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WO2018188551A1 - Médicament pour le traitement de la stéatose hépatique et procédé de traitement - Google Patents

Médicament pour le traitement de la stéatose hépatique et procédé de traitement Download PDF

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WO2018188551A1
WO2018188551A1 PCT/CN2018/082308 CN2018082308W WO2018188551A1 WO 2018188551 A1 WO2018188551 A1 WO 2018188551A1 CN 2018082308 W CN2018082308 W CN 2018082308W WO 2018188551 A1 WO2018188551 A1 WO 2018188551A1
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hcbp6
mice
gene
fatty liver
ginsenoside
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成军
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    • A—HUMAN NECESSITIES
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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Definitions

  • the present invention belongs to the fields of bioengineering and pharmaceuticals, and in particular, to a method for screening for a drug for treating or preventing diseases such as fatty liver by targeting the HCBP6 gene, and a drug obtained by screening by the above method.
  • Fatty liver refers to a lesion of excessive accumulation of fat in liver cells due to various reasons. According to statistics, 80% of liver cancer is caused by viral hepatitis, and fatty liver is recognized as a common cause of occult cirrhosis, becoming the second largest liver disease after viral hepatitis. In recent years, the prevalence of fatty liver in China has been increasing and appearing to be younger. According to statistics, the incidence of fatty liver in China is about 18%, and that in developed cities is higher.
  • Fatty liver is not only an independent disease, but also causes a variety of comorbidities. Long-term disease of fatty liver not only causes liver cirrhosis, but also the blood sugar and blood lipid metabolism of patients will be seriously affected. The causes of fatty liver can be roughly divided into metabolic and viral infections. Metabolic is more common in alcoholic fatty liver, diabetic fatty liver, obese fatty liver, fast weight loss fatty liver, drug-induced fatty liver, pregnancy fatty liver, etc., viral infection is more common in hepatitis C virus (HCV) infection.
  • HCV hepatitis C virus
  • the commonly used hepatoprotective drugs in the clinic include vitamins, drugs that promote liver detoxification, drugs that promote energy metabolism, drugs that promote protein synthesis, drugs that are resistant to fatty liver, and drugs that are resistant to fibrosis. But so far, there is no effective monomeric drug for preventing and treating fatty liver.
  • the treatment of fatty liver depends on the compound Chinese medicine/Chinese patent medicine; or the western medicine often uses protective liver cells, fat-removing drugs and antioxidants, and some Lipid-lowering drugs, such as statins, lipid-lowering drugs, and the like.
  • Hypertriglyceridemia currently lacks effective therapeutic drugs and treatments.
  • About steatohepatitis NASH has become the most common type of liver disease in the world, but in addition to lifestyle adjustments, there is currently no effective drug intervention.
  • HCBP6 plays a negative regulatory role in TC and TG synthesis, or other regulatory mechanisms upstream of it, remains to be further studied.
  • the present invention establishes a cell model in which the HCBP6 gene is overexpressed or silenced, establishes a mouse model of HCBP6 knockout and a zebrafish animal model, and establishes a mouse model of fatty liver. Based on the cell model and animal model, the negative regulation of HCBP6 on TC and TG was verified. Based on the cell model and animal model, a series of ginsenoside monomer compounds, such as Rh2, Rb3, Rc, CK, were obtained.
  • Rh1 or ginseng diol which can significantly up-regulate the expression of HCBP6 gene and achieve inhibition of TC and/or TG synthesis; it has a significant therapeutic effect on the mouse fatty liver model caused by high-fat diet.
  • Rh2, Rb3, Rc, CK, Rh1 or Panaxadiol can inhibit TC and TG synthesis by regulating HCBP6, and treat steatohepatitis, diabetes, and multiple sexual sclerosis, hypertriglyceridemia, and / or cardiovascular and atherosclerosis and other diseases.
  • the present invention provides an animal model of HCBP6 gene knockout, characterized in that the animal is a mouse or a zebrafish.
  • the preparation method of the animal model comprises the following steps:
  • Target gene localization the corresponding gene of human gene HCBP6 in mouse 4833415N24Rik (NM_026126.4);
  • mice Homozygous mice obtained by knockout of HCBP6 gene: F1 mice were selfed, and homozygous HCBP6 knockout mice were obtained.
