WO2019179338A1 - Application of protein in preparing drug for preventing or treating complication of diabetes - Google Patents

Abstract

Disclosed is an application of secretory DSS1 protein in preparing a drug for preventing or treating a complication of diabetes. The secretory DSS1 can bind to advanced glycation end products and block cytotoxicity induced thereby, thereby relieving a symptom in an animal model having a complication of diabetes.

Description

Application of a protein in preparing medicine for preventing and treating diabetic complications Technical field

The invention belongs to the technical field of medicine and relates to the application of the sDSS1 protein in preparing a medicament for preventing and treating diabetic complications.

Background technique

Diabetes is a metabolic disease characterized by chronic hyperglycemia. According to the International Diabetes Federation (IDF) survey data (http://www.diabetesatlas.org), 1 out of every 11 people in the world have diabetes, and the total number of patients in 2017 reached 425 million, including mainland Chinese adults. Diabetes accounts for about 114.4 million, ranking first in the world. The probability of complicated cardiovascular disease in diabetic patients is 2 to 3 times that of non-diabetic patients. The incidence of end-stage renal disease in diabetic patients is 10 times higher than that of non-diabetic patients. Every 30 seconds in the world, some people lose part of their lower limbs or lower limbs because of diabetes. Once a complication occurs, drug treatment is difficult to reverse. Therefore, preventing or delaying the complications of diabetes as soon as possible, protecting the terminal organs involved in diabetes, improving the quality of life of patients and reducing the social and economic burden have become the ultimate goal of treating diabetes.

Long-term elevated blood sugar damages large blood vessels and microvessels and endangers the heart, brain, kidneys, peripheral nerves, eyes, and feet. Urinary microalbumin is the earliest marker of diabetic nephropathy. Urinary microalbumin is also a powerful predictor of cardiovascular complications [1], and its excretion is positively associated with late diabetic complications [2]. Urinary microalbumin is therefore clinically recommended as a marker for early screening for diabetic complications (IDF DIABETES ATLAS Eighth edition 2017). The pathogenesis of diabetic complications is complex. The current research results suggest that metabolic disorders, oxidative stress, hemodynamic changes, chronic low-grade inflammatory reactions, and genetic factors are involved in the progression of the disease [3].

Terminal glycosylation products (AGEs) are stable covalent additions of macromolecules such as proteins, lipids or nucleic acids that spontaneously react with excessive glucose or other reducing monosaccharides under non-enzymatic conditions. Things. It can be considered as an oxidative stress-assisted oxidative and/or glycated complex. On the one hand, circulating AGEs can act on RAGE receptors to cause oxidative stress, inflammation and apoptosis; on the other hand, long-term high glucose and oxidative stress can cause glycosylation and cross-linking of extracellular matrix proteins, making blood vessels Hardening; third, intracellular polysaccharide modification can interfere with a variety of proteins, enzymes or receptors to exert their biological functions [3,4]. Increased levels of AGEs in blood and tissues in patients with type I and type II diabetes [5]. The degree of accumulation of AGEs in tissues was positively correlated with diabetic complications [6]. Therefore, inhibition of AGEs pathway can be one of the ways to prevent or treat diabetic complications [7]. It has been reported that it can interfere with the toxicity of AGEs in various ways, such as inhibition of AGEs production, inhibition of AGEs cross-linking, RAGE receptor antagonists, etc. [7-10]. Representative drugs such as aminoguanidine, Pyridoxamine, TTP488, sRAGE, etc. are currently in clinical research.

The Shfm1 (split hand/split foot malformation type 1) gene is one of the key genes in human crab claw disease. It is highly conserved in evolution and its encoded protein DSS1 is a universal endogenous multifunctional disorder protein. Participate in the process of stable genome, homologous gene recombination, DNA damage repair, RNA splicing, protein degradation and cell proliferation [11]. The results show that the DSS1 protein can be added to the oxidized protein by energy-consuming enzymatic reaction to help the cells clear the oxidized protein [12]. These results show the important role of DSS1 protein in biological activities.

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C. Microalbuminuria, as predictor of late diabetic complications. A prospective study. Medecine interne. 1990;28(2):131-4.

3.Forbes JM, Cooper ME. Mechanisms of diabetic complications. Physiological reviews. 2013;93(1):137-88.

4. Singh VP, Bali A, Singh N, Jaggi AS. Advanced glycation end products and diabetic complications. Korean J Physiol Pharmacol. 2014; 18(1): 1-14.

A,Weber D,Grune T.Advanced glycation end products and oxidative stress in type 2 diabetes mellitus.Biomolecules.2015；5(1):194-222. 5.Nowotny K, Jung T,

A, Weber D, Grune T. Advanced glycation end products and oxidative stress in type 2 diabetes mellitus. Biomolecules. 2015; 5(1): 194-222.

6. Monnier VM, Sell DR, Genuth S. Glycation products as markers and predictors of the progression of diabetic complications. Annals of the New York Academy of Sciences. 2005; 1043(1): 567-81.

7. Younus H, Anwar S. Prevent of non-enzymatic glycosylation (glycation): Implication in the treatment of diabetic complication. International journal of health sciences. 2016; 10(2): 261.

8. Lv M, Chen Z, Hu G, Li Q. Therapeutic strategies of diabetic nephropathy: recent progress and future perspectives. Drug Discov Today. 2015; 20(3): 332-46.

