fold optical microscope.

Islet α cells synthesize and secrete glucagon, primarily distributed in the peripheral regions of the islets.

The results show that the vehicle-administered PBS control group (fig. 18B) had significantly more glucagon positive expression than the vehicle-administered PBS control group (fig. 18C), and that the mean optical density quantitation results were statistically significantly different (fig. 18D), and that the vehicle-administered PBS group was closer to the blank control group (18A) than the vehicle-administered PBS group.

Example 19 plasminogen promotes expression of insulin receptor substrate 2(IRS-2) in 18 week old diabetic mice

7 db/db male mice 18 weeks old and 3 db/m male mice were scored as day 0 and weighed on the day of the start of the experiment, and the db/db mice were randomly divided into two groups according to body weight, 3 plasmin groups and 4 vehicle PBS control groups, and db/m mice were used as normal control groups. Plasminogen or PBS was given starting on day 1. 2mg/0.2 ml/day of human plasminogen is injected into tail vein of plasminogen group, and PBS with the same volume is injected into tail vein of vehicle PBS control group for continuous administration for 35 days. Mice were sacrificed on day 36 and pancreases were fixed in 4% paraformaldehyde. The fixed pancreas tissue is embedded in paraffin after alcohol gradient dehydration and xylene transparence. The thickness of the tissue slice is 3 μm, and the slice is washed with water 1 time after dewaxing and rehydrating. Tissues were circled with PAP pens, incubated with 3% hydrogen peroxide for 15 minutes, and washed 2 times with 0.01MPBS for 5 minutes each. 5% normal sheep blood serum (Vector laboratories, inc., USA) was blocked for 30 minutes; after the time, the goat serum was discarded, and rabbit anti-mouse IRS-2 antibody (Abcam) was added dropwise thereto, incubated overnight at 4 ℃ and washed with 0.01M PBS for 5 minutes each time for 2 times. Goat anti-rabbit igg (hrp) antibody (Abcam) secondary antibody was incubated for 1 hour at room temperature and washed 2 times with 0.01M PBS for 5 minutes each. Color was developed according to DAB kit (Vector laboratories, Inc., USA), 3 washes were followed by hematoxylin counterstaining for 30 seconds and 5 minutes running water wash. Gradient alcohol dehydration, xylene clarity and neutral gum mounting, and sections were viewed under a 200-fold optical microscope.

Insulin Receptor Substrate 2(IRS-2) is a Substrate for the action of activated Insulin Receptor tyrosine kinase, is an important molecule in the Insulin signal transduction pathway and is very important for the survival of islet β cells, IRS-2 has a protective effect on islet β cells when their expression is increased and is crucial for the maintenance of functional islet β cells^[46,47]。

IRS-2 immunohistochemistry results showed that mice given vehicle PBS control group (fig. 19B) had significantly less islet IRS-2 positive expression (arrow) than mice given plasminogen group (fig. 19C) and the statistical difference was very significant (fig. 19D), with the plasminogen group being closer to the blank control group (19A) than the vehicle PBS group. Therefore, the plasminogen can effectively increase the expression of the IRS-2 of the islet cells of the 18-week-old diabetic mouse.

Example 20 plasminogen promotes the expression of islet IRS-2 in 24-25 week old diabetic mice

11 db/db male mice 24-25 weeks old and 5 db/m male mice were scored as day 0 and weighed on the day of the start of the experiment, and the db/db mice were randomly divided into two groups according to body weight, 5 were given to the plasminogen group, 6 were given to the vehicle PBS control group, and the db/m mice were used as normal control groups. Plasminogen or PBS was given starting on day 1. 2mg/0.2 ml/day of human plasminogen is injected into tail vein of plasminogen group, and PBS with the same volume or without any liquid is injected into tail vein of vehicle PBS control group for 31 days. Mice were sacrificed on day 32 and pancreata fixed in 4% paraformaldehyde. The fixed pancreas tissue is embedded in paraffin after alcohol gradient dehydration and xylene transparence. The thickness of the tissue slice is 3 μm, and the slice is washed with water 1 time after dewaxing and rehydrating. Tissues were circled with PAP pens, incubated with 3% hydrogen peroxide for 15 minutes, and washed 2 times with 0.01MPBS for 5 minutes each. 5% normal sheep blood serum (Vector laboratories, inc., USA) was blocked for 30 minutes; after the time, the goat serum was discarded, and rabbit anti-mouse IRS-2 antibody (Abcam) was added dropwise thereto, incubated overnight at 4 ℃ and washed with 0.01M PBS for 5 minutes each time for 2 times. Goat anti-rabbit igg (hrp) antibody (Abcam) secondary antibody was incubated for 1 hour at room temperature and washed 2 times with 0.01M PBS for 5 minutes each. Color was developed according to DAB kit (Vectorlab, Inc., USA), 3 washes were followed by hematoxylin counter staining for 30 seconds and 5 minutes in running water. Gradient alcohol dehydration, xylene clarity and neutral gum mounting, and sections were viewed under a 200-fold optical microscope.

IRS-2 immunohistochemistry results showed that mice given vehicle PBS control group (fig. 20B) had significantly less islet IRS-2 positive expression (arrow) than mice given plasminogen group (fig. 20C) and statistically significant differences (fig. 20D), with the plasminogen group being closer to the normal control group (21A) than the vehicle PBS group. Therefore, the plasminogen can effectively increase the expression of the IRS-2 of the islet cells of the 24-25-week-old diabetic mouse.

Example 21 plasminogen promotes expression of islet IRS-2 in 26 week old diabetic mice

9 db/db male mice 26 weeks old and 3 db/m male mice were scored as day 0 and weighed on the day of the start of the experiment, and the db/db mice were randomly divided into two groups according to body weight, 4 for the plasminogen group, 5 for the vehicle PBS control group, and db/m as the normal control group. Plasminogen or PBS was given starting on day 1. 2mg/0.2 ml/day of human plasminogen is injected into tail vein of plasminogen group, and PBS with the same volume is injected into tail vein of vehicle PBS control group for continuous administration for 35 days. Mice were sacrificed on day 36 and pancreases were fixed in 4% paraformaldehyde. The fixed pancreas tissue is embedded in paraffin after alcohol gradient dehydration and xylene transparence. The thickness of the tissue slice is 3 μm, and the slice is washed with water 1 time after dewaxing and rehydrating. Tissues were circled with PAP pens, incubated with 3% hydrogen peroxide for 15 minutes, and washed 2 times with 0.01MPBS for 5 minutes each. 5% normal sheep blood serum (Vector laboratories, inc., USA) was blocked for 30 minutes; after the time, the goat serum was discarded, and rabbit anti-mouse IRS-2 antibody (Abcam) was added dropwise thereto, incubated overnight at 4 ℃ and washed with 0.01M PBS for 5 minutes each time for 2 times. Goat anti-rabbit igg (hrp) antibody (Abcam) secondary antibody was incubated for 1 hour at room temperature and washed 2 times with 0.01M PBS for 5 minutes each. Color was developed according to DAB kit (Vector laboratories, Inc., USA), 3 washes were followed by hematoxylin counterstaining for 30 seconds and 5 minutes running water wash. Gradient alcohol dehydration, xylene clarity and neutral gum mounting, and sections were viewed under a 200-fold optical microscope.

The results of IRS-2 immunohistochemistry showed that the mice in the vehicle PBS control group (FIG. 21B) had significantly less positive expression of IRS-2 in the islets (arrow) than in the plasminogen group (FIG. 21C); the plasminogen group IRS-2 expression level was close to that of the normal control group mice (FIG. 21A). Therefore, the plasminogen can effectively increase the expression of the IRS-2 of the islet cells of the diabetic mouse with the age of 26 weeks.

Example 22 plasminogen promotion of PLG Activity Normal T1DM expression of mouse islet IRS-2

15 PLG-active normal male mice 9-10 weeks old were randomly divided into three groups, a blank control group, a vehicle-administered PBS control group, and a plasminogen group, each of which was 5 mice. To giveType I diabetes is induced by a single intraperitoneal injection of 200mg/kg STZ (Sigma, cat # S0130) after fasting for 4 hours in a vehicle PBS control group and mice giving plasminogen group^[43]The blank control group was not treated. The administration is started 12 days after the injection and is set as the 1 st day, 1mg/0.1 ml/day of human-derived plasmin is injected into tail vein of plasminogen group, and PBS with the same volume is injected into tail vein of vehicle PBS control group for 28 days continuously. Mice were sacrificed on day 29 and pancreata fixed in 4% paraformaldehyde. The fixed pancreas tissue is embedded in paraffin after alcohol gradient dehydration and xylene transparence. The thickness of the tissue slice is 3 μm, and the slice is washed with water 1 time after dewaxing and rehydrating. Tissues were circled with PAP pens, incubated with 3% hydrogen peroxide for 15 minutes, and washed 2 times with 0.01MPBS for 5 minutes each. 5% normal sheep blood serum (Vector laboratories, inc., USA) was blocked for 30 minutes; after the time, the goat serum was discarded, and rabbit anti-mouse IRS-2 antibody (Abcam) was added dropwise thereto, incubated overnight at 4 ℃ and washed with 0.01M PBS for 5 minutes each time for 2 times. Goat anti-rabbit igg (hrp) antibody (Abcam) secondary antibody was incubated for 1 hour at room temperature, washed 2 times with 0.01MPBS, 5 minutes each. Color was developed according to DAB kit (Vector laboratories, Inc., USA), 3 washes were followed by hematoxylin counterstaining for 30 seconds and 5 minutes running water wash. Gradient alcohol dehydration, xylene clarity and neutral gum mounting, and sections were viewed under a 200-fold optical microscope.

