Application of methyl cholate in hepatic fibrosis

Abstract

The invention relates to application of methyl cholate in hepatic fibrosis, and relates to the technical field of medicines. According to the application of methyl cholate in hepatic fibrosis, the methyl cholate can effectively relieve hepatic fibrosis, relieve expression of alpha-SMA and Collagen-I and restore the liver form, and the methyl cholate can be used as a medicine for treating hepatic fibrosis.

Description

Technical Field

[0001] The invention relates to the field of medical technology, and in particular to application of methyl cholate in liver fibrosis. Background Art

[0002] Hepatic fibrosis (HF) is a pathological process caused by liver cell damage caused by various chronic liver diseases such as alcoholic liver disease, viral hepatitis, and autoimmune liver disease. Improper handling of the self-repair process of liver cells leads to abnormal activation of hepatic stellate cells (HSCs), increased transformation into myofibroblasts, and subsequent deposition of a large amount of extracellular matrix (ECM) in the liver, forming scar tissue that destroys normal liver tissue. It is a key stage in the progression of various chronic liver diseases. The incidence of chronic liver disease in my country is high, which seriously endangers people's health and brings a heavy economic burden. At present, HF treatment is limited to the etiology level and lacks a standard treatment plan. Exploring HF treatment strategies and reversing its progression to cirrhosis or even liver cancer is an important research direction. Therefore, it is very necessary to seek effective anti-hepatic fibrosis drugs and active ingredients. Summary of the invention

[0003] In view of the above problems, the present invention provides the use of cholic acid methyl ester in liver fibrosis. Cholic acid methyl ester can effectively alleviate liver fibrosis, reduce the expression of α-SMA and Collagen-I, restore liver morphology, and can be used as a drug for treating liver fibrosis.

[0004] The present invention provides an application of cholic acid methyl ester in preparing a drug for treating and / or preventing liver fibrosis. The structural formula of the cholic acid methyl ester is as follows:

[0005]

[0006] The molecular weight of the above-mentioned cholic acid methyl ester is 422.6, and the molecular formula is C 25 H 42 O5 is the methyl ester form of bile acid, one of the main bile acids produced by the liver, and is synthesized from cholesterol. The present invention uses zebrafish larvae and activated rat hepatic stellate cells HSC-T6 as experimental models for pharmacodynamics experiments. The experimental results show that methyl cholate can effectively reduce liver fibrosis in zebrafish, reduce the expression of Collagen-I, and restore liver morphology, and can be used as a drug for treating liver fibrosis. In vitro experiments show that methyl cholate can reduce the activation of HSC-T6 cells, reduce the expression of α-SMA and Collagen-I, and can be used as a drug for treating liver fibrosis.

[0007] In one embodiment, the liver fibrosis is caused by chronic liver disease, and the chronic liver disease includes at least one of alcoholic liver disease, viral hepatitis, fatty liver disease, autoimmune liver disease, and hepatolenticular degeneration.

[0008] In one embodiment, the drug for treating and / or preventing liver fibrosis further comprises pharmaceutically acceptable excipients and / or drugs compatible with methyl cholate.

[0009] In one embodiment, the excipients include at least one of a solvent, a disintegrant, a flavoring agent, a colorant, a lubricant, an antioxidant, a preservative, a binder, a filler or a thickener.

[0010] In one embodiment, the dosage form of the drug for treating and / or preventing liver fibrosis includes capsules, granules, tablets, oral liquids, pills, injections, pastes, liposome nanoparticles, sustained-release agents, controlled-release agents or dispersible tablets.

[0011] In one embodiment, the administration route of the drug for treating and / or preventing liver fibrosis includes oral administration or intravenous injection.

[0012] In one embodiment, the working concentration of methyl cholate is ≥20 μM.

[0013] The working concentration is the concentration at which the above-mentioned ingredients can achieve the expected effect when used. It is understandable that those skilled in the art can prepare a mother solution or a stock solution with a higher concentration when preparing the above-mentioned drug containing methyl cholate, and then dilute it when it is used. The mother solution or the stock solution and its concentration are all within the protection scope of the present invention.

[0014] The present invention also provides a medicine for treating and / or preventing liver fibrosis, comprising a medicine active ingredient and excipients, wherein the medicine active ingredient is methyl cholate.