9. Bongarzone S, Savickas V, Luzi F, Gee AD. Targeting the Receptor for Advanced Glycation Endproducts (RAGE): A Medicinal Chemistry Perspective. J Med Chem. 2017; 60(17): 7213-32.

10. Abbas G, Al-Harrasi AS, Hussain H, Hussain J, Rashid R, Choudhary MI. Antiglycation therapy: Discovery of promising antiglycation agents for the management of diabetic complications. Pharmaceutical biology. 2016; 54(2): 198-206 .

SM,Rebula CA,Panse VG,Hartmann-Petersen R.DSS1/Sem1,a multifunctional and intrinsically disordered protein.Trends in biochemical sciences.2016；41(5):446-59. 11.Kragelund BB,

SM, Rebula CA, Panse VG, Hartmann-Petersen R. DSS1/Sem1, a multifunctional and intrinsically disordered protein. Trends in biochemical sciences. 2016;41(5):446-59.

12.Zhang Y, Chang FM, Huang J, Junco JJ, Maffi SK, Pridgen HI, et al. DSSylation, a novel protein modification targets proteins induced by oxidative stress, and facilitates their degradation in cells.Protein&cell.2014;5(2 ): 124-40.

Summary of the invention

In the present invention, the sDSS1 protein provided by our (inventors) can bind to AGEs and reduce the cytotoxicity caused by AGEs. Long-term administration of sDSS1 protein can reduce urinary albumin excretion in diabetic model mice and significantly improve renal filtration function. Therefore, the sDSS1 protein can be clinically prepared for the prevention and treatment of diabetic complications.

The specific technical solutions are as follows:

The use of a protein for the preparation of a medicament for the prevention and treatment of diabetic complications, the use of the sDSS1 protein for the preparation of a medicament for the prevention and treatment of diabetic complications.

Preferably, the diabetic complications include diabetic nephropathy, diabetic ocular complications, diabetic foot, diabetic cardio-cerebral vascular complications or diabetic neuropathy.

Preferably, the diabetic ocular complications include diabetic retinopathy, diabetic macular edema (DME), diabetic cataract or glaucoma.

Preferably, the diabetic cardio-cerebral vascular complications include coronary artery disease (CAD) caused by diabetes, angina pectoris, myocardial infarction, arteriosclerosis, stroke, brain atrophy, peripheral arterial disease (PAD) or congestive heart failure.

Preferably, the diabetic complications include type I diabetes complications and type II diabetes complications.

Preferably, the sDSS1 protein comprises human, chimpanzee, bonobo, gorilla, orangutan, white-cheeked gibbons, golden monkey, rhesus monkey, golden monkey, East African pheasant, Angora simian, white-tailed white-browed monkey, ghost A basic protein formed by any sDSS1 protein sequence of scorpion or porpoise monkey, wherein the amino acid sequence of human sDSS1 is SEQ ID NO: 1, the amino acid sequence of chimpanzee sDSS1 is SEQ ID NO: 2, and the amino acid sequence of porcine chimpanzee sDSS1 is SEQ ID NO: 3, the amino acid sequence of gorilla sDSS1 is SEQ ID NO: 4, the amino acid sequence of orangutan sDSS1 is SEQ ID NO: 5, the amino acid sequence of white cheek gibbon sDSS1 is SEQ ID NO: 6, amino acid of Rhinopithecus sDSS1 The sequence is as SEQ ID NO: 7, the amino acid sequence of rhesus sDSS1 is SEQ ID NO: 8, the amino acid sequence of gilt monkey sDSS1 is SEQ ID NO: 9, and the amino acid sequence of ssDSS1 is SEQ ID NO: 10, Angola The amino acid sequence of the monkey sDSS1 is SEQ ID NO: 11, the amino acid sequence of the white-headed white-breasted monkey sDSS1 is SEQ ID NO: 12, and the amino acid sequence of podophylla sDSS1 is SEQ ID NO: 13. Tailed macaque sDSS1 amino acid sequence as SEQ ID NO: 14.

Preferably, the sDSS1 protein is any first protein having a similarity to the basic protein of 70% or more.

Preferably, the sDSS1 protein is any structural or amino acid sequence characteristic of a polypeptide fragment fused to another polypeptide fragment based on 58 amino acids of the basic protein nitrogen base at the nitrogen terminal or the carbon terminal. Thirty-one second proteins of the same or similar sequence.

Preferably, the sDSS1 protein is any third protein which is based on 58 amino acids of the basic protein nitrogen end, and fuses other amino acid fragments at the nitrogen terminal or the carbon terminal, and the fusion protein can realize transmembrane transport function.

Preferably, the sDSS1 protein is formed by linking any one of a basic protein, a first protein, a second protein and a third protein to the protein itself, a carrier protein, an antibody or other amino acid fragments of any length. Fusion protein.

Preferably, the sDSS1 protein is a protein modification produced by modification based on any one of a basic protein, a first protein, a second protein, and a third protein.

Preferably, the modification of the protein modification is directed to an amino group on the amino acid side chain, a carbonyl group on the amino acid side chain, a nitrogen terminal amino group, a carbon terminal carbonyl group, a cysteine, a tyrosine, a serine, a tryptophan. Specific or non-specific chemical modification of 1-20 sites.