The IRS-2 immunohistochemistry results showed that mice given vehicle PBS control group (fig. 22B) had significantly less islet IRS-2 positive expression (arrow) than mice given vehicle PBS group (fig. 22C), and that the given vehicle PBS group was closer to the blank control group (22A) than the vehicle PBS group. Therefore, the plasminogen can effectively increase the expression of IRS-2 of the PLG active normal mouse islet cells of 9-10 weeks old.

Example 23 plasminogen reduction in islet neutrophil infiltration in 24-26 week old diabetic mice

9 db/db male mice 24-26 weeks old and 3 db/m mice were randomly divided into two groups, 4 mice were given to the plasminogen group, 5 mice were given to the vehicle PBS control group, and db/m mice were used as the normal control group. The day of experiment start was recorded as day 0 weight packet and the day of experiment start was given plasminogen or PBS and recorded as day 1. 2mg/0.2 ml/day of human plasminogen is injected into tail vein of plasminogen group, and PBS with the same volume is injected into tail vein of vehicle PBS control group for continuous administration for 35 days. Mice were sacrificed on day 36 and pancreases were fixed in 4% paraformaldehyde. The fixed pancreas tissue is embedded in paraffin after alcohol gradient dehydration and xylene transparence. The thickness of the tissue slice is 3 μm, and the slice is washed with water 1 time after dewaxing and rehydrating. Tissues were circled with PAP pens, incubated with 3% hydrogen peroxide for 15 minutes, and washed 2 times with 0.01MPBS for 5 minutes each. 5% normal sheep blood serum (vectorlaborators, inc., USA) was blocked for 30 minutes; after the time, the goat serum was discarded, and rabbit anti-mouse neutrophil antibody (Abcam) was added dropwise thereto and incubated overnight at 4 ℃ and washed with 0.01M PBS for 5 minutes each time for 2 times. Goat anti-rabbit igg (hrp) antibody (Abcam) secondary antibody was incubated for 1 hour at room temperature and washed 2 times with 0.01M PBS for 5 minutes each. Color was developed according to DAB kit (Vector laboratories, Inc., USA), 3 washes were followed by hematoxylin counterstaining for 30 seconds and 5 minutes running water wash. Gradient alcohol dehydration, xylene clarity and neutral gum mounting, and sections were viewed under a 200-fold optical microscope.

Central granulocytes are important members of the non-specific cellular immune system and when inflammation occurs, they are attracted to the site of inflammation by chemotactic substances.

The centrogranulocyte immunohistochemistry results showed that the positive expression cells were less in the plasminogen group (fig. 23C) than in the vehicle-PBS control group (fig. 23B), and that the plasminogen group was closer to the normal control group (23A) than the vehicle-PBS group.

Example 24 plasminogen reduction infiltration of islet neutrophils in the T1DM model in mice with impaired PLG Activity

10 male mice with 9-10 weeks old impaired PLG activity were randomly divided into three groups, 3 for the placebo group, 3 for the PBS control group, and 4 for the plasminogen group. Type I diabetes is induced by single intraperitoneal injection of 200mg/kg STZ (sigma S0130) after 4-hour fasting of mice of a vehicle PBS control group and a plasminogen group^[43]Blank control groupNo treatment is done. The administration is started 12 days after the injection and is set as the 1 st day, 1mg/0.1 ml/day of human-derived plasmin is injected into tail vein of plasminogen group, and PBS with the same volume is injected into tail vein of vehicle PBS control group for 28 days continuously. Mice were sacrificed on day 29 and pancreata fixed in 4% paraformaldehyde. The fixed pancreas tissue is embedded in paraffin after alcohol gradient dehydration and xylene transparence. The thickness of the tissue slice is 3 μm, and the slice is washed with water 1 time after dewaxing and rehydrating. Tissues were circled with PAP pens, incubated with 3% hydrogen peroxide for 15 minutes, and washed 2 times with 0.01MPBS for 5 minutes each. 5% normal sheep blood serum (Vector laboratories, inc., USA) was blocked for 30 minutes; after the time, the goat serum was discarded, and rabbit anti-mouse neutrophil antibody (Abcam) was added dropwise thereto and incubated overnight at 4 ℃ and washed with 0.01M PBS for 5 minutes each time for 2 times. Goat anti-rabbit igg (hrp) antibody (Abcam) secondary antibody was incubated for 1 hour at room temperature and washed 2 times with 0.01M PBS for 5 minutes each. Color was developed according to DAB kit (Vector laboratories, Inc., USA), 3 washes were followed by hematoxylin counterstaining for 30 seconds and 5 minutes running water wash. Gradient alcohol dehydration, xylene clarity and neutral gum mounting, sections were observed under a 400 x optical microscope.

The centrogranulocyte immunohistochemistry results showed that positively expressing cells (arrow) were less in the plasminogen group (fig. 24C) than in the vehicle-PBS control group (fig. 24B), and that the plasminogen group was closer to the blank control group (24A) than the vehicle-PBS group.

Example 25 plasminogen reduction of PLG Activity Normal mice infiltration of islet neutrophils in the T1DM model

11 PLG active normal male mice 9-10 weeks old were randomly divided into three groups, 3 mice in the blank control group, 4 mice in the vehicle PBS control group, and 4 mice in the plasminogen group. Type I diabetes is induced by single intraperitoneal injection of 200mg/kg STZ (sigma S0130) after 4-hour fasting of mice of a vehicle PBS control group and a plasminogen group^[43]The blank control group was not treated. The administration is started 12 days after the injection and is set as the 1 st day, 1mg/0.1 ml/day of human-source plasmin is injected into tail vein of plasminogen group, and tail vein of vehicle PBS control group is injectedThe same volume of PBS was injected and the administration continued for 28 days. Mice were sacrificed on day 29 and pancreata fixed in 4% paraformaldehyde. The fixed pancreas tissue is embedded in paraffin after alcohol gradient dehydration and xylene transparence. The thickness of the tissue slice is 3 μm, and the slice is washed with water 1 time after dewaxing and rehydrating. Tissues were circled with PAP pens, incubated with 3% hydrogen peroxide for 15 minutes, washed 2 times with 0.01MPBS, 5 minutes each. 5% normal sheep blood serum (Vector laboratories, inc., USA) was blocked for 30 minutes; after the time, the goat serum was discarded, and rabbit anti-mouse centrogranulocyte antibody (Abcam) was added dropwise thereto, incubated overnight at 4 ℃ and washed with 0.01M PBS for 5 minutes each time for 2 times. Goat anti-rabbit igg (hrp) antibody (Abcam) secondary antibody was incubated for 1 hour at room temperature, washed 2 times with 0.01MPBS, 5 minutes each. Color was developed according to DAB kit (Vector laboratories, Inc., USA), 3 washes were followed by hematoxylin counterstaining for 30 seconds and 5 minutes running water wash. Gradient alcohol dehydration, xylene clarity and neutral gum mounting, sections were observed under a 400 x optical microscope.

The centrogranulocyte immunohistochemistry results showed that positively expressing cells (arrow) were less in the plasminogen group (fig. 25C) than in the vehicle-PBS control group (fig. 25B), and that the plasminogen group was closer to the blank control group (25A) than the vehicle-PBS group.

Example 26 Prolysin promotes the synthesis and secretion of insulin from mice with impaired PLG Activity in the T1DM model

10 male mice with 9-10 weeks old PLG activity-impaired were randomly divided into three groups, 3 mice in the blank control group, 3 mice in the PBS control group, and 4 mice in the plasminogen group. Type I diabetes is induced by single intraperitoneal injection of 200mg/kg STZ (sigma S0130) after 4-hour fasting of mice of a vehicle PBS control group and a plasminogen group^[43]The blank control group was not treated. The administration is started 12 days after the injection and is set as the 1 st day, 1mg/0.1 ml/day of human-derived plasmin is injected into tail vein of plasminogen group, and PBS with the same volume is injected into tail vein of vehicle PBS control group for 28 days continuously. Mice were sacrificed on day 29 and pancreata fixed in 4% paraformaldehyde. After the fixed pancreas tissue is subjected to alcohol gradient dehydration and xylene transparenceAnd (5) carrying out paraffin embedding. The thickness of the tissue slice is 3 μm, and the slice is washed with water 1 time after dewaxing and rehydrating. Tissues were circled with PAP pens, incubated with 3% hydrogen peroxide for 15 minutes, and washed 2 times with 0.01MPBS for 5 minutes each. 5% normal sheep blood serum (Vector laboratories, inc., USA) was blocked for 30 minutes; after the time, the goat serum was discarded, and rabbit anti-mouse insulin antibody (Abcam) was added dropwise thereto, incubated overnight at 4 ℃ and washed with 0.01M PBS for 5 minutes each time for 2 times. Goat anti-rabbit igg (hrp) antibody (Abcam) secondary antibody was incubated for 1 hour at room temperature and washed 2 times with 0.01M PBS for 5 minutes each. Color was developed according to DAB kit (Vector laboratories, Inc., USA), 3 washes were followed by hematoxylin counterstaining for 30 seconds and 5 minutes running water wash. Gradient alcohol dehydration, xylene clarity and neutral gum mounting, and sections were viewed under a 200-fold optical microscope.

