CN117180284A - Application of taurochenodeoxycholic acid in preparation of anti-hepatic fibrosis drugs - Google Patents
Application of taurochenodeoxycholic acid in preparation of anti-hepatic fibrosis drugs Download PDFInfo
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- CN117180284A CN117180284A CN202210598586.0A CN202210598586A CN117180284A CN 117180284 A CN117180284 A CN 117180284A CN 202210598586 A CN202210598586 A CN 202210598586A CN 117180284 A CN117180284 A CN 117180284A
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- liver
- fibrosis
- tcdca
- yap
- hepatic fibrosis
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Abstract
The invention discloses application of taurochenodeoxycholic acid in preparing an anti-hepatic fibrosis medicine. By establishing a rat model of hepatic fibrosis induced by rat bile duct obstruction, blood and liver are collected for respectively carrying out biochemical detection, histopathological detection and related protein PCR detection, and the results show that: the taurochenodeoxycholic acid can inhibit the activation of portal vein fibroblasts, down regulate the expression of YAP and downstream target genes in the fibroblasts, has obviously enhanced anti-fibrosis effect, and can be better applied to clinical anti-hepatic fibrosis treatment.
Description
Technical Field
The invention relates to application of taurochenodeoxycholic acid in preparing anti-hepatic fibrosis medicines, and belongs to the field of pharmacy.
Background
Chronic Liver Disease (CLD) is one of the greatest threats to public health today, with over 2900 thousands of people developing in the european region. The report issued by the lancet in 2018 shows that the Chinese hepatitis B infection rate is as high as 5.1% -10%, about 8000 ten thousand virus carriers exist, and 2000 ten thousand patients are diagnosed. The number of people with fatty liver in China is more than 2 hundred million. The number of chronic liver disease patients in China, including chronic hepatitis, fatty liver and cirrhosis in 2020, may exceed 4.47 million.
Liver fibrosis is a result of chronic liver disease, including cholestatic liver disease (primary sclerosing cholangitis (PSC), primary Biliary Cirrhosis (PBC) and Secondary Biliary Cirrhosis (SBC)) and toxic liver injury (hepatitis b virus (HBV), hepatitis C Virus (HCV), alcoholic liver disease and non-alcoholic steatohepatitis (NASH)). It is characterized by extensive deposition of extracellular matrix (ECM) (Fuji, H, miller, G and Nishio, T, et al role of Mesothelin signaling in Portal Fibroblasts in the pathogenesis of cholestatic liver fibrisis. Front Mol Biosci,2021, 8:790032.). Patients with progressive liver fibrosis due to excessive liver injury may develop cirrhosis and eventually die from liver failure, and liver fibrosis also creates a good microenvironment for the development of tumorigenic nodules by mechanisms that remain under debate. Recent studies have shown that liver fibrosis is a reversible pathology, and cirrhosis is an irreversible pathology, and thus inhibition, prevention and reversal of liver fibrosis are central links in the treatment of various chronic liver diseases.
The development of anti-liver fibrosis drugs is a hotspot in liver disease treatment today. The currently-claimed therapeutic drugs for preventing and treating liver fibrosis mainly comprise the following classes: 1) Chinese medicinal materials and their extracts such as fructus Schisandrae chinensis, artemisinin, curcumin, and rhizoma Gastrodiae; 2) Chemically synthesized drugs such as Senp expression modulator (CN 110755621 a), N- (2-ethoxyphenyl) -N-hydroxyoctanediamide (CN 108379245A), diallyl disulfide-containing (CN 107536830 a); 3) Biological agents, including recombinant proteins, mirnas, and the like, such as BMP9 (CN 106994181 a), BMP7 (CN 101301467 a), miRNA-6766-3p (CN 113730427 a). Although there are many therapeutic agents, no specific drug for liver fibrosis has been found at present, and therefore, finding an exact effective anti-liver fibrosis drug would still be a direction of development in the future.