  • the present invention provides an HCBP6 gene or HCBP6 protein as a drug target in screening and/or preparation for preventing and/or treating fatty liver, steatohepatitis, diabetes, multiple sclerosis, hypertriglyceridemia, hypercholesterolemia Use in disease and/or cardiovascular and cerebrovascular atherosclerosis drugs.
  • the present invention provides a method for screening a drug based on a target of HCBP6 gene or HCBP6 protein drug, which comprises contacting a cell comprising a HCBP6 gene or an HCBP6 protein with a compound to be screened, and screening for promoting expression of the HCBP6 gene or HCBP6 protein.
  • Compound includes methods of incubation, transfection, introduction, and the like.
  • the invention provides an HCBP6 agonist for preventing and/or treating fatty liver, steatohepatitis, diabetes, multiple sclerosis, hypertriglyceridemia, hypercholesterolemia and/or cardiovascular atherosclerosis. Use in medicine.
  • the invention provides a ginsenoside for preparing and preventing or treating fatty liver, steatohepatitis, diabetes, multiple sclerosis, hypertriglyceridemia, hypercholesterolemia and/or cardiovascular and cerebral atherosclerosis drugs.
  • the ginsenoside is selected from the group consisting of Rh2, Rb3, Rc, CK, Rh1 or a combination of one or more of ginseng diols, preferably Rh2, Rb3, Rc, CK.
  • Ginsenoside Rh2, Rb3, Rc, CK, Rh1 or Panaxadiol can inhibit the synthesis and/or accumulation of cholesterol (TC) and/or triglyceride (TG).
  • TC cholesterol
  • TG triglyceride
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising ginsenosides, characterized in that ginsenoside is the only effective pharmaceutical component and the composition further comprises a pharmaceutically acceptable adjuvant.
  • the ginsenoside is selected from the group consisting of Rh2, Rb3, Rc, CK, Rh1 or a combination of one or more of ginseng diols, preferably Rh2, Rb3, Rc, CK.
  • the above pharmaceutical composition can be administered by gastrointestinal, subcutaneous and/or intravenous injection.
  • the invention provides a ginsenoside Rh2, Rb3, Rc, and/or CK for preparing and/or improving fatty liver, steatohepatitis, diabetes, multiple sclerosis, hypertriglyceridemia, high cholesterol Use of blood and healthy cardiovascular foods for cerebral vascular atherosclerosis.
  • the present invention provides a health functional food comprising ginsenoside Rh2, Rb3, Rc, and/or CK and other food common excipients, and does not contain other ginsenoside components.
  • ginsenoside Rh2, Rb3, Rc, CK, Rh1 or ginseng diol may be ginsenoside Rh2, Rb3, Rc, CK, Rh1 or ginsenoside isolated from ginseng which is commercially cultivated or cultivated or extracted in nature. Panaxadiol; or ginsenoside Rh2, Rb3, Rc, CK, Rh1 or ginseng diol isolated from other plants containing ginsenosides; or ginsenoside Rh2, Rb3, Rc, CK transformed from the isolated ginsenoside , Rh1 or Panaxadiol.
  • ginsenoside Rh2, Rb3, Rc, CK, Rh1 or ginseng diol synthesized by a chemical synthesis method or a biological fermentation method may be used as long as the ginsenoside Rh2, Rb3, which exhibits a liver disease prevention or treatment effect of the present invention, Rc, CK, Rh1 or ginseng diol can be used without limitation.
  • liver disease may be selected from the group consisting of hepatitis, cirrhosis, fatty liver, liver dysfunction, and liver cancer, but is not limited thereto.
  • Hepatitis refers to inflammation of liver cells and liver tissues. If the liver cells are repeatedly destroyed and regenerated by chronic hepatitis, the fibrous tissue and regenerative nodules in the liver are increased to evolve into cirrhosis or cirrhosis. If the cirrhosis develops to a certain level or higher, complications such as Hepatic encephalopathy and Esophageal varix can be induced.
  • fatty liver refers to fat that accumulates in the liver more than the proportion (5%) of fat in normal liver.
  • the fatty liver may be alcoholic fatty liver or nonalcoholic fatty liver caused by obesity, rapid weight loss, diabetes, hyperlipidemia or drugs, pregnancy, and the like.
  • the "fatty liver” of the present invention is preferably nonalcoholic fatty liver disease (NAFLD).