Immunohistochemistry results showed that insulin was positively expressed (arrow) more in the plasminogen group (fig. 26C) than in the vehicle-PBS control group (fig. 26B, and the plasminogen group was closer to the blank control group (26A) than the vehicle-PBS group). Indicating that plasminogen can promote synthesis and secretion of insulin in mice with impaired PLG activity in the T1DM model.

Example 27 Synthesis and expression of insulin from mice with Normal PLG Activity in plasminogen-promoted T1DM model

11 PLG active normal male mice 9-10 weeks old were randomly divided into three groups, 3 mice in the blank control group, 4 mice in the vehicle PBS control group, and 4 mice in the plasminogen group. Type I diabetes is induced by single intraperitoneal injection of 200mg/kg STZ (sigma S0130) after 4-hour fasting of mice of a vehicle PBS control group and a plasminogen group^[43]The blank control group was not treated. The administration is started 12 days after the injection and is set as the 1 st day, 1mg/0.1 ml/day of human-derived plasmin is injected into tail vein of plasminogen group, and PBS with the same volume is injected into tail vein of vehicle PBS control group for 28 days continuously. Mice were sacrificed on day 29 and pancreata fixed in 4% paraformaldehyde. The fixed pancreas tissue is embedded in paraffin after alcohol gradient dehydration and xylene transparence. The thickness of the tissue slice is 3 μm, and the slice is washed with water 1 time after dewaxing and rehydrating. PThe tissue was circled with AP pens, incubated with 3% hydrogen peroxide for 15 minutes, washed 2 times with 0.01MPBS, 5 minutes each time. 5% normal sheep blood serum (Vector laboratories, inc., USA) was blocked for 30 minutes; after the time, the goat serum was discarded, and rabbit anti-mouse insulin antibody (Abcam) was added dropwise thereto and incubated overnight at 4 ℃ and washed 2 times with 0.01MPBS for 5 minutes each. Goat anti-rabbit igg (hrp) antibody (Abcam) secondary antibody was incubated for 1 hour at room temperature and washed 2 times with 0.01M PBS for 5 minutes each. Color was developed according to DAB kit (Vector laboratories, Inc., USA), 3 washes were followed by hematoxylin counterstaining for 30 seconds and 5 minutes running water wash. Gradient alcohol dehydration, xylene clarity and neutral gum mounting, and sections were viewed under a 200-fold optical microscope.

Immunohistochemistry results showed that insulin was positively expressed (arrow) more than in vehicle-administered PBS control group (fig. 27B) to plasminogen group (fig. 27C), and that vehicle-administered PBS group was closer to blank control group (27A) than to vehicle-administered PBS group. Indicating that the plasminogen promotes synthesis and expression of insulin of mice with normal PLG activity in the T1DM model.

Example 28 plasminogen promotes expression of the islet pluripotency nuclear transcription factor NF- κ B in the mouse T1DM model with impaired PLG Activity

10 male mice with 9-10 weeks old PLG activity-impaired were randomly divided into three groups, 3 mice in the blank control group, 3 mice in the PBS control group, and 4 mice in the plasminogen group. Type I diabetes is induced by single intraperitoneal injection of 200mg/kg STZ (sigma S0130) after 4-hour fasting of mice of a vehicle PBS control group and a plasminogen group^[43]The blank control group was not treated. The administration is started 12 days after the injection and is set as the 1 st day, 1mg/0.1 ml/day of human-derived plasmin is injected into tail vein of plasminogen group, and PBS with the same volume is injected into tail vein of vehicle PBS control group for 28 days continuously. Mice were sacrificed on day 29 and pancreata fixed in 4% paraformaldehyde. The fixed pancreas tissue is embedded in paraffin after alcohol gradient dehydration and xylene transparence. The thickness of the tissue slice is 3 μm, and the slice is washed with water 1 time after dewaxing and rehydrating. Circling the tissue with PAP pen, incubating with 3% hydrogen peroxide for 15 min, washing with 0.01MPBS for 2 times, each for 5 min. 5% normal sheep blood serum (Vector laboratories, inc., USA) was blocked for 30 minutes; after the time, the goat serum was discarded, and rabbit anti-mouse NF-. kappa.B antibody (Cell Signal) was added dropwise thereto, incubated overnight at 4 ℃ and washed with 0.01M PBS for 5 minutes each time for 2 times. Goat anti-rabbit igg (hrp) antibody (Abcam) secondary antibody was incubated for 1 hour at room temperature and washed 2 times with 0.01M PBS for 5 minutes each. Color was developed according to DAB kit (Vector laboratories, Inc., USA), 3 washes were followed by hematoxylin counterstaining for 30 seconds and 5 minutes running water wash. Gradient alcohol dehydration, xylene clarity and neutral gum mounting, and sections were viewed under a 200-fold optical microscope.

NF-kB as a multi-nuclear transcription factor is activated to participate in the regulation of various genes such as cell proliferation, cell apoptosis, inflammation, immunity and the like^[24]。

The experimental results showed that NF-. kappa.B expression (indicated by arrows) was significantly higher in the plasminogen group (FIG. 28C) than in the vehicle-administered PBS control group (FIG. 28B). Indicating that the plasminogen can promote the expression of the multi-nuclear transcription factor NF-kB.

Example 29 plasminogen promotes expression of the islet pluripotency nuclear transcription factor NF- κ B in 18-week-old diabetic mice

7 male mice, 18 weeks old db/db, were scored as day 0 and weighed on the day of the start of the experiment, and were randomly divided into two groups based on body weight, 3 for the plasminogen group, and 4 for the vehicle PBS control group. Plasminogen or PBS was administered at day 1, human plasminogen 2mg/0.2 ml/day was administered to tail vein of plasminogen group, and PBS of the same volume was administered to tail vein of vehicle PBS control group for 35 days. Mice were sacrificed on day 36 and pancreases were fixed in 4% paraformaldehyde. The fixed pancreas tissue is embedded in paraffin after alcohol gradient dehydration and xylene transparence. The thickness of the tissue slice is 3 μm, and the slice is washed with water 1 time after dewaxing and rehydrating. Tissues were circled with PAP pens, incubated with 3% hydrogen peroxide for 15 minutes, and washed 2 times with 0.01MPBS for 5 minutes each. 5% normal sheep blood serum (Vector laboratories, inc., USA) was blocked for 30 minutes; after the time, the goat serum was discarded, and rabbit anti-mouse NF-. kappa.B antibody (Cell Signal) was added dropwise thereto, incubated overnight at 4 ℃ and washed with 0.01M PBS for 5 minutes each time for 2 times. Goat anti-rabbit igg (hrp) antibody (Abcam) secondary antibody was incubated for 1 hour at room temperature and washed 2 times with 0.01M PBS for 5 minutes each. Color was developed according to DAB kit (Vectorlab, Inc., USA), 3 washes were followed by hematoxylin counter staining for 30 seconds and 5 minutes in running water. Gradient alcohol dehydration, xylene clarity and neutral gum mounting, and sections were viewed under a 200-fold optical microscope.

The experimental results of the present invention showed that NF-. kappa.B expression (arrow) was significantly higher in the plasminogen group (FIG. 29B) than in the vehicle-administered PBS control group (FIG. 29A). Indicating that the plasminogen can promote the expression of the multi-nuclear transcription factor NF-kB.

Example 30 plasminogen promotes expression of the multiple nuclear transcription factor NF- κ B in 26-week-old diabetic mice

9 db/db male mice 26 weeks old and 3 db/m male mice were scored as day 0 and weighed on the day of the start of the experiment, and the db/db mice were randomly divided into two groups according to body weight, 4 for the plasminogen group, 5 for the vehicle PBS control group, and db/m as the normal control group. Plasminogen or PBS was administered starting on day 1 and recorded as day 1, 2mg/0.2 ml/day human plasminogen was administered intravenously to the tail of the plasminogen group, and the same volume of PBS was administered intravenously to the tail of the vehicle PBS control group for 35 consecutive days. Mice were sacrificed on day 36 and pancreases were fixed in 4% paraformaldehyde. The fixed pancreas tissue is embedded in paraffin after alcohol gradient dehydration and xylene transparence. The thickness of the tissue slice is 3 μm, and the slice is washed with water 1 time after dewaxing and rehydrating. Tissues were circled with PAP pens, incubated with 3% hydrogen peroxide for 15 minutes, and washed 2 times with 0.01MPBS for 5 minutes each. 5% normal sheep blood serum (vectorlaborators, inc., USA) was blocked for 30 minutes; after the time, the goat serum was discarded, and rabbit anti-mouse NF-. kappa.B antibody (Cell Signal) was added dropwise thereto, incubated overnight at 4 ℃ and washed with 0.01M PBS for 5 minutes each time for 2 times. Goat anti-rabbit igg (hrp) antibody (Abcam) secondary antibody was incubated for 1 hour at room temperature and washed 2 times with 0.01M PBS for 5 minutes each. Color was developed according to DAB kit (Vector laboratories, Inc., USA), 3 washes were followed by hematoxylin counterstaining for 30 seconds and 5 minutes running water wash. Gradient alcohol dehydration, xylene clarity and neutral gum mounting, and sections were viewed under a 200-fold optical microscope.

