WO2018188551A1

WO2018188551A1 - Medicament for treating fatty liver and treatment method

Info

Publication number: WO2018188551A1
Application number: PCT/CN2018/082308
Authority: WO
Inventors: 成军
Original assignee: 成军
Priority date: 2017-04-12
Filing date: 2018-04-09
Publication date: 2018-10-18
Also published as: CN108686210A; CN112274523A

Abstract

Uses of an HCBP6 gene or HCBP6 protein as a medicament target in screening and/or preparing a medicament for preventing and/or treating fatty liver, steatohepatitis, diabetes, multiple sclerosis, hypertriglyceridemia, hypercholesterolemia, and/or cardiovascular and cerebral atherosclerosis; uses of an HCBP6 agonist in preparing a medicament for preventing and/or treating fatty liver, steatohepatitis, diabetes, multiple sclerosis, hypertriglyceridemia, hypercholesterolemia, and/or cardiovascular and cerebral atherosclerosis; uses of a ginsenoside in preparing a medicament for preventing and/or treating fatty liver, steatohepatitis, diabetes, multiple sclerosis, hypertriglyceridemia, hypercholesterolemia, and/or cardiovascular and cerebral atherosclerosis; and, an HCBP6 gene-knockout animal model and a preparation method therefor.

Description

Medicine and treatment method for treating fatty liver

Technical field

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.

Background technique

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.

At present, 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.

Currently, there are a series of statins for hypercholesterolemia, which are widely used. 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.

Therefore, research on the pathogenesis of fatty liver, and corresponding screening or development of effective drugs to treat fatty liver disease become a common appeal in the field of liver disease treatment. In 2002, the inventors reported the use of yeast two-hybrid (yeast-two hybrid) technology to screen human hepatocyte cDNA libraries for the human gene HCBP6 that binds to HCV core protein. However, the research on the function of HCBP6 has been slow for a long time, and few studies have reported it. On the basis of this, the inventors applied a series of molecular biology techniques to confirm that HCBP6 can significantly inhibit the synthesis of TC and TG, and maintain the stability of intracellular TC and TG environment. Based on the close relationship between lipid metabolism such as TC and TG and cardiovascular and cerebrovascular diseases, the molecular biological mechanism of lipid metabolism is of great significance. However, whether HCBP6 plays a negative regulatory role in TC and TG synthesis, or other regulatory mechanisms upstream of it, remains to be further studied.

Summary of the invention

In view of the above-mentioned defects and gaps in the prior art, 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. Based on the close relationship between lipid metabolism such as TC and TG and cardiovascular and cerebrovascular diseases, 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 specific technical solutions of the present invention are as follows:

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:

1) Target gene localization: the corresponding gene of human gene HCBP6 in mouse 4833415N24Rik (NM_026126.4);

2) TALEN design and construction: the gene 4833415N24Rik exon 3 is used as a TALEN gene editing target;

3) Gene editing: the mouse fertilized egg is injected into the mRNA edited by TALEN, and the fertilized egg after injection is transplanted into the body of the pseudo-pregnant mother;

4) Obtaining positive F0 mice: feeding transplant recipient mothers and identifying positive F0 mice;

5) Obtaining germline-inherited F1 hybrid mice: the positive mice obtained in step 4) were mated with wild mice to obtain F1 hybrid mice;

6) 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. The contacting 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. In the use, 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).

The present invention provides 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.

In the present invention, 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. Alternatively, 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.

The above "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).

The term "prevention" as used in the present invention 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.

The term "treating" as used in the present invention 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.

The term "pharmaceutically acceptable carrier/excipient" as used in the present invention 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. In addition, other common additives 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.

In the present invention, 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. However, in accordance with the purpose of the present invention, 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. In addition, 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.

The term "administering" as used in the present invention 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.

The inventors systematically screened the water-soluble extract of Korean ginseng. It was first discovered that the chemical constituents of ginsenoside Rh2, Rb3, Rc, CK, Rh1 or ginseng diol could significantly increase the expression level of HCBP6 gene and achieve TC and/or TG. This invention was accomplished by significant inhibition of synthesis and effective reduction of blood glucose levels, both in cell models and animal models. 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.

DRAWINGS

Figure 1. Overexpression and silencing effect of HCBP6 in L02 and HepG2 cells: a. HCBP6 overexpression; b. HCBP6 gene interference.