  • prevention may refer to all the actions of an individual to administer the ginsenoside Rh2, Rb3, Rc, CK, Rh1 or ginseng diol of the present invention to inhibit or delay the onset of liver disease.
  • treating may be directed to a liver disease suspected individual to administer the one or a combination of the ginsenoside Rh2, Rb3, Rc, CK, Rh1 or ginseng diol composition to improve liver disease symptoms or to benefit All behaviors of symptom relief.
  • the pharmaceutical composition of the present invention can be used as a single preparation, and can be prepared by adding a recognized drug having a therapeutic effect of liver disease to a composite preparation, which can be formulated by using a pharmaceutically acceptable carrier or excipient. It is prepared in a unit volume form or loaded into a multi-volume container.
  • pharmaceutically acceptable carrier/excipient may refer to a carrier or diluent which neither irritates the organism nor hinders the biological activity and properties of the injected compound.
  • the type of the carrier which can be used in the present invention is not particularly limited, and any carrier which is commonly used in the art and which is pharmaceutically acceptable can be used.
  • Non-limiting examples of the carrier include saline, sterilized water, Ringer's solution, buffered saline, albumin injection solution, glucose solution, maltodextrin solution, glycerin, ethanol, and the like. These can be used individually or in mixture of 2 or more types.
  • a diluent such as an antioxidant, a buffer, and/or a bacteriostatic agent may be added as needed, and a diluent, a dispersant, a surfactant, a binder, a lubricant, or the like may be added and formulated into an aqueous solution, Injectable dosage forms, pills, capsules, granules or tablets for suspensions, emulsions, and the like are used.
  • the pharmaceutical composition of the present invention may comprise any one or a combination of pharmaceutically effective amounts of ginsenoside Rh2, Rb3, Rc, CK, Rh1 or ginseng diol.
  • the term "pharmaceutically effective amount” means an amount sufficient to treat a disease in a reasonable benefit/risk ratio applicable to medical treatment, and is usually administered in an amount of from 1 to several times per day in an amount of from 0.001 to 1000 mg. /kg, preferably from 0.05 to 200 mg/kg, more preferably from 0.1 to 100 mg/kg.
  • the particular therapeutically effective amount for a particular patient is preferably a particular composition, such as the type and extent of the response to be achieved, whether other formulations are used, the age, weight, general health of the patient, Various factors, such as sex and diet, time of administration, route of administration and secretion rate of the composition, duration of treatment, use with a particular composition, or drugs used simultaneously, are employed in different ways than similar factors disclosed in the medical arts.
  • the pharmaceutical composition of the present invention can be administered as a single therapeutic agent, or can be used in combination with other therapeutic agents, or can be administered sequentially or simultaneously with conventional therapeutic agents.
  • single administration or multiple administration may be employed. It is important to apply an amount that does not induce side effects and can achieve maximum effect in a minimum amount in consideration of the elements, which can be easily determined by those skilled in the art.
  • administering means that the pharmaceutical composition of the present invention is introduced into a patient by some appropriate method, and the administration route of the composition of the present invention may be oral or non-oral, as long as the target tissue can be reached. kind of path.
  • the mode of administration of the pharmaceutical composition of the present invention is not particularly limited, and a method generally used in the art can be employed. As a non-limiting manner of the mode of administration, the composition can be administered orally or parenterally.
  • the pharmaceutical composition of the present invention can be prepared into various dosage forms in accordance with the mode of administration.
  • the frequency of administration of the composition of the present invention is not particularly limited and may be administered once a day or in multiple portions.
  • Ginsenoside Rh2, Rb3, Rc, CK, Rh1 or Panaxadiol is widely found in nature. It can be directly used as a medicine without side effects. It is developed to prevent and/or treat fatty liver, steatohepatitis, diabetes, multiple sclerosis, high glycerol. Triglyceride, hypercholesterolemia, cardiovascular and cerebrovascular atherosclerosis and / or other important sources of lipid, carbohydrate metabolism drugs.
  • FIG. 1 Overexpression and silencing effect of HCBP6 in L02 and HepG2 cells: a. HCBP6 overexpression; b. HCBP6 gene interference.
  • Figure 4.a HCBP6 protein expression levels after HPCD and cholesterol injection; b. HCBP6 mRNA levels after HPCD and cholesterol treatment.