The experimental results showed that the expression of NF-. kappa.B (arrow) was significantly higher in the plasminogen-administered group (FIG. 30C) than in the vehicle-administered PBS control group (FIG. 30B), and that the plasminogen-administered group was closer to the normal control group (30A) than the vehicle-administered PBS group. Indicating that the plasminogen can promote the expression of the multi-nuclear transcription factor NF-kB in relatively old (26 weeks old) diabetic mice.

Example 31 plasminogen promotes expression of islet TNF- α in 24-25 week old diabetic mice

11 db/db male mice 24-25 weeks old and 5 db/M male mice, which were marked as day 0 and weighed on the start of the experiment, the db/db mice were randomly divided into two groups according to body weight, 5 plasminogen groups were given, 6 vehicle PBS control groups were given, db/M mice were used as normal control groups, plasminogen or PBS was given on day 1, 2mg/0.2 ml/day of human plasminogen was given to the tail vein of the plasminogen groups, the tail vein of the vehicle PBS control groups was given either the same volume of PBS or no liquid, 31 days were given continuously, mice were sacrificed on day 32, pancreas was fixed in 4% paraformaldehyde, pancreas was incubated with alcohol gradient dehydration and xylene clearing, paraffin embedding, tissue section thickness was 3 μ M, section dewaxing was washed with water 1 time, tissue was circled with 3% goat serum and washed with water, 3% ethyl alcohol, 15 minutes, 0.01MPBS 2 times, 5 minutes each time, 5% normal goat serum was washed with 5% PBS (normal serum, rabbit serum was washed with water for 1 minute, 5 minutes, rabbit serum after the wash with 3% alcohol gradient, rabbit serum was added, and the rabbit serum was washed with ethyl alcohol, 2 minutes, 5 minutes, and the rabbit serum was removed after the rabbit serum was washed with ethyl alcohol wash, the rabbit serum was added with water for 15 minutes, the rabbit serum was removed with the rabbit serum after the rabbit serum was added with the rabbit serum, the rabbit serum was washed with the rabbit serum after the rabbit serum was washed with the rabbit serum was added with the rabbit serum for 5 minutes after the rabbit serum was added with the rabbit serum after the rabbit serum was added with the rabbit.

Tumor necrosis factor α (Tumor)Necrosis Factor-α，TNFα) is mainly produced by activated monocytes/macrophages, an important proinflammatory factor^[48]。

The experimental results show that the positive expression of TNF- α in the plasminogen group (FIG. 31C) is obviously higher than that in the vehicle-fed PBS control group (FIG. 31B), and the plasminogen group is closer to the normal control group (31A) than the vehicle-fed PBS group, which indicates that the plasminogen can promote the expression of TNF- α in 24-25 weeks old diabetic mice.

Example 32 plasminogen promotes expression of islet TNF- α in 26 week old diabetic mice

9 db/db male mice 26 weeks old and 3 db/M male mice were assigned day 0 and weighed on the day of start of the experiment, db/db mice were randomly divided into two groups according to body weight, 4 plasminogen groups were administered, 5 vehicle PBS control groups were administered, db/M mice served as normal control groups, day 1 plasminogen or PBS was administered, 2mg/0.2 ml/one/day of human plasminogen was administered to the tail vein of the plasminogen groups, the tail vein of the vehicle PBS control groups was administered with the same volume of PBS or without any liquid, 35 days of continuous administration, mice were sacrificed on day 36, pancreas was fixed in 4% paraformaldehyde, PAP-embedded tissue was purified by alcohol gradient dehydration and xylene, tissue sections were paraffin embedded at a thickness of 3 μ M, water washed 1 after rewetting and rewetting, tissue enclosed by pen with 3% hydrogen peroxide solution for 15 minutes, 0.01MPBS 2 times, 5 minutes each time, 5% normal blood serum (PBS, 5% serum after rewetting and rat), and serum was washed with water for 1 min after rinsing with water, 2 minutes, 3% alcohol gradient, 5 minutes, 2 minutes of rabbit serum after incubating, 2 minutes of rabbit serum (abcambium), and after rinsing).

The results of the study showed that the positive expression of TNF- α was significantly higher in the plasminogen-administered group (FIG. 32C) than in the vehicle-administered PBS control group (FIG. 32B), and that the plasminogen-administered group was closer to the normal control group (32A) than the vehicle-administered PBS group.

Example 33 plasminogen promotion of expression of islet TNF- α in a T1DM model in mice with impaired PLG Activity

9-10 weeks old 7 male mice with impaired PLG activity were randomly divided into two groups, 3 for PBS control group and 4 for plasminogen group. Type I diabetes was induced by a single intraperitoneal injection of 200mg/kg STZ (sigma S0130) after 4 hours fasting in two groups of mice^[43]After 12 days of injection, the administration is started and set as day 1, 1mg/0.1 ml/day of human plasmin is injected into the tail vein of plasminogen group, the same volume of PBS is injected into the tail vein of PBS control group for 28 days of continuous administration, the mouse is sacrificed on day 29, pancreas is fixed in 4% paraformaldehyde, fixed pancreas tissue is paraffin-embedded after alcohol gradient dehydration and xylene clearing, tissue section thickness is 3 μ M, the section is dewaxed and rehydrated and then washed with water 1 time, PAP pen is circled out of the tissue, incubated with 3% hydrogen peroxide for 15 minutes, washed with 0.01MPBS for 2 times, 5 minutes each, 5% normal serum (Vector laboratories, inc., USA) for 30 minutes, after the time, the sheep serum is discarded, rabbit anti-mouse antibody TNF- α (Abcam) is added dropwise at 4 ℃, washed with 0.01M for 2 times, 5 minutes each, goat anti-rabbit antibody (hrp) for 1 hour, light anti-mouse antibody (Abcam) is added dropwise, washed with water for 1 hour, and washed with alcohol twice as a wash with water, 5 minutes, and then the supernatant is washed with alcohol under a neutral microscope for 2 minutes, and then the sections are washed with water for 5 minutes.

The experimental results show that the positive expression of TNF- α in the plasminogen group (FIG. 33B) is significantly higher than that in the vehicle-fed PBS control group (FIG. 33A), indicating that plasminogen can promote the expression of TNF- α in the T1DM model of mice with PLG activity impaired.

Example 34 plasminogen reduction of damaged islets in mice with impaired PLG Activity in T1DM model

10 male mice with 9-10 weeks old PLG activity-impaired were randomly divided into three groups, 3 mice in the blank control group, 3 mice in the PBS control group, and 4 mice in the plasminogen group. Type I diabetes is induced by single intraperitoneal injection of 200mg/kg STZ (sigma S0130) after 4-hour fasting of mice of a vehicle PBS control group and a plasminogen group^[43]The blank control group was not treated. The administration is started 12 days after the injection and is set as the 1 st day, 1mg/0.1 ml/day of human-derived plasmin is injected into tail vein of plasminogen group, and PBS with the same volume is injected into tail vein of vehicle PBS control group for 28 days continuously. Mice were sacrificed on day 29 and pancreata fixed in 4% paraformaldehyde. The fixed pancreas tissue is embedded in paraffin after alcohol gradient dehydration and xylene transparence. The thickness of the tissue slice is 3 μm, and the slice is washed with water 1 time after dewaxing and rehydrating. Tissues were circled with PAP pens, incubated with 3% hydrogen peroxide for 15 minutes, and washed 2 times with 0.01MPBS for 5 minutes each. 5% normal sheep blood serum (Vector laboratories, inc., USA) was blocked for 30 minutes; after the time was up, the sheep serum was discarded, goat anti-mouse IgM (HRP) antibody (Abcam) was added dropwise and incubated at room temperature for 1 hour, and washed with 0.01M PBS for 2 times for 5 minutes each. Color was developed according to DAB kit (Vector laboratories, Inc., USA), 3 washes were followed by hematoxylin counterstaining for 30 seconds and 5 minutes running water wash. Gradient alcohol dehydration, xylene clarity and neutral gum mounting, and sections were viewed under a 200-fold optical microscope.

The IgM antibody plays an important role in the process of eliminating apoptotic and necrotic cells, and the level of the IgM antibody in the damaged part of tissues and organs is positively correlated with the damage degree^[49,50]. Therefore, detecting the level of local IgM antibodies of a tissue organ can reflect the damage condition of the tissue organ.