[0015] In one embodiment, the drug reduces the expression of α-SMA and Collagen-I, reduces the activation of HSC-T6 cells to restore liver morphology.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention provides application of cholic acid methyl ester in liver fibrosis. Cholic acid methyl ester can effectively alleviate liver fibrosis, reduce the expression of α-SMA and Collagen-I, restore liver morphology, and can be used as a drug for treating liver fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1Graph showing the CCK8 detection results of the HSC-T6 cell line control group and the concentration drug administration groups (0, 5, 10, 20, 50, 100 μM) in the example.

[0019] Figure 2 The WB, RT-PCR, and immunofluorescence detection diagrams of α-SMA and Collagen-I of the HSC-T6 cell line control group and the drug-treated group in the embodiment; wherein, Figure 2 The upper left part of the upper part shows the WB results of α-SMA and Collagen-I, and the upper right part shows the RT-PCR results. Figure 2 The lower part is the immunofluorescence detection image.

[0020] Figure 3 These are the H&E pathological staining results of the zebrafish control group (Control), model group (0.06% TAA), low-concentration drug administration group (6.25 μmol / L), medium-concentration drug administration group (12.5 μmol / L) and high-concentration drug administration group (25 μmol / L) described in the present invention.

[0021] Figure 4 It is the Collagen-I immunofluorescence staining image of the zebrafish control group (Control), model group (0.06% TAA), low concentration administration group (6.25 μmol / L), medium concentration administration group (12.5 μmol / L) and high concentration administration group (25 μmol / L) of the present invention. DETAILED DESCRIPTION

[0022] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0024] Unless otherwise specified, the reagents, materials, and equipment used in this example are all commercially available; the experimental methods are all conventional experimental methods in the art unless otherwise specified.

[0025] Example

[0026] 1. Experimental materials and methods.

[0027] Cholic acid methyl ester was purchased from MCE with a purity of ≥98%, CAS No.: 631-01-6.

[0028] Rat hepatic stellate cells: HSC-T6 cell line.

[0029] Wild-type zebrafish (AB strain) and a transgenic strain with liver-specific eGFP expression [Tg(lfabp10α-eGFP)] and AB strain wild-type fish.

[0030] 2. Effect of methyl cholate on the growth and proliferation of HSC-T6 cells.

[0031] HSC-T6 cells in the logarithmic growth phase were evenly plated on a 96-well (3000-5000 cells / well) culture plate. After incubation in a cell culture incubator overnight, the culture medium of each well was discarded, and then 100 μL of cholic acid methyl ester solution of different concentrations was added to each well. The sample concentration was set to 0, 5, 10, 20, 50, 100 μM, and 5 replicates were placed in a cell culture incubator for incubation for 24h and 48h. After the incubation, 10 μL of CCK8 solution was added to each well, and then placed in a cell culture incubator for incubation in dark for 2-4h, and then the absorbance of each well was detected by a full-wavelength microplate reader, and the wavelength was set to 450nm. The survival rate of cells in each well was calculated according to the following formula: Cell Viability (%) = As / Ab * 100%, where As and Ab represent the absorbance of sample treatment and blank control absorbance, respectively.

[0032] Results: Figure 1 As shown, after drug treatment for 24 or 48 hours, methyl cholate has an inhibitory effect on the growth of HSC-T6 cells within a certain concentration range, proving that it has a certain effect of inhibiting the activation of hepatic stellate cells.

[0033] 3. Effect of methyl cholate on activation of HSC-T6 cells.

[0034] 1. WB: Extract protein samples and then separate them by polyacrylamide gel electrophoresis (PAGE) to separate the proteins according to their molecular weight. The separated proteins are transferred to polyvinylidene fluoride membrane (PVDF membrane) by electrotransfer technology. Block nonspecific binding sites (using milk or BSA, etc.), and then add primary antibodies for α-SMA and Collagen-I for incubation. After the primary antibody binds to the protein, it is incubated with the secondary antibody. Finally, the immunoreaction product is detected by chemiluminescence to determine the presence and expression of α-SMA and Collagen-I before and after drug treatment. The concentration of MC is 25μM.