Preferably, the method for modifying the protein modification comprises glycosylation modification, fatty acid modification, acylation modification, Fc fragment fusion, albumin fusion, polyethylene glycol modification, dextran modification, heparin modification, polyvinylpyrrolidone modification , polyamino acid modification, polysialic acid modification, chitosan and its derivative modification, lectin modification, sodium alginate modification, carbomer modification, polyvinylpyrrolidone modification, hydroxypropyl methylcellulose modification, hydroxypropyl One or more of a cellulose modification, acetylation modification, formylation modification, phosphorylation modification, methylation modification or sulfonation modification, and other pharmaceutically acceptable polypeptide/protein drug modification methods.

Preferably, the sDSS1 protein is one based on amino acids other than the 20 essential amino acids based on the amino acid sequence of any of the basic protein, the first protein, the second protein, and the third protein. A non-natural amino acid replacement protein substituted with 31 arbitrary amino acid positions.

Preferably, the amino acid substitution of the non-natural amino acid replacement protein comprises hydroxyproline, hydroxylysine, selenocysteine, D-form amino acid or synthetic non-natural amino acid and derivatives thereof.

Preferably, the sDSS1 protein is formed by combining a basic protein, a first protein, a second protein, a third protein, a fusion protein, a protein modification or a non-natural amino acid substitute with a pharmaceutically acceptable drug carrier. Part or all of the complex.

Preferably, the pharmaceutical carrier comprises one or more of an enteric coating formulation, a capsule, a microsphere/capsule, a liposome, a microemulsion, a double emulsion, a nanoparticle, a magnetic particle, a gelatin or a gel.

Preferably, the sDSS1 protein targets the individual's own sDSS1 protein, and affects the level of the individual's own sDSS1 protein by the exogenous drug.

Preferably, the drug is a drug target of a sDSS1 protein, a gene of sDSS1 protein, a regulatory element of a gene of sDSS1 or a gene of sDSS1.

Preferably, the drug modulates the amount of sDSS1 protein in the blood by affecting protease/peptidase activity in the blood.

Preferably, the drug is a chemical small molecule drug, an antibody, a polypeptide/protein drug, a nucleic acid drug or a nano drug.

Preferably, the sDSS1 protein is any one of a basic protein, a first protein, a second protein, a third protein, a fusion protein, a protein modification, an unnatural amino acid substitute, a complex, and a drug. a combination of two or more of the ingredients.

Preferably, the sDSS1 protein is any one of a basic protein, a first protein, a second protein, a third protein, a fusion protein, a protein modification, an unnatural amino acid substitute, a complex, and a drug. One, two or more of the ingredients in combination with a pharmaceutically acceptable excipient.

Preferably, the sDSS1 protein is a protein obtained by introducing a nucleotide sequence encoding any one of a basic protein, a first protein, a second protein, a third protein and a fusion protein into an expression system by expression system. .

Preferably, the expression system is eukaryotic expression plasmid vector, adenovirus, adeno-associated virus, lentivirus, retrovirus, baculovirus, herpes virus, pseudorabies virus, ZFN gene editing technology, TALEN gene editing technology, CRISPR/Cas gene editing technology and other medically available gene editing techniques or viral vectors.

Preferably, the sDSS1 protein is any one of a basic protein, a first protein, a second protein, a third protein, and a fusion protein obtained by the transplanted cell in an individual.

Preferably, the cell is a stem cell, a precursor cell or an adult cell of any one of humans.

Preferably, the stem cells are embryonic stem cells, induced pluripotent stem cells, cells obtained by transdifferentiation, or stem cells derived from primary culture, pluripotent or pluripotent stem cells differentiated from mother cells.

Preferably, the sDSS1 protein is an sDSS1 protein introduced into an individual by serum or interstitial fluid infusion.

Preferably, the sDSS1 protein is any one of a basic protein, a first protein, a second protein, and a third protein obtained in an individual by transplanting tissues or organs.

Preferably, in tissue or organ transplantation, the tissue is a whole organ or part of a tissue block of the brain, liver, kidney, spleen, pancreatic islet, or blood, fat, muscle, bone marrow, skin.

Preferably, the prophylactic agent comprises a basic protein, a first protein, a second protein, a third protein, a fusion protein, a protein modification, a non-natural amino acid replacement protein, a complex, a drug combination, an expression system, Protein, tissue, organ, body fluid, tissue fluid protein drug, peptide drug, nucleic acid drug, chemical small molecule drug, cell product, commercial transplantation tissue, injection, lyophilized powder, health care product or food additive.

Preferably, the therapeutic drug comprises a basic protein, a first protein, a second protein, a third protein, a fusion protein, a protein modification, a non-natural amino acid substitution protein, a complex, a drug combination, an expression system, Protein, tissue, organ, body fluid, tissue fluid protein drug, peptide drug, nucleic acid drug, chemical small molecule drug, cell product, commercial transplantation tissue, injection, lyophilized powder, health care product or food additive.

The use of a protein in a nursing device for improving diabetic complications, which is the use of any of the above-mentioned methods in the preparation of a medicament for preventing and treating diabetes complications, and then using the prepared medicament for improving medical equipment related to diabetes complications care Performance.

Preferably, the medical device comprises blood collection equipment and consumables, blood purification equipment and consumables, blood purification equipment auxiliary equipment and consumables, body fluid processing equipment and consumables, kidney dialysis equipment and consumables, peritoneal dialysis equipment and consumables, blood perfusion One or more of a device, an infusion set, an insufflator, a sustained release device, and an artificial kidney.

Features and/or benefits of the present invention are:

1. The sDSS1 protein provided by the present invention binds to AGEs and effectively alleviates the cytotoxicity caused by AGEs.