Figure 2. Results of intracellular TC/TG assay after overexpression or silencing of HCBP6.

Figure 3. Detection of intracellular SREBP2/HMGCR/SREBP1c/FASN protein expression levels after overexpression or silencing of HCBP6.

Figure 4.a. HCBP6 protein expression levels after HPCD and cholesterol injection; b. HCBP6 mRNA levels after HPCD and cholesterol treatment.

Figure 5.a. 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 6. Body weight record of control and high fat diet fed experimental group model mice.

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 8. HE staining results show the degree of hepatic steatosis in mice of different experimental groups.

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).

Figure 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.

Figure 11. Control group and high-fat diet feeding experimental group model mouse inflammatory response comparison: a. ALT, AST, ALP; b. IL-6, TNF-α.

Figure 12. Protein expression of HCBP6 in liver tissue of control group and high-fat diet-fed mice.

Figure 13. Changes in mRNA levels of HCBP6 and lipid synthesis and breakdown genes in control and high-fat diet-fed mice.

Figure 14. Immunohistochemistry results show a decrease in the expression of HCBP6 in liver tissue of fatty liver mice.

Figure 15.a. 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).

Figure 17.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).

Figure 18.a. After adding different concentrations of CK, the intracellular TC content decreased; b. After adding different concentrations of CK, the intracellular HCBP6 expression increased (protein level).

Figure 19.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.

Figure 21.a. 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.

Figure 22.a. 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.

Figure 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.

Figure 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.

Figure 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.

Figure 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.

detailed description

Example 1: HCBP6 regulates the synthesis and accumulation of TC and TG at the cellular level

1. Verify HCBP6 overexpression and silencing effect

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).

2. Overexpression/silencing of HCBP6 on intracellular TC/TG

After transient transfection of pHCBP6 or si-HCBP6 in L02 and HepG2 cell lines, the levels of TC/TG in each group were measured. The results showed that the intracellular TC/TG content decreased when HCBP6 was overexpressed; the intracellular TC/TG content increased after silencing HCBP6, the difference was statistically significant (*P<0.05, **P<0.01, n=3) (Fig. 2) .

3. Effect of overexpression/silencing of HCBP6 on intracellular SREBP2/HMGCR/SREBP1c/FASN protein levels

To verify that HCBP6 can reduce the mechanism of intracellular cholesterol synthesis, after transient transfection of pHCBP6 or si-HCBP6 in L02 and HepG2 cell lines, 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. Expression of the marker proteins SREBP1c and FASN in the triester 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.

(1) After treatment with hydroxypropyl-β-cyclodextrin (HPCD) (Sigma, C0926), the total cholesterol level in the cells decreased, and HCBP6 decreased correspondingly at the protein level, and the SREBP2 as a control increased accordingly. Statistical differences (Figure A in Figure 4b); however, at the mRNA level, there was a trend toward a decrease in HCBP6 levels, but there was no statistical difference (Figure A in Figure 4b).

(2) After treatment with Cholesterol (Sigma, C3045) in the overloaded cells, HCBP6 increased correspondingly at the protein level after the increase of total cholesterol in the cells, and the SREBP2 as a control decreased correspondingly. Differences (panel B in Figure 4a); there was a trend toward elevated levels of HCBP6 at the mRNA level, but there was no statistical difference (panel B in Figure 4a).

Example 2: Construction of a HCBP6 knockout mouse model

1. The human gene HCBP6 is in mouse 4833415N24Rik (NM_026126.4), which is located on the mouse X chromosome;

2. TALEN design and construction, the gene 4833415N24Rik exon 3 as a TALEN genome editing target;

3. Mouse fertilized egg pronucleus injection specific mRNA (TALEN);

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;

5. Obtain homozygous mice with HCBP6 gene knockout: F1 mice were selfed, and homozygous HCBP6 knockout mice were obtained. The tail genotype identification confirmed the germline inheritance:

(1) The genomic DNA was extracted from the tail of HCBP6 knockout mice, HCBP6 was amplified by PCR, and the products were verified by gel electrophoresis.

(2) The liver tissue of HCBP6 knockout mice was taken, protein was extracted, and the expression of HCBP6 was detected by Western Blot.