The results of the study showed that positive IgM expression was significantly lower in the plasminogen group (fig. 34C) than in the vehicle PBS control group (fig. 34B), which was closer to the blank control group (34A) than in the vehicle PBS group. Indicating that plasminogen can reduce IgM expression, suggesting that plasminogen can reduce islet damage in the mouse T1DM model with impaired PLG activity.

Example 35 plasminogen reduction apoptosis in islet cells of 24-25 week old diabetic mice

11 db/db male mice 24-25 weeks old and 5 db/m male mice were scored as day 0 and weighed on the day of the start of the experiment, and the db/db mice were randomly divided into two groups according to body weight, 5 were given to the plasminogen group, 6 were given to the vehicle PBS control group, and the db/m mice were used as normal control groups. Plasminogen or PBS was administered starting on day 1, human plasminogen 2mg/0.2 ml/day was administered to tail vein of plasminogen group, and the vehicle PBS control group was administered with the same volume of PBS or without any liquid to tail vein for 31 consecutive days. Mice were sacrificed on day 32 and pancreata fixed in 4% paraformaldehyde. The fixed pancreas tissue is embedded in paraffin after alcohol gradient dehydration and xylene transparence. The thickness of the tissue slice is 3 μm, and the slice is washed with water 1 time after dewaxing and rehydrating. The tissues were circled with PAP pen, the proteinase K working solution was added dropwise to cover the tissues, incubated at room temperature for 7min, and washed 3 times with 0.01M PBS, each for 3 min. The TUNEL kit (Roche) reagent 1 and reagent 2 mixture (5:45) was added dropwise, incubated at 37 ℃ for 40min, and washed 3 times with 0.01M PBS, each for 3 minutes. 3% hydrogen peroxide solution (hydrogen peroxide: methanol 1:9) in methanol was added dropwise thereto, and the mixture was incubated at room temperature for 20 minutes in the dark, and washed 3 times with 0.01M PBS for 3 minutes each. Adding reagent 3 of the tunel kit dropwise, incubating at the constant temperature of 37 ℃ for 30min, washing for 3 times with 0.01MPBS, developing with DAB kit (Vector laboratories, Inc., USA), washing for 3 times, performing hematoxylin counterstaining for 30 seconds, and washing for 5 minutes with running water. Gradient alcohol dehydration, xylene clarity and neutral gum mounting, and sections were viewed under a 200-fold optical microscope.

TUNEL staining can be used to detect fragmentation of nuclear DNA during late apoptosis in tissue cells.

The results of this experiment show that the number of positive cells (indicated by arrows) was significantly less for the plasminogen group (FIG. 35C) than for the vehicle PBS control group (FIG. 35B). TUNEL positive staining was very low in the normal control group (fig. 35A). The apoptosis rate of the normal control group is about 8 percent, the apoptosis rate of the vehicle PBS group is about 93 percent, and the apoptosis rate of the plasminogen group is about 16 percent. Thus showing that the plasminogen group can obviously reduce the apoptosis of the islet cells of the diabetic mice.

Example 36 plasminogen reduces serum fructosamine levels in 26 week old diabetic mice

9 db/db male mice 26 weeks old, scored day 0 on the day of start of the experiment and weighed, randomly divided into two groups based on body weight, 4 for the plasminogen group and 5 for the vehicle PBS control group. 2mg/0.2 mL/L/day of human plasminogen was injected into tail vein of plasminogen group, and the same volume of PBS was injected into tail vein of vehicle PBS control group. Plasminogen or PBS was administered starting on day 1 for 35 consecutive days. Mice were sacrificed on day 36 and serum fructosamine concentrations were measured. The fructosamine concentration was detected using fructosamine detection kit (Nanjing institute of technology, A037-2).

The test results show that the concentration of fructosamine in serum given to the plasminogen group is obviously lower than that of the vehicle-given PBS control group, and the statistical difference is close to significant (P is 0.06) (FIG. 36). Indicating that the plasminogen can reduce the blood sugar fructosamine of the diabetic mice of 26 weeks old.

Example 37 plasminogen enhances glucose-decomposing ability of T1DM model mice

8 male mice, 9-10 weeks old, C57, were randomized into two groups, 4 each, vehicle PBS control and plasminogen. T1DM was induced by a single intraperitoneal injection of 200mg/kg Streptozotocin (STZ) (sigma S0130) 4 hours after fasting in vehicle PBS control group and plasminogen group mice [43 ]. The administration was started 12 days after STZ injection and was set to day 1, the tail vein of plasminogen group was injected with 1mg/0.1 ml/day of human-derived plasmin, and the tail vein of vehicle PBS control group was injected with the same volume of PBS. After a continuous administration for 19 days, on the 20 th day after the mice were fasted for 6 hours, 20% glucose was perfused at 2g/kg body weight, and after 60 minutes, blood was collected from orbital venous plexus and the supernatant was centrifuged to determine blood glucose using a glucose assay kit (shanghai rongsheng 361500).

The results showed that the blood glucose of vehicle-administered PBS control mice was significantly higher than that of plasminogen mice, and the statistical difference was significant (P ═ 0.04) (fig. 37). Therefore, the plasminogen can improve the glucose decomposition capability of the T1DM mouse, thereby reducing the blood sugar.

Example 38 plasminogen improves insulin secretion in T1DM model mice

6 male mice, 9-10 weeks old, C57, were randomly divided into two groups, 3 each for vehicle PBS control and plasminogen. T1DM was induced by a single intraperitoneal injection of 200mg/kg Streptozotocin (STZ) (sigma S0130) after 4 hours fasting in two groups of mice^[43]. The administration was started 12 days after STZ injection and was set to day 1, the tail vein of plasminogen group was injected with 1mg/0.1 ml/day of human-derived plasmin, and the tail vein of vehicle PBS control group was injected with the same volume of PBS. After fasting for 6 hours on day 21, mice were bled from the venous plexus of the eyeball, centrifuged to collect the supernatant, and the serum insulin concentration was measured using an insulin detection kit (Mercodia AB) according to the instructions for use, for 20 consecutive days.

The results showed that the insulin concentration was significantly lower in the vehicle-administered PBS control mice than in the plasminogen-administered mice, and the statistical difference was nearly significant (P ═ 0.08) (fig. 38). Indicating that the plasminogen can promote the secretion of the insulin of the T1DM mouse.

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sequence listing

<110> Shenzhen Rizhen Life sciences research institute Limited

<120> a novel method for treating diabetes

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Ile Ile Ile Arg Met Arg Asp Val Val Leu Phe Glu Lys Lys Val Tyr

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Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser Glu Gly Leu

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Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln Gly Pro Trp

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Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys Asp Ile Leu

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Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn Tyr Asp Gly

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Gln Ser Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe Pro Asn Lys

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Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala Val Thr Val

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Ser Gly His Thr Cys Gln His Trp Ser Ala Gln Thr Pro His Thr His

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Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp Glu Asn Tyr

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Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp Cys His Thr Thr Asn

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Ser Gln Val Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys Asp Ser Ser

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Pro Val Ser Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro Glu Leu Thr

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Pro Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser Tyr Arg Gly

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Thr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser Trp Ser Ser

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Met Thr Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr Pro Asn Ala

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Gly LeuThr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp Lys Gly Pro

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Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr Cys Asn Leu

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Lys Lys Cys Ser Gly Thr Glu Ala Ser Val Val Ala Pro Pro Pro Val

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Val Leu Leu Pro Asp Val Glu Thr Pro Ser Glu Glu Asp Cys Met Phe

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Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala Thr Thr Val Thr Gly

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Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro His Arg His Ser Ile

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Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu Glu Lys Asn Tyr Cys

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Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr Thr Thr Asn

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Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys Ala Ala Pro

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Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys Pro Gly

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Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro Trp Gln

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Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly Thr Leu

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Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu Lys Ser

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Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln Glu Val

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Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu Phe Leu

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Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser Pro Ala

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Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro Asn Tyr

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Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly Glu Thr

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Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro Val

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Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly Arg Val

705 710 715 720

Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr Asp Ser

725 730 735

Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys

740 745 750

Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro

755 760 765

Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp Ile

770 775 780

Glu Gly Val Met Arg Asn Asn

785 790

<210>3

<211>2433

<212>DNA

<213> nucleic acid sequence of native plasminogen (derived from swiss prot) containing signal peptide