[0035] 2. RT-PCR: The total RNA of T6 cells in each group was extracted by Trizol method, and 2 μg of total RNA was reverse transcribed into cDNA (20 μl volume), which was diluted 5 times and stored at -20°C for later use. The PCR amplification reaction system was 20 μl, and the conditions were set as follows: 95°C pre-denaturation for 5 min; 40 PCR cycles (95°C denaturation for 10 seconds, 60°C annealing for 60 seconds). After correction with internal reference GAPDH, the relative expression of the target gene was calculated by 2-ΔΔCT method. Three replicate wells were set for each group, the concentration of MC was 25 μM, and the experiment was repeated 3 times.

[0036] 3. Immunofluorescence: During subculture, cells were inoculated into a culture dish with cell slides placed in advance. After treatment with methyl cholate (MC-L concentration was 6.25 μM, MC-M concentration was 12.5 μM, and MC-H concentration was 25 μM), the cells were fixed, the cells were permeabilized, and then blocked with blocking solution. The primary antibody was incubated at room temperature for 1 hour or at 4°C overnight. The secondary antibody was incubated at room temperature in the dark for 1 hour, and then the slides were sealed and observed under a fluorescence microscope.

[0037] Results: Figure 2 As shown, we used WB, IF and RT-PCR to detect the changes in the protein and mRNA levels of α-SMA and Collagen-I. Compared with the blank group, the expression of α-SMA and Collagen-I in HSC-T6 cells in the group adding methyl cholate solution was significantly reduced (P < 0.01) and was concentration-dependent.

[0038] 4. Zebrafish modeling and drug administration.

[0039] One day in advance, transgenic zebrafish [Tg(lfabp10α-eGFP)] zebrafish and AB strain wild fish were selected and placed in mating boxes in a 1:1 ratio of male to female, and the partitions were removed for mating on the second day; the eggs were collected after spawning, and the transgenic zebrafish eggs and AB fish eggs that specifically expressed green fluorescence in the liver were collected using a 180 μm pore filter, and the eggs were washed 2-3 times with culture water; the embryos were placed in a 90 mm dish filled with methylene blue culture water at a density of 50-60 eggs / dish, and foreign matter and unfertilized eggs were removed; at 2 dpf, zebrafish larvae and AB strain larvae with fluorescent livers were randomly selected and randomly divided into a control group, a model group, a DMSO group, a low-concentration drug administration group, a medium-concentration drug administration group, and a high-concentration drug administration group, and placed in a six-well plate, with 30 eggs / well in each group.

[0040] Except for the control group, 8 mL of fish culture system water containing 0.06% thioacetamide (TAA) was added to each group and the exposure was continued for 6 days. On the third day of exposure, the low-concentration drug administration group, the medium-concentration drug administration group and the high-concentration drug administration group were added with 0.06% TAA modeling system water containing 6.25 μM, 12.5 μM and 25 μM cholic acid methyl ester, respectively, and the DMSO group was added with 0.06% TAA modeling system water containing 0.1% DMSO. The control group and the model group did not receive any treatment, and the death of zebrafish was recorded every day. The modeling was terminated 3 days after drug administration, and the above-mentioned zebrafish fry were collected.

[0041] 5. Effect of methyl cholate on the liver tissue morphology of zebrafish.

[0042] The above-mentioned zebrafish fry were fixed with 4% polyformaldehyde (PFA) at a mass concentration and kept in a refrigerator at 4°C overnight. The next day, they were dehydrated with 80% ethanol for 2 hours, 90% ethanol for 2 hours, and 95% ethanol overnight. The next day, they were dehydrated with 100% ethanol I for 0.5 hours, 100% ethanol II for 0.5 hours, and 100% ethanol III for hours. They were treated with xylene for 0.5 hours, paraffin I for 0.5 hours, paraffin II for 0.5 hours, and paraffin III for 1 hour. The zebrafish embryos were taken out of the paraffin, placed in an embedding box, poured with paraffin, embedded, and then sliced ​​on a microtome, with the slice thickness set to 4 μm. The sliced ​​thin slices were gently placed in a 60°C water bath. After they were fully developed, they were transferred to a coated slide and placed in a 60°C constant temperature box for drying for about 2 hours.