Cholestatic fibrosis is caused by chronic cholestatic injury, hepatocyte apoptosis, duct proliferation, inflammation and myofibroblast activation (lazardis, K N and LaRusso, n.primary scanning chol technologies, N Engl J Med,2016,375 (25): 2501-2502.). Both activated portal fibroblasts (activated portal fibroblasts, afcs) and activated hepatic stellate cells (activated Hepatic stellate cells, afcs) can produce myofibroblasts that drive cholestatic fibrosis. Despite extensive research, the origin of hepatic myofibroblasts and the effect on cholestatic fibrosis remain controversial. Several studies in experimental models of cholestatic fibrosis indicate that affs play an important role in the pathogenesis of cholestatic fibrosis, suggesting that affs may be a major target for anti-fibrotic therapy (Dranoff, J a and Wells, r.g. Portal fibrilasts: underappreciated mediators of biliary fibrins. Hepatology,2010,51 (4): 1438-1444.). Under physiological conditions, portal fibroblasts contain a small population of cells surrounding the portal vein to maintain the integrity of the portal vein. Liver injury from hepatotoxicity activates hepatic stellate cells to myofibroblasts, while cholestatic liver injury activates hepatic stellate cells and portal fibroblasts. At the beginning of Bile Duct Ligation (BDL), bile duct Blockage (BA) induced injury, afvs account for 70% of myofibroblasts, while as fibrosis progresses, afcs are increasing, so afcs are the "first responders" to fibrous liver injury caused by cholestasis (Fuji, H, miller, G and nisio, T, et al, the role of Mesothelin signaling in Portal Fibroblasts in the pathogenesis of cholestatic liver fibrisis. Front Mol Biosci,2021, 8:790032.). The above studies indicate that inhibition of portal fibroblast activation is the primary research strategy for cholestatic liver fibrosis.
In recent years, bile acids have received increasing attention as drugs for treating liver fibrosis. Ursodeoxycholic acid (Ursodeoxycholic acid, UDCA) has been used as the first anti-cholestasis drug for about 20 years, and is the first-line therapeutic drug for cholestatic liver disease, primary Biliary Cirrhosis (PBC), recommended by current guidelines at home and abroad, but some patients are intolerant to UDCA or unresponsive to treatment thereof. The efficacy of 6E-CDCA (also known as obeticholic acid, OCA) against nonalcoholic steatohepatitis is being studied in clinical stage III, and has been approved by the FDA in the United states for marketing as a second line therapeutic against PBC in month 5 of 2016. However, the results of several clinical studies showed that: compared to placebo, OCA did not significantly improve the liver fibrosis index in patients (Sumida, Y, yoneda, M and Ogawa, Y, et al Current and new pharmacotherapy options for non-impulse steatoheat Opin Pharmacother,2020,21 (8): 953-967.). One important reason for clinical failure of UDCA, OCA is that the expression level of FXR, the target of action, is significantly down-regulated in the liver of patients with liver injury.
Earlier, it was reported that high doses of femoral intravenous TCDCA induced cholestasis in rat liver resulting in acute cholestatic liver injury (Fukumoto, Y, murakami, F and Tateishi, a, et al effects of secretin on TCDCA-or TDCA-induced cholestatic liver injury in the rate, hepatol Res,2002,22 (3): 214-222.). The effect of TCDCA is closely related to the administration mode, the dosage of TCDCA is lower, and the damage stimulation to the liver is reduced by the method of gastric lavage, so that the acute cholestasis damage is avoided, and the treatment effect is achieved.
Yes-associated protein (YAP) is the major effector of the Hippo pathway and plays an important role in controlling hepatic cell fate and hepatic stellate cell activation. Upon activation, YAP translocation into the nucleus promotes transcription of downstream target genes, most of which are associated with liver fibrosis, such as connective tissue growth factor (connective tissue growth factor, CTGF). CTGF induces synthesis and secretion of extracellular matrix components, especially fibrous collagen (Lu, Z N, niu, W X and Zhang, N, et al, pantoprazole ameliorates liver fibrosis and suppresses hepatic stellate cell activation in bile duct ligation rats by promoting YAP degradation. Acta Pharmacol Sin,2021,42 (11): 1808-1820.). Previous studies have shown that YAP stimulates pro-fibrotic gene expression in the early stages of hepatic stellate cell activation (Mannaerts, I, leite, S B and Verhulst, S, et al, the Hippo pathway effector YAP controls mouse hepatic stellate cell activation.j Hepatol,2015,63 (3): 679-688), whereas afvs also play a major role in the early stages of cholestatic hepatic fibrosis, leading to subsequent hepatic stellate cell activation. Furthermore, elevated YAP expression is closely related to bile duct proliferation and fibrosis. Thus, inhibition of YAP overexpression in YAP, particularly affs, is a potential treatment for liver fibrosis.