Identification results:

(1) Sequencing results showed that the bases of HCBP6 mice were deleted to different degrees (not a multiple of 3) (Fig. 5a);

(2) Western Blot results showed that the expression of HCBP6 protein in two HCBP6 knockout mice was significantly reduced, indicating that the HCBP6 knockout mouse model was successfully constructed (Fig. 5b).

Example 3: Construction of a fatty liver mouse model

1. 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:

Note: WT, wild type mice; KO, knockout mice; Chow, normal diet feeding; HFD, high fat diet feeding.

2. Construction of fatty liver model:

(1) Control group mice (Chow): given normal diet feeding (Huaqi Kang company conventional mouse feed);

(2) Fatty liver model group (HFD): feeding with high-fat diet (Whitby Technology Development (Beijing) Co., Ltd.);

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.

3. Specimen collection 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:

(1) HE staining to observe the pathological changes of liver tissue and the degree of steatosis;

(2) Q-PCR detection of changes in the expression of inflammatory factors IL-6 and TNF-α in liver tissue;

(3) Q-PCR detection of changes in HCBP6 and lipid synthesis (SREBP2, HMGCR, SREBP1c, FASN)/decomposition related genes (ATGL, HSL) and glucokinase (GCK) mRNA levels in liver tissues;

(4) Comparison of HCBP6 protein expression in liver tissue;

(5) Immunohistochemical method was used to detect the change in the expression level of HCBP6.

4. Results:

(1) The results showed that the weight of mice in the fatty liver model group was significantly higher than that in the control group; the weight of HCBP6 knockout mice in the fatty liver model group was higher than that in the wild group, and there was statistical difference (Fig. 6);

(2) The results showed that there was no significant difference in the food intake of the mice; the blood glucose of the mice in the fatty liver model group was higher than that of the control group, and the blood glucose of the HCBP6 knockout mice was higher than that of the wild type mice; the 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. Plasma was isolated and tested. The results showed that CHO, LDL-C, HDL-C, LDL-C/HDL-C increased in the mouse model of fatty liver induced by high-fat diet, and wild type Compared with mice, HCBP6 knockout mice had increased CHO and increased LDL-C (Figure 7);

(3) We observed the hepatic steatosis in mice by pathological HE staining. The results showed that the liver tissue of the normal diet group was intact, and the hepatocytes were centered on the central vein and arranged radially around the cytoplasm. No lipid droplet formation was observed. After induction of high-fat diet, there were many different fat vacuoles in the liver tissue of wild-type mice. The structure of hepatic lobule was still visible, while the liver cells of HCBP6 knockout mice were enlarged. There are round or oval vacuoles in the nucleus, some of which push the nucleus to one side, and the degree of fatty liver is significantly heavier than that of wild-type mice (Fig. 8);

(4) In order to further clarify whether the mouse has abnormal glucose metabolism, we performed a glucose tolerance test. The results showed that after giving normal diet, wild-type mice and HCBP6 knockout mice had peak blood glucose 15 to 30 minutes after intraperitoneal injection of glucose. After 2 hours, blood glucose decreased significantly, indicating that the body load of mice was given to normal diet. Good ability, no abnormal glucose metabolism; after administration of high-fat diet, blood glucose increased sharply after injection of glucose in wild-type mice and HCBP6 knockout mice, peaked at 30 min, and 2 h after glucose administration. It is still at a high level (Fig. 9), indicating that after induction of a high-fat diet, the glucose tolerance of the mice is impaired and abnormal glucose metabolism occurs, while the blood glucose level of HCBP6 knockout mice is restored more than that of wild-type mice. Slow, and statistically significant (Figure 9), indicating that HCBP6 knockout mice are more severely impaired in glucose tolerance after induction of a high-fat diet;

(5) 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. Studies have found that brown adipose tissue plays an important role in maintaining systemic glucose homeostasis. At the same time, 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. Therefore, 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. In this experiment, we used mouse rectal temperature to represent the body temperature of mice. The experimental results showed that there was no significant difference in body temperature between mice in the conventional feeding environment. When the mice were in a cold environment, the body temperature of the mice decreased significantly. After induction of high-fat diet, 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). 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). After the HCBP6 gene was knocked out, 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.