2. The sDSS1 protein provided by the present invention significantly reduces urinary microalbumin levels in one of the markers of diabetic complications in db diabetic model mice.

3. The sDSS1 protein provided by the present invention improves the compensatory increase in glomerular filtration rate in db diabetic model mice, improves renal function and reduces blood glycosylated hemoglobin levels.

4. The sDSS1 protein provided by the present invention is an endogenous protein or a derivative thereof of human and other primates, has a relatively small molecular weight, low immunogenicity, and has a natural protein degradation mechanism in vivo, and therefore, clinical application The probability of causing a significant immune response or other toxic side effects is not high, safe and reliable.

In summary, the present invention provides a method for the prevention and treatment of diabetic complications by using sDSS1 protein. The sDSS1 protein can be combined with AGEs to reduce cell damage caused by AGEs by molecular, cellular and animal level experiments. sDSS1 protein can effectively reduce the excretion of urinary microalbumin in db diabetic mice, improve renal function, reduce glycosylated hemoglobin levels, and alleviate disease symptoms. The sDSS1 protein has the potential to be used clinically to prepare drugs for the prevention and treatment of diabetic complications.

DRAWINGS

The invention is further described in detail below with reference to the accompanying drawings, in which

Figure 1A-1B. Figure 1A. Molecular level experiments show that sDSS1 interacts with AGEs. The AGEs protein or AGEs protein was incubated with sDSS1 and stained with Coomassie blue. The results showed that AGEs could interact with sDSS1 protein and the SDSS1 protein band became lighter (L3vs L2, L6vs L5). Figure 1B. AGEs protein or AGEs protein and sDSS1 incubation products were separated by SDS-PAGE and labeled with anti-AGEs antibody, and developed in the darkroom by developer and fixer. The results showed that AGEs protein strips recognized by sDSS1 interacted with AGEs. The band is lighter and the degree of lightening is proportional to the concentration of sDSS1 protein, showing a significant concentration dependence.

Figure 2. Cytotoxicity caused by sDSS1 protein blocking AGEs. Adding 50μM AGEs to rat kidney cell culture can significantly reduce the cell viability level. After adding different concentrations of sDSS1 protein, the cell viability is recovered. As the concentration of sDSS1 protein increases, the cell vigor increases gradually, and the effect shows a typical dose. Dependence effect. The 75 μM sDSS1 protein completely blocked the decrease in cell viability caused by 50 μM AGEs. Data were analyzed by ANOVA, #, blank control group vs only AGEs group, *, all AGEs and sDSS1 groups were added vs only AGEs group; *, p-value<0.05; **, p-value<0.01;### , p-value<0.001; ****, p-value<0.0001.

Figures 3A-3B. Molecular level experiments show that sDSS1 interacts with CML-BSA. Figure 3A. SDS-PAGE separation and staining with Coomassie brilliant blue showed that CML-BSA interacted with sDSS1 protein, and the band at the corresponding position of sDSS1 protein became lighter (L3 vs L2, L5 vs L4). Figure 3B. Western blotting shows that the amount of CML-BSA protein recognized by the antibody after sDSS1 interacts with CML-BSA is reduced, showing a shallow band. And the degree of lightening is directly proportional to the concentration of sDSS1 protein, showing a significant concentration dependence.

Figure 4A-4B. sDSS1 protein shields cytotoxicity caused by CML-BSA. Figure 4A. 33 μg/mL CML-BSA was added to rat kidney cell culture, and the state of the cells was observed under a microscope and photographed. When 33μg/mL CML-BSA was added to the culture medium, the number of cells was significantly reduced, the cells became round, and the intercellular connections disappeared. As the concentration of sDSS1 protein was increased, the cell status gradually returned to normal. Figure 4B. By measuring cell viability, the addition of 33 μg/mL CML-BSA in rat kidney cell culture can significantly reduce the cell viability level. After adding different concentrations of sDSS1 protein, the cell viability is recovered, and as the concentration of sDSS1 protein increases, Cell viability gradually increases. The 30 μM sDSS1 protein can substantially block the decrease in cell viability caused by 33 μg/mL CML-BSA, and this effect exhibits a typical dose-dependent effect. The data was analyzed by ANOVA, #, blank control group vs only CML-BSA group, *, all added CML-BSA and sDSS1 group vs only CML-BSA group; #, *, p-value<0.05;##,* *, p-value<0.01; ###, ***, p-value<0.001.

Figure 5. sDSS1 improves urinary albumin levels in diabetic mice. The urinary albumin excretion of db mouse urinary albumin was increased compared with wild littermate mice. After sDSS1 administration, the urinary albumin excretion symptoms were improved. The 10 mpk (mg protein per kilogram body weight) dose group The effect of prolonged administration time was increased, and the amount of urinary albumin excretion was significantly reduced after 40 days of administration. Data were analyzed by ANOVA, *P<0.05, **P<0.01 vs.db/db.

Figure 6. sDSS1 improves symptoms of diabetes complicated with kidney disease. The glomerular filtration function of db mice was compensatoryly increased compared to wild littermates. After administration of sDSS1, sDSS1 was able to dose-dependently alleviate this compensatory increase. Data were analyzed by ANOVA, *P<0.05, **P<0.01 vs.db/db.