  • FIG. 5 HCBP6 knockout mice were sequenced and identified; b. HCBP6 knockout mice showed a significant decrease in HCBP6 protein expression in liver, heart and kidney tissues.
  • Figure 7 Changes in food intake, blood glucose, fat content, and blood lipids in the control group and the high-fat diet feeding group: a. 5 weeks of food intake; b. 12 weeks of blood glucose; c. 8 weeks of fat content; d. Weekly blood lipids.
  • Figure 9 Comparison of glucose tolerance in mice in the control and high-fat diet feeding experimental groups: a. intraperitoneal injection of glucose glucose dynamics; b. overall blood glucose levels in mice (area under the curve AUC).
  • FIG. 10 Comparison of thermoregulatory functions in mice in the control group and the high-fat diet feeding experimental group: a-b. changes in body temperature after cold stimulation; c. thermographic images of body temperature.
  • Control group and high-fat diet feeding experimental group model mouse inflammatory response comparison: a. ALT, AST, ALP; b. IL-6, TNF- ⁇ .
  • FIG. 15a After adding different concentrations of Rh2, the levels of TC and TG in the cells decreased; b. After adding different concentrations of Rh2, the expression of HCBP6 in the cells increased (mRNA and protein levels); c. After Rh2 treatment of HepG2 cells, The change of HCBP6 expression at different time points.
  • Figure 16.a After adding different concentrations of Rb3, the intracellular TC, TG content decreased; b. After adding different concentrations of Rb3, the intracellular HCBP6 expression increased (protein level).
  • FIG.a After adding different concentrations of Rc, the intracellular TC content decreased, TG did not change; b. After adding different concentrations of Rc, the intracellular HCBP6 expression increased (protein level).
  • FIG.a After adding different concentrations of Rh1, there was no change in intracellular TC and a decrease in TG content; b. After adding different concentrations of Rh1, the expression of HCBP6 in the cells increased (protein level).
  • Figure 20 a. Intracellular TC content decreased after addition of different concentrations of ginseng diol; b. Increased intracellular HCBP6 expression (protein level) after addition of different concentrations of ginseng diol.
  • FIG. 21 After adding different concentrations of Rt5, there was no change in intracellular TC; b. There was no change in the expression level of HCBP6 protein in cells.
  • FIG. 22 After adding different concentrations of F11, there was no change in intracellular TC; b. There was no change in the expression level of HCBP6 protein in cells.
  • Figure 23 a. Increased intracellular TC levels after addition of different concentrations of R1; b. Reduced expression of HCBP6 protein levels in cells.
  • Figure 24 Changes in body weight, liver tissue HE and body fat content after administration of Rh2: a. body weight; b. body fat; c. liver tissue HE staining.
  • FIG. 25 Glucose tolerance in mice following administration of Rh2: a. blood glucose; b. fasting blood glucose; c. AUC.
  • Figure 26 Changes in biochemical markers in mice following administration of Rh2: a. ALT, AST, ALP; b. CHO, TG, HDL-C, LDL-C.
  • Figure 27 Changes in body weight, liver tissue HE staining and body fat content after administration of Rb3: a. body weight; b. body fat; c. liver tissue HE staining.
  • FIG. 28 Glucose tolerance in mice following administration of Rb3: a. blood glucose; b. fasting blood glucose; c. AUC.
  • Figure 29 Changes in biochemical markers in mice following administration of Rb3: a. ALT, AST, ALP; b. CHO, TG, HDL-C, LDL-C.
  • Figure 30 Changes in body weight, liver tissue HE staining and body fat content after administration of CK: a. body weight; b. body fat; c. liver tissue HE staining.
  • FIG. 31 Glucose tolerance in mice following administration of CK: a. blood glucose; b. fasting blood glucose; c. AUC.
  • Figure 32 Changes in biochemical parameters of mice after administration of CK: a. ALT, AST, ALP; b. CHO, TG, HDL-C, LDL-C.
  • Figure 33 Changes in body weight, liver tissue HE staining and body fat content after administration of Rc: a. body weight; b. body fat; c. liver tissue HE staining.
  • FIG. 34 Mice glucose tolerance after Rc administration: a. blood glucose; b. fasting blood glucose; c. AUC.
  • Figure 35 Changes in biochemical markers in mice following administration of Rc: a. ALT, AST, ALP; b. CHO, TG, HDL-C, LDL-C.