(6) In order to detect the degree of liver cell damage in mice, we detected the transaminase in mouse plasma. The results showed that ALT and AST in the plasma of HCBP6 knockout mice were slightly elevated compared with wild-type mice after administration of a high-fat diet, but there was no statistical difference (Fig. 11a). We further extracted mouse tissue RNA and detected the expression of inflammatory factors in liver tissue by Q-PCR. The results showed that IL-6 and TNF-α expression was increased in liver tissues of HCBP6 knockout mice after administration of a high-fat diet (Fig. 11b). The above results indicate that the inflammatory response in mice is aggravated after HCBP6 knockout, suggesting that HCBP6 may have an inhibitory effect on inflammation.

(7) Western Blot results showed that the expression of HCBP6 in the fatty liver model group was decreased (Fig. 12). The results of Q-PCR showed no significant changes in lipid synthesis related genes in each group. Fatty liver model group HCBP6 and lipid breakdown related gene ATGL HSL expression was significantly reduced and statistically significant (Figure 13).

(8) Immunohistochemical results showed that compared with the normal diet group, the expression of HCBP6 in the liver tissue of the high fat diet fed fatty liver model group was significantly decreased, indicating that HCBP6 is related to liver lipid metabolism (Fig. 14).

The above results show that: 1. The non-alcoholic fatty liver mouse model was successfully constructed. 2. 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.

Example 4: Drug Screening for Treatment of Fatty Liver

1.Rh2 cell experiment:

1) Experimental method:

(1) Rh2 was added to HepG2 cell line at different concentrations (0, 1, 2.5, 5, 10, 25 μM), and the expression of TC, TG and HCBP6 in cells was detected 48 h later.

(2) 50 μM 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.

2) Results:

(1) After adding different concentrations of Rh2, the levels of TC and TG in the cells decreased (Fig. 15a); the expression levels of HCBP6 in mRNA and protein levels increased to different degrees (Fig. 15b).

(2) The level of intracellular HCBP6 increased with the duration of Rh2 (12h, 24h, 36h and 48h), and increased most at 48h after treatment (Fig. 15c).

2. Cell experiments of Rb3, Rc, CK, Rh1, ginseng diol, Rt5, F11 and R1:

1) Experimental method:

(1) Add different concentrations (0, 1, 5, 10, 50, 100 μM) of Rb3, Rc, CK, Rh1, ginseng diol, Rt5, F11 or R1 in HepG2 cell line, and measure intracellular TC after 48h. And / or changes in the expression levels of TG and HCBP6.

2) Results:

(1) After adding different concentrations of Rb3, the levels of TC and TG in the cells were decreased (Fig. 16a); the expression level of HCBP6 protein in the cells was increased (Fig. 16b).

(2) After adding different concentrations of Rc, the intracellular TC content decreased, TG did not change (Fig. 17a); the intracellular HCBP6 protein level expression increased (Fig. 17b).

(3) After adding different concentrations of CK, the intracellular TC content was decreased (Fig. 18a); the expression level of intracellular HCBP6 protein was increased (Fig. 18b).

(4) After adding different concentrations of Rh1, there was no change in intracellular TC and a decrease in TG content (Fig. 19a); the expression level of intracellular HCBP6 protein was increased (Fig. 19b).

(5) After adding different concentrations of ginseng diol, the intracellular TC content decreased (Fig. 20a); the expression level of intracellular HCBP6 protein increased (Fig. 20b).

(6) After adding different concentrations of Rt5, there was no change in intracellular TC (Fig. 21a); there was no change in the expression level of HCBP6 protein in cells (Fig. 21b).

(7) After adding different concentrations of F11, there was no change in intracellular TC (Fig. 22a); there was no change in the expression level of HCBP6 protein in cells (Fig. 22b).

(8) After adding different concentrations of R1, the intracellular TC content increased (Fig. 23a); the expression level of intracellular HCBP6 protein was decreased (Fig. 23b).

In conclusion, 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. The role of NAFLD.

3. Rh2, CK, Rc, Rb3 animal experiments:

1) Experimental method:

Note: 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.

All data were analyzed using SPSS 13.0 software. Quantitative data were expressed as mean ± standard deviation; t test was used to compare the mean of the two samples, and P < 0.05 was used as the difference significance threshold.