Figure 7. sDSS1 improves glycated hemoglobin levels in diabetic mice. The blood glycosylated hemoglobin of db mice was significantly increased compared to wild littermate mice, and the dose of 10 mpk decreased the ratio of glycated hemoglobin by about 1.3% after administration of sDSS1. Data were analyzed by ANOVA, *P<0.05, ****P<0.0001 vs.db/db.

detailed description

The preferred embodiments of the present invention are described and illustrated in the following examples, which are not intended to limit the scope of the invention. All ranges of the invention are defined by the claims.

The experimental methods used in the following examples are routine experimental methods unless otherwise specified.

The sDSS1 protein used in the following examples is self-produced and quality controlled by the company. The purity of the tested protein is greater than 95%. Endotoxin and other impurity residues meet the standards and can be used in animal experiments without causing significant animal toxicity.

The materials and reagents in the following examples, except for the sDSS1 protein, are commercially available.

Example 1. The sDSS1 protein interacts with AGEs.

1.1. Experimental materials and methods

Materials: Bovine serum albumin (Aladdin, A104912), ribose (Aladdin, D1608050), anti-AGEs antibody was purchased from TransGenic, 50489-M08H.

METHODS: Bovine serum albumin and ribose were incubated in PBS for 14 days to prepare AGEs protein. 2 μg of AGEs, 4 μg of sDSS1, 2 μg of AGEs, 4 μg of sDSS1, 2 μg of AGEs, 8 μg of sDSS1, 2 μg of AGEs and 8 μg of sDSS1 were respectively added to a 1.5 mL EP tube, and reacted at 37 ° C overnight. The incubation product was added to the loading buffer, mixed, and denatured at 100 ° C for 10 minutes to prepare a sample for loading. The samples were separated by polyacrylamide gel electrophoresis (SDS-PAGE), and the PAGE gel was stained with Coomassie blue to reveal the protein bands.

2 μg of AGEs, 2 μg of AGEs and 4 μg of sDSS1, 2 μg of AGEs and 10 μg of sDSS1, 2 μg of AGEs and 20 μg of sDSS1 were respectively added to a 1.5 mL EP tube, and reacted at 37 ° C overnight. The incubation product was added to the loading buffer, mixed, and denatured at 100 ° C for 10 minutes to prepare a sample for loading. The samples were separated by polyacrylamide gel electrophoresis (SDS-PAGE), separated, transferred to a PVDF membrane, and blocked with 5% skim milk. Primary anti-AGEs were then performed overnight at 4 °C. After washing with TBST, horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibody was added for incubation. Subsequently, the membrane was washed with TBST, the EVL supersensitive luminescent liquid was used to rinse the PVDF membrane, and the X-ray film was developed and fixed to show the target protein band.

1.2. Experimental results

It can be seen on the stained PAGE gel that the obtained AGEs protein has a molecular weight of 75 KD-150 KD, forming a diffuse band (L1, Fig. 1A). When the AGEs were mixed with the sDSS1 protein, it was observed that the light color of the sDSS1 protein was light, suggesting that AGEs interacted with the sDSS1 protein and decreased the number of sDSS1 proteins. In the Westen-Blot experiment, when the AGEs were mixed with the sDSS1 protein, the two bands of the AGEs protein were significantly lighter, and as the sDSS1 ratio increased, the bands became lighter and lighter (L2). -L4, Figure 1B). These results indicate that the sDSS1 protein can interact with AGEs.

Example 2. sDSS1 protein blocks cytotoxicity caused by AGEs.

2.1. Experimental materials and methods

MATERIALS: NRK-52E cell line (Chinese Academy of Sciences, Culture Collection Committee Cell Bank, catalog number: GNR 8), DMEM medium (HyClone, AC10210629), SpectraMax Plus 384 multi-function microplate reader (Molecular Devices), cell proliferation / Toxicity test kit (Dojindo, CK04).

Methods: NRK-52E cells were seeded into 96-well plates at 2x10 ⁴ cells per well. After adhering to the cells overnight, the cells were replaced with serum-free medium and starved for 24 hours. Each group was divided into blank medium, 50 μM AGEs, 50 μM AGEs and 25 μM sDSS1, 50 μM AGEs and 50 μM sDSS1, 50 μM AGEs and 75 μM sDSS1, 50 μM AGEs and 100 μM sDSS1. After 48 hours, cell viability assay was performed using the cell proliferation toxicity test kit. .

2.2. Experimental results

Cell viability assay showed that when only 10 μM AGEs were added to NRK-52E cells, the level of cell activity was significantly reduced, only about 20% of control cells. When the sDSS1 protein was added to the medium, the cell vigor gradually increased. In the range of 25 μM-75 μM, as the concentration of sDSS1 protein increased, the cell activity increased more and more in a concentration-dependent manner (Fig. 2). These results indicate that the sDSS1 protein is able to block the decrease in cell viability caused by AGEs.

Example 3 sDSS1 can interact with CML-BSA.

3.1 Experimental materials and methods

Materials: CML-BSA was purchased from Cell Biolab (STA-314); anti-AGEs antibody was purchased from TransGenic (50489-M08H).

Method: 1 μg of CML-BSA was mixed with 0.5, 5 μg of sDSS1. A separate CML-BSA was also included in comparison with sDSS1. After all samples were incubated at 37 °C overnight in an EP tube, the incubation product was added to the loading buffer, mixed, and denatured at 100 ° C for 10 minutes to prepare a sample for loading. The samples were separated by polyacrylamide gel electrophoresis (SDS-PAGE), and the PAGE gel was stained with Coomassie blue to reveal the protein bands.