  • Example 1 HCBP6 regulates the synthesis and accumulation of TC and TG at the cellular level
  • the pHCBP6 was transiently transfected into the cells cultured in the six-well plate, and the total protein was extracted after 48 hours.
  • the band of 21kDa was detected by the primary antibody against HCBP6, and the expression of HCBP6 was significantly increased in the experimental group compared with the control group. .
  • the same experimental method verified the interference effect of si-HCBP6 chemically synthesized by Shanghai Jima Company. Western blot showed that it could interfere with more than 60% of endogenous HCBP6 protein compared with the control group (see Figures 1a and 1b).
  • HCBP6 can reduce the mechanism of intracellular cholesterol synthesis
  • total protein was extracted and Western blot was used to detect the marker proteins SREBP 2 and HMGCR and glycerol in the cholesterol synthesis pathway.
  • the results showed that overexpression of HCBP6 significantly down-regulated the expression of SREBP2/HMGCR/SREBP1c/FASN protein (Fig. 3). After silencing the expression of HCBP6 in cells, the expression of SREBP2/HMGCR/SREBP1c/FASN protein was significantly increased (Fig. 3).
  • 4.HCBP6 can sense changes in total cholesterol levels in cells and oscillate to maintain cellular cholesterol homeostasis.
  • the human gene HCBP6 is in mouse 4833415N24Rik (NM_026126.4), which is located on the mouse X chromosome;
  • the F0 generation mouse was obtained and the F1 generation was propagated: the mother of the transplant recipient was born and F0 was born; the chimeric mouse F0 was mated with the wild mouse, and the F1 hybrid mouse of the germline inheritance was obtained;
  • mice F1 mice were selfed, and homozygous HCBP6 knockout mice were obtained.
  • the tail genotype identification confirmed the germline inheritance:
  • genomic DNA was extracted from the tail of HCBP6 knockout mice, HCBP6 was amplified by PCR, and the products were verified by gel electrophoresis.
  • Example 3 Construction of a fatty liver mouse model
  • Fatty liver model animal grouping 20 6-8 weeks C57BL/6J male mice and 20 6-8 weeks HCBP6 knockout male mice (C57BL/6J strain) were randomly divided into the following 4 groups:
  • WT wild type mice
  • KO knockout mice
  • Chow normal diet feeding
  • HFD high fat diet feeding.
  • Control group mice (Chow): given normal diet feeding (Huaqi Kang company conventional mouse feed);
  • Fatty liver model group feeding with high-fat diet (Whitby Technology Development (Beijing) Co., Ltd.);
  • mice The body weight changes of the mice were recorded during the modeling period, and the mice's food intake, body fat content, fasting blood glucose, GTT, body temperature and other indicators were measured.
  • RNA and protein were collected and analysis: After 12 weeks of modeling, blood was taken, plasma was separated, and liver function indexes AST, ALT and HDL, LDL, CHO and TG were detected. Fresh liver tissue, brown fat and kidney were taken from the mice, and some were fixed in 10% formalin, embedded in paraffin, sliced, and a part of liquid nitrogen was frozen to extract total RNA and protein. For subsequent experiments:
  • mice were analyzed by the nuclear magnetic resonance component analysis technique. Analysis of adipose tissue, lean tissue and free water showed that the fat content of wild-type mice and HCBP6 knockout mice increased significantly after induction of high-fat diet, with significant statistical differences, and Compared with wild-type mice, the increase of fat content in HCBP6 knockout mice is more obvious; the levels of CHO, TG, HDL-C and LDL-C in plasma represent the blood lipids of the body. Therefore, we take blood by eyeball.
  • Brown fat is the body tissue responsible for breaking down the white fat that causes obesity, which can be converted into carbon dioxide, water and heat, accelerate the body's metabolism and promote white fat consumption.
  • brown adipose tissue plays an important role in maintaining systemic glucose homeostasis.
  • brown adipose tissue is also a major source of non-thrombotic heat production in mammals and plays an important role in maintaining animal body temperature and energy balance. Under the stimulation of cold, brown fat can be activated, causing the decomposition and oxidation of lipids in brown fat cells, generating a lot of heat to maintain the stability of body temperature.
  • mice we examined the body temperature of mice in a low temperature environment to observe whether brown fat was activated, and indirectly judged the glucose metabolism of mice.
  • the experimental results showed that there was no significant difference in body temperature between mice in the conventional feeding environment.