<400>3

atggaacata aggaagtggt tcttctactt cttttatttc tgaaatcagg tcaaggagag 60

cctctggatg actatgtgaa tacccagggg gcttcactgt tcagtgtcac taagaagcag 120

ctgggagcag gaagtataga agaatgtgca gcaaaatgtg aggaggacga agaattcacc 180

tgcagggcat tccaatatca cagtaaagag caacaatgtg tgataatggc tgaaaacagg 240

aagtcctcca taatcattag gatgagagat gtagttttat ttgaaaagaa agtgtatctc 300

tcagagtgca agactgggaa tggaaagaac tacagaggga cgatgtccaa aacaaaaaat 360

ggcatcacct gtcaaaaatg gagttccact tctccccaca gacctagatt ctcacctgct 420

acacacccct cagagggact ggaggagaac tactgcagga atccagacaa cgatccgcag 480

gggccctggt gctatactac tgatccagaa aagagatatg actactgcga cattcttgag 540

tgtgaagagg aatgtatgca ttgcagtgga gaaaactatg acggcaaaat ttccaagacc 600

atgtctggac tggaatgcca ggcctgggac tctcagagcc cacacgctca tggatacatt 660

ccttccaaat ttccaaacaa gaacctgaag aagaattact gtcgtaaccc cgatagggag 720

ctgcggcctt ggtgtttcac caccgacccc aacaagcgct gggaactttg tgacatcccc 780

cgctgcacaa cacctccacc atcttctggt cccacctacc agtgtctgaa gggaacaggt 840

gaaaactatc gcgggaatgt ggctgttacc gtgtccgggc acacctgtca gcactggagt 900

gcacagaccc ctcacacaca taacaggaca ccagaaaact tcccctgcaa aaatttggat 960

gaaaactact gccgcaatcc tgacggaaaa agggccccat ggtgccatac aaccaacagc 1020

caagtgcggt gggagtactg taagataccg tcctgtgact cctccccagt atccacggaa 1080

caattggctc ccacagcacc acctgagcta acccctgtgg tccaggactg ctaccatggt 1140

gatggacaga gctaccgagg cacatcctcc accaccacca caggaaagaa gtgtcagtct 1200

tggtcatcta tgacaccaca ccggcaccag aagaccccag aaaactaccc aaatgctggc 1260

ctgacaatga actactgcag gaatccagat gccgataaag gcccctggtg ttttaccaca 1320

gaccccagcg tcaggtggga gtactgcaac ctgaaaaaat gctcaggaac agaagcgagt 1380

gttgtagcac ctccgcctgt tgtcctgctt ccagatgtag agactccttc cgaagaagac 1440

tgtatgtttg ggaatgggaa aggataccga ggcaagaggg cgaccactgt tactgggacg 1500

ccatgccagg actgggctgc ccaggagccc catagacaca gcattttcac tccagagaca 1560

aatccacggg cgggtctgga aaaaaattac tgccgtaacc ctgatggtga tgtaggtggt 1620

ccctggtgct acacgacaaa tccaagaaaa ctttacgact actgtgatgt ccctcagtgt 1680

gcggcccctt catttgattg tgggaagcct caagtggagc cgaagaaatg tcctggaagg 1740

gttgtagggg ggtgtgtggc ccacccacat tcctggccct ggcaagtcag tcttagaaca 1800

aggtttggaa tgcacttctg tggaggcacc ttgatatccc cagagtgggt gttgactgct 1860

gcccactgct tggagaagtc cccaaggcct tcatcctaca aggtcatcct gggtgcacac 1920

caagaagtga atctcgaacc gcatgttcag gaaatagaag tgtctaggct gttcttggag 1980

cccacacgaa aagatattgc cttgctaaag ctaagcagtc ctgccgtcat cactgacaaa 2040

gtaatcccag cttgtctgcc atccccaaat tatgtggtcg ctgaccggac cgaatgtttc 2100

atcactggct ggggagaaac ccaaggtact tttggagctg gccttctcaa ggaagcccag 2160

ctccctgtga ttgagaataa agtgtgcaat cgctatgagt ttctgaatgg aagagtccaa 2220

tccaccgaac tctgtgctgg gcatttggcc ggaggcactg acagttgcca gggtgacagt 2280

ggaggtcctc tggtttgctt cgagaaggac aaatacattt tacaaggagt cacttcttgg 2340

ggtcttggct gtgcacgccc caataagcct ggtgtctatg ttcgtgtttc aaggtttgtt 2400

acttggattg agggagtgat gagaaataat taa 2433

<210>4

<211>810

<212>PRT

<213> amino acid sequence of native plasminogen (derived from swiss prot) containing signal peptide