The above protein samples were separated by SDS-PAGE, transferred to a PVDF membrane, and blocked with 5% skim milk. Then, anti-AGEs were performed and overnight at 4 °C. The membrane was washed sequentially, the secondary antibody was added, the membrane was washed, the color developed, developed, and fixed to show the protein band of interest.

When the affinity constant of CML-BSA with sDSS1 or sDSS1 mutant 1 (sDSS1-M1, see SEQ ID NO: 15) was detected using an Octet Biomolecular Interaction Instrument (ForteBIO, RED96), an NTA sensor (ForteBIO, 18-5101) was used. The sDSS1 or sDSS1-M1 protein was coupled as a test substance, and the analyte CML-BSA was diluted to 0.596 μM, 0.298 μM, 0.149 μM, 0.0745 μM, 0.0373 μM, and 0.0186 μM, respectively, and the buffer was used as a blank control. Detect and analyze the data according to the parameters and procedures recommended by the instrument.

3.2 Experimental results

CML is one of the representatives of the AGEs complex. When CML-BSA was mixed with sDSS1 protein, it was observed that the sDSS1 protein band became lighter (Fig. 3A). Subsequent Western-Blot results showed that the two bands corresponding to the CML-BSA protein were significantly lighter, and as the sDSS1 ratio increased, the band color became lighter (Fig. 3B). Therefore, western blot results indicate that sDSS1 protein can interact with CML-BSA and reduce the number of AGE proteins recognized by antibodies. The results of the Octet molecular interrogation test showed that the affinity constant KD of sDSS1 protein and CML-BSA was 5.26×10 ^-7 , and the binding constant was 3.71×10 ³ , and the dissociation constant was 1.95×10 ^-3 ; while sDSS1-M1 The binding of the protein to CML-BSA is more tight, and the affinity constant KD is 2.96×10 ^-8 , wherein the binding constant is 9.35×10 ³ and the dissociation constant is 2.77×10 ^-4 . Therefore, the sDSS1 protein can interact with CML-BSA, and the affinity of mutant 1 is higher.

Example 4. sDSS1 protein shields cytotoxicity caused by CML-BSA.

4.1. Experimental materials and methods

Materials: NRK-52E cell line, DMEM medium (HyClone, AC10210629), SpectraMax Plus 384 multi-function microplate reader (Molecular Devices), cell proliferation/toxicity test kit (Dojindo, CK04), CML-BSA (Cell Biolabs) , STA-314).

Methods: NRK-52E cells were seeded into 96-well plates at 2x10 ⁴ cells per well. The cells were starved for 24 hours after adherence. Each group was separately added with blank medium, 33 μg/mL CML-BSA, 33 μg/mL CML-BSA and 3 μM sDSS1, 33 μg/mL CML-BSA and 10 μM sDSS1, 33 μg/mL CML-BSA and 30 μM sDSS1. After completion, the cells were continued for 48 hours. The cell morphology was observed under the microscope of the completed cells, and the cell viability level test was performed using the cell proliferation toxicity test kit.

4.2. Experimental results

When only 33μg/mL CML-BSA was added to NRK-52E cells, the number of cells was significantly reduced under the microscope, the cells became round and the intercellular connections disappeared. The spindle shape of normal NRK-52E cells in the blank group was significant. Difference (Figure 4A). And the cell viability test showed that the level of cell activity was significantly reduced, only about 30% of the control cells (Fig. 4B). When the sDSS1 protein was added to the medium, the morphology and viability of the cells were saved. When 3μM sDSS1 protein was added, the cell viability increased slightly, and the cell morphology did not improve significantly, but the cell number increased. When the concentration of sDSS1 protein increased to 10μM-30μM, the cell activity increased more and more, and the cell morphology under microscope It has also returned to a normal state, which is concentration dependent. These results indicate that sDSS1 protein can block the cytotoxicity caused by CML-BSA and protect cell viability.

Example 5. sDSS1 can alleviate complications of db diabetic mice

5.1. Experimental materials and methods

The db/db mice of C57BLKS/J background were purchased from Nanjing University-Nanjing Institute of Biomedical Research, male. Mice were grouped according to body weight and blood glucose after adaptive feeding, followed by intravenous administration of solvent control or sDSS1 protein every two days. Monitor both weight and diet. At 21 days of administration, the mice were placed in a metabolic cage and urine was collected for 24 hours to measure the urinary albumin concentration. After 37 days of administration, FITC-labeled inulin was intravenously administered, and plasma inulin concentrations were measured at 3/7/10/15/35/70 minutes, respectively, and glomerular filtration rate was calculated. At 40 days of administration, the mice were again placed in a metabolic cage and urine was collected for 24 hours to measure the urinary albumin concentration. The mice were sacrificed after 6 weeks of administration, and plasma, heart, eyeball, kidney, and brain were collected. The mouse urinary albumin assay kit was purchased from Bethyl Laboratories, Inc. The mouse glycated hemoglobin Elisa kit was purchased from Shanghai Langton Biotechnology Co., Ltd. (Cat. No. BPE20512).