  • the body temperature of the mice decreased significantly.
  • the body temperature of HCBP6 knockout mice was significantly lower than that of wild-type mice, and the body temperature was statistically different at 1h and 3h after cold stimulation (Fig. 10a, b).
  • Fig. 10c From the thermographic map, we can more intuitively observe that the body temperature of HCBP6 knockout mice induced by high-fat diet decreased significantly after cold stimulation (Fig. 10c).
  • the mice could not maintain the stability of body temperature under cold stimulation, suggesting that the brown fat may not be activated or dysfunctional, resulting in a weakening of the regulation of glucose homeostasis.
  • HCBP6 has a role in regulating the homeostasis of glycolipid metabolism. It suggests that HCBP6 may become a new target for the treatment of NAFLD. In the next part of the experiment, we will use HCBP6 as a target to find potential drugs that may treat NAFLD.
  • Rh2 was added to HepG2 cell line at different concentrations (0, 1, 2.5, 5, 10, 25 ⁇ M), and the expression of TC, TG and HCBP6 in cells was detected 48 h later.
  • Rh2 50 ⁇ M Rh2 was added to HepG2 cell line, and total RNA and protein were extracted after 12h, 24h, 36h and 48h, respectively, and the expression of HCBP6 was detected.
  • ginsenoside monomers Rh2, Rb3, Rc, CK and Rh1 can up-regulate the expression of HCBP6 protein and inhibit the synthesis of total cholesterol and/or triglyceride in cells, suggesting that Rh2, Rb3, Rc, CK and Rh1 may improve.
  • WT wild-type mice
  • Chow normal diet feeding
  • HFD high-fat diet feeding
  • LFD low-fat diet feeding
  • GTT glucose tolerance test
  • ITT insulin tolerance test.
  • the results of HE staining showed that the liver tissue of the normal diet group was intact and no lipid droplets were formed in the cytoplasm.
  • the mouse liver tissue cytoplasm was induced by the high-fat diet alone. A large number of round or oval vacuoles were observed, lipids were obviously accumulated, and the fatty liver model was successfully constructed.
  • Rh2 the lipid droplets in the liver tissue of the mice were reduced compared with the control, and the fat in the mouse liver tissue cells was empty. The vesicles were significantly reduced, and the degree of fatty liver was significantly lower than that of the untreated group (Fig. 24c).
  • mice showed that there was no significant change in plasma AST, ALT, ALP, CHO, TG, HDL-C and LDL-C after treatment with Rb3 (Fig. 29a-b).
  • ginsenoside Rh2 can improve the pathological condition and glucose tolerance of liver tissue in mice, and ginsenoside CK is better than Rh2 in improving the pathology of liver tissue in mice. It can reduce the fasting blood glucose and plasma cholesterol content of mice, but the improvement of glucose tolerance in mice is not as obvious as that of Rh2, while the improvement of glucose and lipid metabolism in mice by Rb3 and Rc is weaker than that of Rh2 and CK.

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Abstract

La présente invention concerne des utilisations d'un gène HCBP6 ou d'une protéine HCBP6 en tant que cible de médicament dans le criblage et/ou la préparation d'un médicament pour la prévention et/ou le traitement de la stéatose hépatique, la stéatohépatite, le diabète, la sclérose en plaques, l'hypertriglycéridémie, l'hypercholestérolémie et/ou l'athérosclérose cardiovasculaire et cérébrale ; l'utilisation d'un agoniste de HCBP6 dans la préparation d'un médicament pour la prévention et/ou le traitement de la stéatose hépatique, la stéatohépatite, le diabète, la sclérose en plaques, l'hypertriglycéridémie, l'hypercholestérolémie, et/ou l'athérosclérose cardiovasculaire et cérébrale ; l'utilisation d'un ginsénoside dans la préparation d'un médicament pour la prévention et/ou le traitement de la stéatose hépatique, la stéatohépatite, le diabète, la sclérose en plaques, l'hypertriglycéridémie, l'hypercholestérolémie, et/ou l'athérosclérose cardiovasculaire et cérébrale ; et un modèle animal knock-out pour le gène HCBP6 et son procédé de préparation.
PCT/CN2018/082308 2017-04-12 2018-04-09 Médicament pour le traitement de la stéatose hépatique et procédé de traitement Ceased WO2018188551A1 (fr)

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