<400>4

Met Glu His Lys Glu Val Val Leu Leu Leu Leu Leu Phe Leu Lys Ser

1 5 10 15

Gly Gln Gly Glu Pro Leu Asp Asp Tyr Val Asn Thr Gln Gly Ala Ser

20 25 30

Leu Phe Ser Val Thr Lys Lys Gln Leu Gly Ala Gly Ser Ile Glu Glu

35 40 45

Cys Ala Ala Lys Cys Glu Glu Asp Glu Glu Phe Thr Cys Arg Ala Phe

50 55 60

Gln Tyr His Ser Lys Glu Gln Gln Cys Val Ile Met Ala Glu Asn Arg

65 70 75 80

Lys Ser Ser Ile Ile Ile Arg Met Arg Asp Val Val Leu Phe Glu Lys

85 90 95

Lys Val Tyr Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg

100 105 110

Gly Thr Met Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser

115 120 125

Ser Thr Ser Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser

130 135 140

Glu Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln

145 150 155 160

Gly Pro Trp Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys

165 170 175

Asp Ile Leu Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn

180 185 190

Tyr Asp Gly Lys Ile Ser Lys Thr Met Ser Gly Leu Glu Cys Gln Ala

195 200 205

Trp Asp Ser Gln Ser Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe

210 215 220

Pro Asn Lys Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu

225 230 235 240

Leu Arg Pro Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu

245 250 255

Cys Asp Ile Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr

260 265 270

Tyr Gln Cys Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala

275 280 285

Val Thr Val Ser Gly His Thr Cys Gln His Trp Ser Ala Gln Thr Pro

290 295 300

His Thr His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp

305 310 315 320

Glu Asn Tyr Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp Cys His

325 330 335

Thr Thr Asn Ser Gln Val Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys

340 345 350

Asp Ser Ser Pro Val Ser Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro

355 360 365

Glu Leu Thr Pro Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser

370 375 380

Tyr Arg Gly Thr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser

385 390 395 400

Trp Ser Ser Met Thr Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr

405 410 415

Pro Asn Ala Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp

420 425 430

Lys Gly Pro Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr

435 440 445

Cys Asn LeuLys Lys Cys Ser Gly Thr Glu Ala Ser Val Val Ala Pro

450 455 460

Pro Pro Val Val Leu Leu Pro Asp Val Glu Thr Pro Ser Glu Glu Asp

465 470 475 480

Cys Met Phe Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala Thr Thr

485 490 495

Val Thr Gly Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro His Arg

500 505 510

His Ser Ile Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu Glu Lys

515 520 525

Asn Tyr Cys Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr

530 535 540

Thr Thr Asn Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys

545 550 555 560

Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys

565 570 575

Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp

580 585 590

Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly

595 600 605

Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu

610 615 620

Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His

625 630 635 640

Gln Glu Val Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg

645 650 655

Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser

660 665 670

Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser

675 680 685

Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp

690 695 700

Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln

705 710 715 720

Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn

725 730 735

Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly

740 745 750

Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu

755 760 765

Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys

770 775 780

Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val

785 790 795 800

Thr Trp Ile Glu Gly Val Met Arg Asn Asn

805 810

<210>5

<211>2145

<212>DNA

<213> LYS77-PLG (Lys-plasminogen) nucleic acid sequences

<400>5

aaagtgtatc tctcagagtg caagactggg aatggaaaga actacagagg gacgatgtcc 60

aaaacaaaaa atggcatcac ctgtcaaaaa tggagttcca cttctcccca cagacctaga 120

ttctcacctg ctacacaccc ctcagaggga ctggaggaga actactgcag gaatccagac 180

aacgatccgc aggggccctg gtgctatact actgatccag aaaagagata tgactactgc 240

gacattcttg agtgtgaaga ggaatgtatg cattgcagtg gagaaaacta tgacggcaaa 300

atttccaaga ccatgtctgg actggaatgc caggcctggg actctcagag cccacacgct 360

catggataca ttccttccaa atttccaaac aagaacctga agaagaatta ctgtcgtaac 420

cccgataggg agctgcggcc ttggtgtttc accaccgacc ccaacaagcg ctgggaactt 480

tgtgacatcc cccgctgcac aacacctcca ccatcttctg gtcccaccta ccagtgtctg 540

aagggaacag gtgaaaacta tcgcgggaat gtggctgtta ccgtgtccgg gcacacctgt 600

cagcactgga gtgcacagac ccctcacaca cataacagga caccagaaaa cttcccctgc 660

aaaaatttgg atgaaaacta ctgccgcaat cctgacggaa aaagggcccc atggtgccat 720

acaaccaaca gccaagtgcg gtgggagtac tgtaagatac cgtcctgtga ctcctcccca 780

gtatccacgg aacaattggc tcccacagca ccacctgagc taacccctgt ggtccaggac 840

tgctaccatg gtgatggaca gagctaccga ggcacatcct ccaccaccac cacaggaaag 900

aagtgtcagt cttggtcatc tatgacacca caccggcacc agaagacccc agaaaactac 960

ccaaatgctg gcctgacaat gaactactgc aggaatccag atgccgataa aggcccctgg 1020

tgttttacca cagaccccag cgtcaggtgg gagtactgca acctgaaaaa atgctcagga 1080

acagaagcga gtgttgtagc acctccgcct gttgtcctgc ttccagatgt agagactcct 1140

tccgaagaag actgtatgtt tgggaatggg aaaggatacc gaggcaagag ggcgaccact 1200

gttactggga cgccatgcca ggactgggct gcccaggagc cccatagaca cagcattttc 1260

actccagaga caaatccacg ggcgggtctg gaaaaaaatt actgccgtaa ccctgatggt 1320

gatgtaggtg gtccctggtg ctacacgaca aatccaagaa aactttacga ctactgtgat 1380

gtccctcagt gtgcggcccc ttcatttgat tgtgggaagc ctcaagtgga gccgaagaaa 1440

tgtcctggaa gggttgtagg ggggtgtgtg gcccacccac attcctggcc ctggcaagtc 1500

agtcttagaa caaggtttgg aatgcacttc tgtggaggca ccttgatatc cccagagtgg 1560

gtgttgactg ctgcccactg cttggagaag tccccaaggc cttcatccta caaggtcatc 1620

ctgggtgcac accaagaagt gaatctcgaa ccgcatgttc aggaaataga agtgtctagg 1680

ctgttcttgg agcccacacg aaaagatatt gccttgctaa agctaagcag tcctgccgtc 1740

atcactgaca aagtaatccc agcttgtctg ccatccccaa attatgtggt cgctgaccgg 1800

accgaatgtt tcatcactgg ctggggagaa acccaaggta cttttggagc tggccttctc 1860

aaggaagccc agctccctgt gattgagaat aaagtgtgca atcgctatga gtttctgaat 1920

ggaagagtcc aatccaccga actctgtgct gggcatttgg ccggaggcac tgacagttgc 1980

cagggtgaca gtggaggtcc tctggtttgc ttcgagaagg acaaatacat tttacaagga 2040

gtcacttctt ggggtcttgg ctgtgcacgc cccaataagc ctggtgtcta tgttcgtgtt 2100

tcaaggtttg ttacttggat tgagggagtg atgagaaata attaa 2145

<210>6

<211>714

<212>PRT

<213> LYS77-PLG (Lys-plasminogen) amino acid sequence

<400>6

Lys Val Tyr Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg

1 5 10 15

Gly Thr Met Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser

20 25 30

Ser Thr Ser Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser

35 40 45

Glu Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln

50 55 60

Gly Pro Trp Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys

65 70 75 80

Asp Ile Leu Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn

85 90 95

Tyr Asp Gly Lys Ile Ser Lys Thr Met Ser Gly Leu Glu Cys Gln Ala

100105 110

Trp Asp Ser Gln Ser Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe

115 120 125

Pro Asn Lys Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu

130 135 140

Leu Arg Pro Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu

145 150 155 160

Cys Asp Ile Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr

165 170 175

Tyr Gln Cys Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala

180 185 190

Val Thr Val Ser Gly His Thr Cys Gln His Trp Ser Ala Gln Thr Pro

195 200 205

His Thr His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp

210 215 220

Glu Asn Tyr Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp Cys His

225 230 235 240

Thr Thr Asn Ser Gln Val Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys

245 250 255

Asp Ser Ser Pro Val Ser Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro

260 265 270

Glu Leu Thr Pro Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser

275 280 285

Tyr Arg Gly Thr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser

290 295 300

Trp Ser Ser Met Thr Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr

305 310 315 320

Pro Asn Ala Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp

325 330 335

Lys Gly Pro Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr

340 345 350

Cys Asn Leu Lys Lys Cys Ser Gly Thr Glu Ala Ser Val Val Ala Pro

355 360 365

Pro Pro Val Val Leu Leu Pro Asp Val Glu Thr Pro Ser Glu Glu Asp

370 375 380

Cys Met Phe Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala Thr Thr

385 390 395 400

Val Thr Gly Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro His Arg

405 410 415

His Ser Ile Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu Glu Lys

420 425 430

Asn Tyr Cys Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr

435 440 445

Thr Thr Asn Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys

450 455 460

Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys

465 470 475 480

Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp

485 490 495

Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly

500 505 510

Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu

515 520 525

Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His

530 535 540

Gln Glu Val Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg

545 550 555 560

Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser

565 570 575

Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser

580 585 590

Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp

595 600 605

Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln

610 615 620

Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn

625 630 635 640

Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly

645 650 655

Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu

660 665 670

Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys

675 680 685

Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val

690 695 700

Thr Trp Ile Glu Gly Val Met Arg Asn Asn

705 710

<210>7

<211>1245

<212>DNA

<213> Delta-plg (Delta-plasminogen) nucleic acid sequence

<400>7

gagcctctgg atgactatgt gaatacccag ggggcttcac tgttcagtgt cactaagaag 60

cagctgggag caggaagtat agaagaatgt gcagcaaaat gtgaggagga cgaagaattc 120

acctgcaggg cattccaata tcacagtaaa gagcaacaat gtgtgataat ggctgaaaac 180

aggaagtcct ccataatcat taggatgaga gatgtagttt tatttgaaaa gaaagtgtat 240

ctctcagagt gcaagactgg gaatggaaag aactacagag ggacgatgtc caaaacaaaa 300

aatggcatca cctgtcaaaa atggagttcc acttctcccc acagacctag attctcacct 360

gctacacacc cctcagaggg actggaggag aactactgca ggaatccaga caacgatccg 420

caggggccct ggtgctatac tactgatcca gaaaagagatatgactactg cgacattctt 480

gagtgtgaag aggcggcccc ttcatttgat tgtgggaagc ctcaagtgga gccgaagaaa 540

tgtcctggaa gggttgtagg ggggtgtgtg gcccacccac attcctggcc ctggcaagtc 600

agtcttagaa caaggtttgg aatgcacttc tgtggaggca ccttgatatc cccagagtgg 660

gtgttgactg ctgcccactg cttggagaag tccccaaggc cttcatccta caaggtcatc 720

ctgggtgcac accaagaagt gaatctcgaa ccgcatgttc aggaaataga agtgtctagg 780

ctgttcttgg agcccacacg aaaagatatt gccttgctaa agctaagcag tcctgccgtc 840

atcactgaca aagtaatccc agcttgtctg ccatccccaa attatgtggt cgctgaccgg 900

accgaatgtt tcatcactgg ctggggagaa acccaaggta cttttggagc tggccttctc 960