5.2. Experimental results

According to the 24h urinary albumin test results, db diabetic mice showed increased urinary albumin excretion compared with their littermate wild control mice, suggesting impaired renal function. At 21 days of dosing, sDSS1 had a trend to improve urinary albumin excretion, but there were no statistical differences. At 37 days of administration, sDSS1 10mpk significantly improved urinary albumin excretion, and the effect of sDSS1 10mpk was stronger than that of 3mpk (Figure 5). The glomerular filtration rate function was tested and the results showed that db mice had a compensatory increase in glomerular filtration function compared to their wild control mice. The sDSS1 proteins 3mpk and 10mpk alleviated the increased glomerular filtration rate in db mice (Fig. 6). Blood glycated hemoglobin data showed that the 10 mpk dose reduced the glycated hemoglobin ratio by about 1.3%, suggesting that sDSS1 can affect glycosylated hemoglobin levels (Figure 7). Summarizing these data, sDSS1 can improve the symptoms of renal complications in db diabetic mice, and this improvement has a time-dependent and dose-effect relationship.

Claims (35)

Use of a protein for the preparation of a medicament for preventing and treating diabetes complications, characterized in that the application is to use the sDSS1 protein for the preparation of a medicament for preventing and treating diabetic complications. The use according to claim 1, wherein the diabetic complications include diabetic nephropathy, diabetic ocular complications, diabetic foot, diabetic cardio-cerebral vascular complications or diabetic neuropathy. The use according to claim 2, wherein the diabetic ocular complications include diabetic retinopathy, diabetic macular edema (DME), diabetic cataract or glaucoma. The use according to claim 2, wherein the diabetic cardio-cerebral vascular complications include coronary artery disease (CAD) caused by diabetes, angina pectoris, myocardial infarction, arteriosclerosis, stroke, brain atrophy, and peripheral arterial disease ( PAD) or congestive heart failure. The use according to claim 1, wherein said diabetic complications include type I diabetes complications and type II diabetes complications. The use according to claim 1, wherein the sDSS1 protein comprises a human, a chimpanzee, a bonobo, a gorilla, an orangutan, a white-cheeked gibbon, a Rhesus monkey, a rhesus monkey, a golden monkey, an East African ape, A basic protein formed by any sDSS1 protein sequence of Angora marmoset, white-tailed white-browed macaque, podophyllum or porpoise monkey, wherein the amino acid sequence of human sDSS1 is SEQ ID NO: 1, and the amino acid sequence of chimpanzee sDSS1 is SEQ ID NO: 2. , the amino acid sequence of the chimpanzee sDSS1 is SEQ ID NO: 3, the amino acid sequence of gorilla sDSS1 is SEQ ID NO: 4, the amino acid sequence of the orangutan sDSS1 is SEQ ID NO: 5, and the amino acid sequence of the white-cheeked gibbon sDSS1 is SEQ. ID NO: 6, the amino acid sequence of Rhinopithecus sDSS1 is SEQ ID NO: 7, the amino acid sequence of rhesus sDSS1 is SEQ ID NO: 8, and the amino acid sequence of Rhinopithecus sDSS1 is SEQ ID NO: 9, amino acid of East African 狒狒sDSS1 The sequence is as SEQ ID NO: 10, the amino acid sequence of Angora simian sDSS1 is SEQ ID NO: 11, and the amino acid sequence of leucocephalus sDSS1 is SEQ ID NO: 12, scorpion sDSS The amino acid sequence of 1 is SEQ ID NO: 13, and the amino acid sequence of porpoise monkey sDSS1 is SEQ ID NO: 14. The use according to claim 6, wherein the sDSS1 protein is any first protein having a similarity to the basic protein of 70% or more. The use according to claim 6, wherein the sDSS1 protein is any polypeptide fragment which is fused to other polypeptide fragments at the nitrogen terminal or the carbon terminal based on 58 amino acids of the basic protein nitrogen end. A second protein having structural or amino acid sequence characteristics identical or similar to the 31 sequences of the carbon end of the basal protein. The use according to claim 6, wherein the sDSS1 protein is based on any of the 58 amino acids of the basic protein nitrogen end, and the other amino acid fragments are fused at the nitrogen terminal or the carbon terminal, and the fusion protein can realize the cross. The third protein of membrane transport function. The use according to any one of claims 6-9, wherein the sDSS1 protein utilizes any one of a basic protein, a first protein, a second protein, and a third protein, and the protein itself A fusion protein formed by ligation of a carrier protein, antibody or other amino acid fragment of any length. The use according to any one of claims 6 to 9, wherein the sDSS1 protein is produced based on modification of any one of a basic protein, a first protein, a second protein, and a third protein. Protein modification. The protein modification according to claim 11, wherein the modification of the protein modification is directed to an amino group on an amino acid side chain, a carbonyl group on an amino acid side chain, a nitrogen terminal amino group, a carbon terminal carbonyl group, and a cysteine. Specific or non-specific chemical modification of 1-20 sites by amino acid, tyrosine, serine, tryptophan. The polypeptide/protein modification according to claim 11, wherein the modification method of the protein modification comprises glycosylation modification, fatty acid modification, acylation modification, Fc fragment fusion, albumin fusion, polyethylene glycol Modification, dextran modification, heparin modification, polyvinylpyrrolidone modification, polyamino acid modification, polysialic acid modification, chitosan and its derivative modification, lectin modification, sodium alginate modification, carbomer modification, polyethylene Pyrrolidone modification, hydroxypropyl methylcellulose modification, hydroxypropyl cellulose modification, acetylation modification, formylation modification, phosphorylation modification, methylation modification or sulfonation modification, and other pharmaceutically useful polypeptide/protein drugs One or more of the modification methods. The use according to any one of claims 6 to 10, wherein the sDSS1 protein is an amino acid sequence using any one of a basic protein, a first protein, a second