[0043] The paraffin sections prepared above were dewaxed in xylene I, II, and III for 3 minutes each, anhydrous ethanol I and II for 2 minutes each, 95% ethanol, 90% ethanol, and 80% ethanol for 1 minute each, stained with hematoxylin for 10 minutes, rinsed with water, differentiated with 1% hydrochloric acid alcohol for 15 seconds, soaked in water for 10 minutes, stained with eosin for 3 minutes, 80% ethanol and 90 ethanol for 10 seconds each, 95% ethanol for 1-2 minutes, anhydrous ethanol I, II, and III for 3 minutes each, xylene I, II, and III for 3 minutes each, and finally sealed with neutral gum and photographed under a microscope.

[0044] Results: Figure 3 As shown in the figure, the liver tissue structure of the model group was disordered, the liver cells were swollen, degenerated, with a large number of vacuoles, and nodular substances were formed in some parts. The liver tissue structure of zebrafish in the high-concentration drug administration group was normal, and the liver cells were arranged neatly.

[0045] 6. Effect of methyl cholate on the expression of Collagen-I in zebrafish with liver fibrosis.

[0046] Liver-specific fluorescent transgenic zebrafish [Tg(lfabp10α-eGFP)] were collected and fixed with 4% paraformaldehyde (PFA) and kept in a 4°C refrigerator overnight. The next day, they were dehydrated with 30% sucrose for 3 days until all the fish sank to the bottom of the tube. The dehydrated zebrafish were taken out with tweezers, embedded with OCT, cooled and solidified in a -80°C refrigerator, and sliced ​​on a frozen microtome with a slice thickness of 14 μm. The sliced ​​slices were stored in a -20°C refrigerator.

[0047] The frozen sections were washed with distilled water, permeabilized with 0.1% Triton X-100 PBS at room temperature for 1 hour, washed with PBS 3 times, 5 minutes each time, blocked with 5% blocking solution (PBS + 5% goat serum + 0.1% Triton X-100) at room temperature for 2 hours, Collagen-I (1:200 dilution), and kept in a refrigerator at 4°C overnight. On the second day, washed with PBS 3 times, 5 minutes each time, incubated with fluorescent rabbit secondary antibody at room temperature, away from light for 2 hours, washed with PBS 3 times, 5 minutes each time, stained with DAPI in the dark for 10 minutes, washed with PBS 3 times, 5 minutes each time, dried, sealed with anti-fluorescence quencher, and photographed.

[0048] Results: Figure 4 As shown, thioacetamide (TAA) induced a large amount of expression of Collagen-I. Compared with the model group, the high-concentration administration group significantly reduced the expression of Collagen-I in the liver.

[0049] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0050] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A use of methyl cholate in the preparation of a drug for treating and / or preventing liver fibrosis, wherein the structural formula of the methyl cholate is as follows:

2. The use according to claim 1, characterized in that: The liver fibrosis is caused by chronic liver disease, and the chronic liver disease includes at least one of alcoholic liver disease, viral hepatitis, fatty liver disease, autoimmune liver disease, and hepatolenticular degeneration.

3. The use according to claim 1, characterized in that: The drug for treating and / or preventing liver fibrosis also includes pharmaceutically acceptable adjuvants and / or drugs compatible with methyl cholate.

4. The use according to claim 3, characterized in that: The auxiliary materials include at least one of solvents, disintegrants, flavoring agents, colorants, lubricants, antioxidants, preservatives, adhesives, fillers or thickeners.

5. The use according to claim 1, characterized in that: The dosage forms of the drug for treating and / or preventing liver fibrosis include capsules, granules, tablets, oral liquids, pills, injections, pastes, liposome nanoparticles, sustained-release agents, controlled-release agents or dispersible tablets.

6. The use according to claim 5, characterized in that: The administration routes of the drug for treating and / or preventing liver fibrosis include oral administration or intravenous injection.

7. The use according to any one of claims 1 to 6, characterized in that: The working concentration of methyl cholate is ≥20 μM.

8. A drug for treating and / or preventing liver fibrosis, characterized in that: The invention comprises active pharmaceutical ingredients and auxiliary materials, wherein the active pharmaceutical ingredient is methyl cholate.

9. The drug according to claim 8, characterized in that The drug reduces the expression of α-SMA and Collagen-I, reduces the activation of HSC-T6 cells, and restores the liver morphology.

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