aaggaagccc agctccctgt gattgagaat aaagtgtgca atcgctatga gtttctgaat 1020

ggaagagtcc aatccaccga actctgtgct gggcatttgg ccggaggcac tgacagttgc 1080

cagggtgaca gtggaggtcc tctggtttgc ttcgagaagg acaaatacat tttacaagga 1140

gtcacttctt ggggtcttgg ctgtgcacgc cccaataagc ctggtgtcta tgttcgtgtt 1200

tcaaggtttg ttacttggat tgagggagtg atgagaaata attaa 1245

<210>8

<211>414

<212>PRT

<213> Delta-plg (Delta-plasminogen) amino acid sequence

<400>8

Glu Pro Leu Asp Asp Tyr Val Asn Thr Gln Gly Ala Ser Leu Phe Ser

1 5 10 15

Val Thr Lys Lys Gln Leu Gly Ala Gly Ser Ile Glu Glu Cys Ala Ala

20 25 30

Lys Cys Glu Glu Asp Glu Glu Phe Thr Cys Arg Ala Phe Gln Tyr His

35 40 45

Ser Lys Glu Gln Gln Cys Val Ile Met Ala Glu Asn Arg Lys Ser Ser

50 55 60

Ile Ile Ile Arg Met Arg Asp Val Val Leu Phe Glu Lys Lys Val Tyr

65 70 75 80

Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg Gly Thr Met

85 90 95

Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser Ser Thr Ser

100 105 110

Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser Glu Gly Leu

115 120 125

Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln Gly Pro Trp

130 135 140

Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys Asp Ile Leu

145 150 155 160

Glu Cys Glu Glu Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val

165 170 175

Glu Pro Lys Lys Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala His

180 185 190

Pro His Ser Trp Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met

195 200 205

His Phe Cys Gly Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala

210 215 220

Ala His Cys Leu Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile

225 230 235 240

Leu Gly Ala His Gln Glu Val Asn Leu Glu Pro His Val Gln Glu Ile

245 250 255

Glu Val Ser Arg Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu

260 265 270

Leu Lys Leu Ser Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala

275 280 285

Cys Leu Pro Ser Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe

290 295 300

Ile Thr Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu

305 310 315 320

Lys Glu Ala Gln Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr

325 330 335

Glu Phe Leu Asn Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His

340 345 350

Leu Ala Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu

355 360 365

Val Cys Phe Glu Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp

370 375 380

Gly Leu Gly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val

385 390 395 400

Ser Arg Phe Val Thr Trp Ile Glu Gly Val Met Arg Asn Asn

405 410

<210>9

<211>1104

<212>DNA

<213> Mini-plg (Small plasminogen) nucleic acid sequence

<400>9

gtcaggtggg agtactgcaa cctgaaaaaa tgctcaggaa cagaagcgag tgttgtagca 60

cctccgcctg ttgtcctgct tccagatgta gagactcctt ccgaagaaga ctgtatgttt 120

gggaatggga aaggataccg aggcaagagg gcgaccactg ttactgggac gccatgccag 180

gactgggctg cccaggagcc ccatagacac agcattttca ctccagagac aaatccacgg 240

gcgggtctgg aaaaaaatta ctgccgtaac cctgatggtg atgtaggtgg tccctggtgc 300

tacacgacaa atccaagaaa actttacgac tactgtgatg tccctcagtg tgcggcccct 360

tcatttgatt gtgggaagcc tcaagtggag ccgaagaaat gtcctggaag ggttgtaggg 420

gggtgtgtgg cccacccaca ttcctggccc tggcaagtca gtcttagaac aaggtttgga 480

atgcacttct gtggaggcac cttgatatcc ccagagtggg tgttgactgc tgcccactgc 540

ttggagaagt ccccaaggcc ttcatcctac aaggtcatcc tgggtgcaca ccaagaagtg 600

aatctcgaac cgcatgttca ggaaatagaa gtgtctaggc tgttcttgga gcccacacga 660

aaagatattg ccttgctaaa gctaagcagt cctgccgtca tcactgacaa agtaatccca 720

gcttgtctgc catccccaaa ttatgtggtc gctgaccgga ccgaatgttt catcactggc 780

tggggagaaa cccaaggtac ttttggagct ggccttctca aggaagccca gctccctgtg 840

attgagaata aagtgtgcaa tcgctatgag tttctgaatg gaagagtcca atccaccgaa 900

ctctgtgctg ggcatttggc cggaggcact gacagttgcc agggtgacag tggaggtcct 960

ctggtttgct tcgagaagga caaatacatt ttacaaggag tcacttcttg gggtcttggc 1020

tgtgcacgcc ccaataagcc tggtgtctat gttcgtgttt caaggtttgt tacttggatt 1080

gagggagtga tgagaaataa ttaa 1104

<210>10

<211>367

<212>PRT

<213> Mini-plg (plasminogen) amino acid sequence

<400>10

Val Arg Trp Glu Tyr Cys Asn Leu Lys Lys Cys Ser Gly Thr Glu Ala

1 5 10 15

Ser Val Val Ala Pro Pro Pro Val Val Leu Leu Pro Asp Val Glu Thr

20 25 30

Pro Ser Glu Glu Asp Cys Met Phe Gly Asn Gly Lys Gly Tyr Arg Gly

35 40 45

Lys Arg Ala Thr Thr Val Thr Gly Thr Pro Cys Gln Asp Trp Ala Ala

5055 60

Gln Glu Pro His Arg His Ser Ile Phe Thr Pro Glu Thr Asn Pro Arg

65 70 75 80

Ala Gly Leu Glu Lys Asn Tyr Cys Arg Asn Pro Asp Gly Asp Val Gly

85 90 95

Gly Pro Trp Cys Tyr Thr Thr Asn Pro Arg Lys Leu Tyr Asp Tyr Cys

100 105 110

Asp Val Pro Gln Cys Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln

115 120 125

Val Glu Pro Lys Lys Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala

130 135 140

His Pro His Ser Trp Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly

145 150 155 160

Met His Phe Cys Gly Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr

165 170 175

Ala Ala His Cys Leu Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val

180 185 190

Ile Leu Gly Ala His Gln Glu Val Asn Leu Glu Pro His Val Gln Glu

195 200 205

Ile Glu Val Ser Arg Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala

210 215 220

Leu Leu Lys Leu Ser Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro

225 230 235 240

Ala Cys Leu Pro Ser Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys

245 250 255

Phe Ile Thr Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu

260 265 270

Leu Lys Glu Ala Gln Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg

275 280 285

Tyr Glu Phe Leu Asn Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly

290 295 300

His Leu Ala Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro

305 310 315 320

Leu Val Cys Phe Glu Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser

325 330 335

Trp Gly Leu Gly CysAla Arg Pro Asn Lys Pro Gly Val Tyr Val Arg

340 345 350

Val Ser Arg Phe Val Thr Trp Ile Glu Gly Val Met Arg Asn Asn

355 360 365

<210>11

<211>750

<212>DNA

<213> Micro-plg (microplasminogen) nucleic acid sequence

<400>11

gccccttcat ttgattgtgg gaagcctcaa gtggagccga agaaatgtcc tggaagggtt 60

gtaggggggt gtgtggccca cccacattcc tggccctggc aagtcagtct tagaacaagg 120

tttggaatgc acttctgtgg aggcaccttg atatccccag agtgggtgtt gactgctgcc 180

cactgcttgg agaagtcccc aaggccttca tcctacaagg tcatcctggg tgcacaccaa 240

gaagtgaatc tcgaaccgca tgttcaggaa atagaagtgt ctaggctgtt cttggagccc 300

acacgaaaag atattgcctt gctaaagcta agcagtcctg ccgtcatcac tgacaaagta 360

atcccagctt gtctgccatc cccaaattat gtggtcgctg accggaccga atgtttcatc 420

actggctggg gagaaaccca aggtactttt ggagctggcc ttctcaagga agcccagctc 480

cctgtgattg agaataaagt gtgcaatcgc tatgagtttc tgaatggaag agtccaatcc 540

accgaactct gtgctgggca tttggccgga ggcactgaca gttgccagggtgacagtgga 600

ggtcctctgg tttgcttcga gaaggacaaa tacattttac aaggagtcac ttcttggggt 660

cttggctgtg cacgccccaa taagcctggt gtctatgttc gtgtttcaag gtttgttact 720

tggattgagg gagtgatgag aaataattaa 750

<210>12

<211>249

<212>PRT

<213> Micro-plg (microplasminogen) amino acid sequence

<400>12

Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys

1 5 10 15

Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro

20 25 30

Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly

35 40 45

Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu

50 55 60

Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln

65 70 75 80

Glu Val Asn LeuGlu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu

85 90 95

Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser

100 105 110

Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro

115 120 125

Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly

130 135 140

Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu

145 150 155 160

Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly

165 170 175

Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr

180 185 190

Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys

195 200 205

Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala

210 215 220

Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr

225 230 235 240

Trp Ile Glu Gly Val Met Arg Asn Asn

245

<210>13

<211>684

<212>DNA

<213> nucleic acid sequence of serine protease (Domain)

<400>13

gttgtagggg ggtgtgtggc ccacccacat tcctggccct ggcaagtcag tcttagaaca 60

aggtttggaa tgcacttctg tggaggcacc ttgatatccc cagagtgggt gttgactgct 120

gcccactgct tggagaagtc cccaaggcct tcatcctaca aggtcatcct gggtgcacac 180

caagaagtga atctcgaacc gcatgttcag gaaatagaag tgtctaggct gttcttggag 240

cccacacgaa aagatattgc cttgctaaag ctaagcagtc ctgccgtcat cactgacaaa 300

gtaatcccag cttgtctgcc atccccaaat tatgtggtcg ctgaccggac cgaatgtttc 360

atcactggct ggggagaaac ccaaggtact tttggagctg gccttctcaa ggaagcccag 420

ctccctgtga ttgagaataa agtgtgcaat cgctatgagt ttctgaatgg aagagtccaa 480

tccaccgaac tctgtgctgg gcatttggcc ggaggcactg acagttgcca gggtgacagt 540

ggaggtcctc tggtttgctt cgagaaggac aaatacattt tacaaggagt cacttcttgg 600

ggtcttggct gtgcacgccc caataagcct ggtgtctatg ttcgtgtttc aaggtttgtt 660

acttggattg agggagtgat gaga 684

<210>14

<211>228

<212>PRT

<213> amino acid sequence of serine protease (Domain)

<400>14

Val Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro Trp Gln Val

1 5 10 15

Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly Thr Leu Ile

20 25 30

Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu Lys Ser Pro

35 40 45

Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln Glu Val Asn

50 55 60

Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu Phe Leu Glu

65 70 75 80

Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser Pro Ala Val

8590 95

Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro Asn Tyr Val

100 105 110

Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly Glu Thr Gln

115 120 125

Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro Val Ile

130 135 140

Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly Arg Val Gln

145 150 155 160

Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr Asp Ser Cys

165 170 175

Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys Tyr

180 185 190

Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro Asn

195 200 205

Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp Ile Glu

210 215 220

Gly Val Met Arg

225

Claims (10)

1. A method of lowering blood glucose in a diabetic subject comprising administering to the subject an effective amount of plasminogen.

2. The method of claim 1, wherein the blood glucose is selected from one or more of the following: serum glucose levels, serum fructosamine levels, serum glycated hemoglobin levels.

3. The method of claim 2, wherein the blood glucose is serum glucose level.

4. The method of any one of claims 1-3, wherein the diabetes is T1DM or T2 DM.

5. A method of increasing glucose tolerance in a diabetic subject comprising administering to the subject an effective amount of plasminogen.

6. The method of claim 5, wherein the diabetes is T2 DM.

7. A method of promoting postprandial blood glucose lowering in a diabetic subject comprising administering to the subject an effective amount of plasminogen.

8. The method of claim 7, wherein the plasminogen is administered 30 minutes to 1.5 hours before the subject meals.

9. The method of claim 8, wherein the plasminogen is administered 30 minutes to 1 hour before the subject meals.

10. A method of promoting glucose utilization in a diabetic subject comprising administering to the subject an effective amount of plasminogen.