Disclosure of Invention
The invention aims to provide application of taurochenodeoxycholic acid (TCDCA) and salt thereof in preparing anti-hepatic fibrosis drugs.
The applicant establishes a rat model of hepatic fibrosis induced by rat bile duct obstruction, collects blood and liver for biochemical detection, histopathological detection and related protein PCR detection respectively, and the results show that:
(1) TCDCA significantly reduces serum bile acid content of the rat model, and the effect is better than UDCA, which indicates that TCDCA can be better used for liver injury;
(2) TCDCA obviously reduces the hydroxyproline content in the liver collagen tissue of the rat model, and the sirius red staining result also shows that the TCDCA can obviously reduce the liver fibrosis area, and the effect is better than UDCA, which indicates that the TCDCA can be better used for liver fibrosis;
(3) The results of real-time RT-PCR show that TCDCA significantly reduces the expression of rat model liver fibrosis gene alpha-SMA protein.
(4) The results of real-time RT-PCR showed that bile duct blockage resulted in YAP overactivation, while downstream target gene CTGF expression was significantly upregulated, while TCDCA inhibited YAP activation and CTGF expression was significantly downregulated.
(5) Immunohistochemical results showed that large areas of co-staining occurred with large amounts of Thy1 (PFs markers) and YAP signals after bile duct obstruction, while TCDCA co-stained Thy1 with YAP only in small portions around the portal vein, indicating that TCDCA inhibited YAP activation in PFs.
The results prove that the TCDCA can down regulate the expression of YAP and downstream target genes in the fibroblast by inhibiting the activation of the portal fibroblast, further has a remarkably enhanced anti-fibrosis effect, and can be better applied to the clinical anti-hepatic fibrosis treatment.
Wherein the TCDCA and salts thereof include natural sources, semisynthetic, and fully chemically synthesized products. The structural formula of TCDCA is as follows:
the taurochenodeoxycholic acid and the salt thereof comprise soluble salts formed by taurochenodeoxycholic acid and various cations such as sodium ions, potassium ions, ammonia ions and the like, and particularly soluble salts formed by sulfonyl groups in taurochenodeoxycholic acid molecules and various cations.
The medicament can be administered by oral administration, injection and the like, and the invention is preferably an oral medicament.
The beneficial effects of the invention are as follows:
one important reason for clinical failure of bile acids such as UDCA, 6E-CDCA (also known as obeticholic acid, OCA) is that the expression level of FXR as an action target point in the liver of a patient suffering from liver injury is significantly reduced, so that the drug cannot significantly activate FXR pathway and further exert an anti-fibrosis effect. Therefore, the invention realizes the inhibition of liver fibrosis through a non-FXR pathway, and researches show that TCDCA can inhibit the activation of YAP in portal vein fibroblasts and the expression of target genes downstream of YAP, thereby inhibiting the liver fibrosis process. TCDCA exerts a significantly enhanced anti-fibrotic effect in the hepatic fibrosis state induced by rat bile duct obstruction, providing a new strategy for hepatic fibrosis treatment targeting portal fibroblasts.
Drawings
Fig. 1: the total bile acid content in the serum of each group of rats was measured using the TBA kit. Using GraphPad Prism 8 software single factor anova test, P <0.05 was represented.
Fig. 2: hydroxyproline content in liver tissue of each group of rats was measured using a hydroxyproline kit. Using GraphPad Prism 8 software single factor anova test, P <0.05 was represented.
Fig. 3: effect of TCDCA on liver fibrosis area in model rats. (A) Sirius red staining patterns of liver sections of rats in each group are 100 times visual field; and (B) counting the positive area of the sirius red staining chart. Using GraphPad Prism 8 software single factor anova test, P <0.05 is represented and P <0.05 is represented.
Fig. 4: effects of TCDCA on liver fibrosis rat liver α -SMA gene expression levels. (A) Detecting the expression level of the alpha-SMA gene of the liver tissue of each group of rats by real-time fluorescence quantitative PCR; (B) Western Blot detects the expression level of alpha-SMA protein in liver tissue of each group of rats. Using GraphPad Prism 8 software single factor anova test, P <0.05 is represented and P <0.01 is represented.