protein, and a third protein. A non-natural amino acid replacement protein substituted with 1-31 arbitrary amino acid positions of amino acids other than the 20 essential amino acids. The use according to claim 14, wherein the amino acid substitution of the non-natural amino acid replacement protein comprises hydroxyproline, hydroxylysine, selenocysteine, D-type amino acid or synthetic non- Natural amino acids and their derivatives. The use according to any one of claims 6 to 15, wherein the sDSS1 protein is a basic protein, a first protein, a second protein, a third protein, a fusion protein, a protein modification or a non- Part or all of a complex of a natural amino acid substitute with a pharmaceutically acceptable pharmaceutical carrier. The use according to claim 16, wherein the pharmaceutical carrier comprises an enteric coating preparation, a capsule, a microsphere/capsule, a liposome, a microemulsion, a double emulsion, a nanoparticle, a magnetic particle, a gelatin or a gel. One or more of them. The use according to claim 6, wherein the sDSS1 protein targets the individual's own sDSS1 protein and affects the level of the individual's own sDSS1 protein by the exogenous drug. The use according to claim 18, wherein the drug is a drug target of a sDSS1 protein, a gene of sDSS1 protein, a regulatory element of a gene of sDSS1 or a gene of sDSS1. The use according to claim 18, wherein said drug modulates the amount of sDSS1 protein in the blood by affecting protease/peptidase activity in the blood. The use according to any one of claims 18 to 20, characterized in that the drug is a chemical small molecule drug, an antibody, a polypeptide/protein drug, a nucleic acid drug or a nano drug. The use according to any one of claims 6 to 21, wherein the sDSS1 protein is a basic protein, a first protein, a second protein, a third protein, a fusion protein, a protein modification, or a non- A pharmaceutical combination of two or more of any one of natural amino acid substitutes, complexes, and drugs. The use according to any one of claims 6 to 21, wherein the sDSS1 protein is a basic protein, a first protein, a second protein, a third protein, a fusion protein, a protein modification, and an unnatural one. A combination of one, two or more of any one of an amino acid substitute, a complex, and a drug with a pharmaceutically acceptable excipient. The use according to any one of claims 6 to 10, wherein the sDSS1 protein encodes a basic protein, a first protein, a second protein, a third protein, and a fusion protein by an expression system. A nucleotide sequence of a protein is introduced into the body and the obtained protein is expressed. The use according to claim 24, wherein the expression system is a eukaryotic expression plasmid vector, an adenovirus, an adeno-associated virus, a lentivirus, a retrovirus, a baculovirus, a herpes virus, a pseudorabies virus, ZFN gene editing technology, TALEN gene editing technology, CRISPR/Cas gene editing technology and other medically available gene editing techniques or viral vectors. The use according to any one of claims 6 to 10, wherein the sDSS1 protein is a basic protein, a first protein, a second protein, a third protein and a fusion obtained in a human body by transplanting cells. Any protein in the protein. The use according to claim 26, wherein said cell is a stem cell, a precursor cell or an adult cell of any one of humans. The use according to claim 27, wherein the stem cells are embryonic stem cells, induced pluripotent stem cells, transdifferentiated cells, or stem cells derived from primary culture, pluripotently differentiated from mother cells or Single energy stem cells. The use according to claim 6, wherein the sDSS1 protein is an sDSS1 protein introduced into an individual by serum or interstitial fluid infusion. The use according to claim 6, wherein the sDSS1 protein is a basic protein, a first protein, a second protein, and a third protein obtained in an individual by transplanting tissues or organs. protein. The use according to claim 30, wherein in the tissue or organ transplantation, the tissue is a whole organ or a part of a tissue block of the brain, liver, kidney, spleen, islet, or blood, fat, muscle, bone marrow, skin. The use according to any one of claims 1 to 31, wherein the prophylactic agent comprises a basic protein, a first protein, a second protein, a third protein, a fusion protein, a protein modification, and a non- Natural amino acid substitution protein, complex, drug combination, expression system, protein, tissue, organ, body fluid, tissue fluid protein drug, peptide drug, nucleic acid drug, chemical small molecule drug, cell product, commercial transplantation tissue, injection, frozen Dry powder, health supplements or food additives. The use according to claims 1 to 31, characterized in that the therapeutic drug comprises a basic protein, a first protein, a second protein, a third protein, a fusion protein, a protein modification, an unnatural amino acid. Alternative protein, complex, drug combination, expression system, protein, tissue, organ, body fluid, tissue fluid protein drug, peptide drug, nucleic acid drug, chemical small molecule drug, cell product, commercial transplantation tissue, injection, lyophilized powder , health products or food additives. The use of a protein for improving a diabetic complications care device, characterized in that it is an application for preparing a medicament for preventing and treating diabetes complications by using the application according to any one of claims 6-17, and then preparing the drug Used to improve the performance of medical devices related to the care of diabetic complications. The use of the protein according to claim 34 for improving a diabetic complications care device, characterized in that the medical device comprises a blood collection device and a consumable, a blood purification device and a consumable, a blood purification device auxiliary device and a consumable, One or more of body fluid treatment equipment and consumables, kidney dialysis equipment and consumables, peritoneal dialysis equipment and consumables, blood perfusion devices, infusion devices, inhalation devices, sustained release devices, artificial kidneys.

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