2) Rh2 results:

(1) Body weight results showed that after 10 weeks of modeling, compared with LFD and Chow group, the weight of mice in HFD group increased significantly, which was statistically significant (Fig. 24a); after ginsenoside Rh2 intervention; and high fat diet group Compared with the mice, there was no significant change in body weight. Compared with the HFD+Chow group, the HFD+Chow+Rh2 mice lost weight (Fig. 24a). Similarly, compared with the high-fat diet mice, Rh2 was given. There was no significant change in the body fat content of the mice (Fig. 24b).

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. Compared with the normal diet group, 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. After the intervention of 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). The above results indicated that ginsenoside Rh2 had no effect on body weight and body fat content in mice, but it could improve the liver tissue steatosis in mice.

(2) The results showed that there was no significant difference in the amount of food consumed by the mice; after 10 weeks of modeling, the blood glucose of the mice in the high-fat diet group was increased compared with the normal diet group.

After glucose stimulation, the blood glucose of the mice in the normal diet group increased rapidly, peaked at 15 minutes, and then decreased rapidly. At 2 hours, the blood glucose returned to normal levels. Compared with wild-type mice, the mice were given a high-fat diet. Fasting blood glucose was significantly increased. After glucose stimulation, blood glucose increased rapidly and reached a peak at around 15-30 minutes. With the prolongation of time, blood glucose decreased slowly and remained at a high level 2 hours after stimulation. After Rh2 treatment, Compared with the untreated high-fat diet group, the fasting blood glucose of the mice was decreased, and the glucose recovery rate was accelerated after glucose stimulation, which was statistically different (Fig. 25a-c). This result indicates that ginsenoside Rh2 has an effect of improving blood sugar levels.

(3) Biochemical results showed that after administration of high-fat diet, plasma AST, ALT, ALP increased, cholesterol, high-density lipoprotein and low-density lipoprotein increased significantly. After treatment with Rh2, plasma AST, ALT Cholesterol decreased, but there was no statistical difference, HDL-C decreased and the difference was significant (Fig. 26a-b).

3) Rb3 results:

(1) After the intervention of ginsenoside Rb3, the body weight and fat content of the mice were not significantly changed compared with the mice in the high-fat diet group (Fig. 27a-b); the HE staining results of the liver tissue of the mice showed that the normal diet group was small. The lobular structure of the rat liver tissue was intact, and no lipid droplets were formed in the cytoplasm. After induction of the high-fat diet, a large number of lipid droplets were formed in the liver tissue cells, and after Rb3 treatment, the lipid accumulation was reduced and scattered (Fig. 27c). . This result indicates that ginsenoside Rb3 can improve the liver histopathology of non-alcoholic fatty liver mice.

(2) The results of glucose tolerance test in mice showed that after treatment with Rb3, the fasting blood glucose of the mice decreased, and after the intraperitoneal injection of glucose, there was no significant change in blood glucose of the mice compared with the untreated group (Fig. 28a-c). It is indicated that ginsenoside Rb3 can only improve the fasting blood glucose level in mice.

(3) Biochemical results of 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).

4) CK results:

(1) After administration of high-fat diet, the body weight and fat content of the mice increased significantly. After treatment with CK, there was no significant difference in body weight and fat content between the mice treated with the high-fat diet (Fig. 30a-b). HE staining results showed that the lipid accumulation in the liver tissue of the mice in the high-fat diet group was obvious, and a large number of steat-denatured hepatocytes were observed. After CK intervention, the liver cells with fatty degeneration in the liver tissue were significantly reduced (Fig. 30c). . This result indicates that ginsenoside CK can improve the liver histopathology of non-alcoholic fatty liver mice.

(2) The results of glucose tolerance test in mice showed that the fasting blood glucose of the mice was significantly increased after induction of high-fat diet. After CK treatment, the blood glucose was significantly decreased compared with the untreated group (Fig. 31a-b); intraperitoneal injection After glucose, the blood glucose of the three groups increased, and the blood sugar of the mice in the high-fat diet group increased significantly, and the blood sugar recovered slowly. At 2 hours after the injection, the blood glucose level was still high, and after the CK treatment, the blood sugar recovered faster. The tolerance was improved but there was no statistical difference (Figures 31a and c). The results of this part of the experiment indicate that ginsenoside CK helps to improve fasting blood glucose levels in mice.

(3) The results of biochemical tests in mice showed that plasma AST, ALT, and ALP levels decreased slightly compared with untreated high-fat diet mice after CK treatment, but there was no statistical difference (Fig. 32a). After treatment with CK, plasma cholesterol and high-density lipoprotein levels decreased and were statistically different (Fig. 32b). It is indicated that ginsenoside CK has the effect of lowering plasma cholesterol levels.

5) Rc results:

(1) After Rc treatment, there was no significant change in body weight and fat content in mice compared with the high-fat diet group (Fig. 33a-b); liver HE staining results showed: mice in the untreated group with high-fat diet In contrast, lipid accumulation was slightly reduced in liver tissue after Rc intervention (Fig. 33c).

(2) The results of the glucose tolerance test in mice showed that there was no significant change in fasting blood glucose compared with the untreated group after Rc treatment (Fig. 34b); after intraperitoneal injection of glucose, compared with the high fat diet group. After Rc treatment, the glucose tolerance of the mice was improved to some extent (Fig. 34a and c).

(3) Biochemical test results of mice showed that there was no significant difference in plasma AST, ALT, ALP, CHO, TG, HDL-C and LDL-C compared with untreated high-fat diet mice after Rc treatment. (Fig. 35a-b). The above results indicate that ginsenoside Rc can improve glucose tolerance to some extent.

In summary, we found that in non-alcoholic fatty liver mice, 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.

The above is only the preferred embodiment of the present invention, and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., which are included in the spirit and scope of the present invention, should be included in the present invention. Within the scope of protection.

Claims

HCBP6 gene or HCBP6 protein as a drug target in screening and / or preparation for prevention and / or treatment of fatty liver, steatohepatitis, diabetes, multiple sclerosis, hypertriglyceridemia, hypercholesterolemia and / or heart and brain Use in vascular atherosclerosis drugs.
Use of an HCBP6 agonist for the preparation of a prophylactic and/or therapeutic fatty liver drug, steatohepatitis, diabetes, multiple sclerosis, hypertriglyceridemia, hypercholesterolemia and/or cardiovascular and cerebrovascular atherosclerosis.
Use of ginsenosides for the preparation of a medicament for preventing and/or treating fatty liver, steatohepatitis, diabetes, multiple sclerosis, hypertriglyceridemia, hypercholesterolemia and/or cardiovascular and cerebrovascular atherosclerosis, The ginsenoside is selected from the group consisting of one or more of Rh2, Rb3, Rc, CK, Rh1 or ginseng diol.
The use according to claim 3, wherein the ginsenoside is a combination of one or more of Rh2, Rb3, Rc, and/or CK.
Use according to claim 3 or 4, characterized in that ginsenosides are capable of inhibiting the synthesis and/or accumulation of cholesterol (TC) and/or triglycerides (TG).
A pharmaceutical composition comprising ginsenoside characterized in that ginsenoside is the only effective pharmaceutical component, and the composition further comprises a pharmaceutically acceptable carrier/auxiliary, wherein the ginsenoside is selected from the group consisting of Rh2, Rb3, Rc, CK, Rh1 Or a combination of any one or more of the ginseng diols, preferably Rh2, Rb3, Rc, and/or CK.
The pharmaceutical composition according to claim 6, which is administered by gastrointestinal, subcutaneous and/or intravenous injection.
A method of treating fatty liver, characterized in that a pharmaceutically effective amount of the pharmaceutical composition according to claim 6 or 7 is administered to a patient.
An animal model of HCBP6 gene knockout characterized in that the animal is a mouse or a zebrafish.
A method for preparing a mouse model of HCBP6 gene knockout, comprising the steps of:

1) Target gene localization: the corresponding gene of human gene HCBP6 in mouse 4833415N24Rik (NM_026126.4);

2) TALEN design and construction: the gene 4833415N24Rik exon 3 is used as a TALEN gene editing target;

3) Gene editing: the mouse fertilized egg is injected into the mRNA edited by TALEN, and the fertilized egg after injection is transplanted into the body of the pseudo-pregnant mother;

4) Obtaining positive F0 mice: feeding transplant recipient mothers and identifying positive F0 mice;

5) Obtaining germline-inherited F1 hybrid mice: the positive mice obtained in step 4) were mated with wild mice to obtain F1 hybrid mice;

6) Homozygous mice obtained by knockout of HCBP6 gene: F1 mice were selfed, and homozygous HCBP6 knockout mice were obtained.