Fig. 5: effects of TCDCA on liver fibrosis rat liver Hippo-YAP pathway and downstream target gene expression levels. (A) The Mst1 gene and YAP and the downstream target genes CTGF, CYR61, ankrd1 and Birc5 gene expression levels in the liver tissue Hippo-YAP pathway of each group of rats are detected by real-time fluorescence quantitative PCR. (B) Western Blot detects YAP protein expression levels in liver tissue of each group of rats. Conventional one-way analysis of variance test in GraphPad Prism 8 software, P <0.01 and P <0.001 were used.
Fig. 6: immunohistochemical staining of the Thy1 and YAP proteins in liver sections of rats of each group, left panel x 100, right panel x 200; dark brown is positive signal, blue is hematoxylin lined nuclei, dashed box represents central vein, solid box represents enlarged field of view, black arrow indicates positive signal.
Detailed Description
In order to facilitate the implementation of the present invention by a person skilled in the art, the following detailed description of the technical solution of the present invention is provided with reference to specific embodiments. It should be understood that the following specific examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. The materials and reagents used in the examples were obtained from commercial sources unless otherwise specified.
Given the important role of activated portal fibroblasts in the pathogenesis of cholestatic fibrosis, in some embodiments of the invention, a protocol is provided for portal fibroblasts (portal fibroblasts, PFs) to inhibit activation, providing a new strategy for cholestatic liver fibrosis treatment. In the initial stage of cholestatic liver fibrosis, a large number of portal fibroblasts are activated to activate hepatic stellate cells and weight liver fibrosis progress, so that TCDCA is used for cholestatic liver fibrosis by inhibiting the activation of portal fibroblasts and has important significance.
The invention firstly proves that TCDCA can obviously reduce serum transaminase and liver hydroxyproline level and expression of liver fibrosis gene alpha-SMA in liver fibrosis induced by rat bile duct obstruction, and the effect is superior to ursodeoxycholic acid (Ursodeoxycholic acid, UDCA) and tauroursodeoxycholic acid (Tauroursodeoxycholic acid, TUDCA). Further studies indicate that TCDCA can inhibit PFs activation and YAP activation of PFs, improving liver fibrosis status. The comprehensive test results show that the technical scheme of the invention has better effect on the treatment of hepatic fibrosis.
Examples:
1 Experimental method
1.1 Liver fibrosis induced by TCDCA Sodium treatment on rat biliary obstruction
SD rats (weight 250+/-30 g) are adaptively fed for one week before the experiment, the room temperature is 22-26 ℃, the relative humidity is 40-60%, the brightness is alternated for 12 hours, and the rats can drink water and eat freely. The experiment was divided into SHAM (SHAM), model (BA), UDCA (ba+udca), TUDCA (ba+tudca) and TCDCA Sodium (ba+tcdca) with 5 animals per group.
Animals were induced to anesthetize and maintained with isoflurane. Laparotomy was performed through a midline incision. Using microsurgical techniques, the common bile duct above the pancreatic manifold was cannulated with 1 silicon catheter and secured in place with 8-0 nylon wire leaving the bile duct branches to the caudal and right lobes intact. The common bile duct is ligated at the distal end of the cannula site. The microinjector was connected to the catheter, and the injection of absolute ethanol mixed with black ink was stopped after about 30 seconds, as clearly seen by the liver indicating the coloration of the ink. The catheter was clamped for 5 minutes and then unclamped. After surgery, animals were allowed to eat and drink ad libitum. After monitoring animals 7 days after the initial surgery and injection, rats in the dosing group were given the corresponding drug treatment at a dose of 15mg/kg/Day and were gavaged. Rats were euthanized after 6 weeks. Blood and liver were collected.
1.2 determination of the content of TBA in blood and HYP in liver tissue
Total Bile Acid (TBA) is a sensitive and effective liver function test, and the content of serum bile acid is trace under physiological conditions, and when liver cell damage or intrahepatic and extrahepatic obstruction occurs, the metabolism of bile acid is abnormal, and the level of total bile acid is increased. Clinically, it is mainly used for diagnosing liver lesions and cholestasis syndrome.
Hydroxyproline (HYP) is one of the imino acids, is one of the main components of collagen tissue, and is a unique amino acid in collagen, and is used as a gold index for detecting liver fibrosis.
The operations were all performed according to the kit instructions.
1.3 liver sirius red staining
The sirius red staining can lead the collagen to be permanently stained, and can be used as a special staining method for pathological sections, and the sirius red staining can specifically show collagen tissues. In clinical pathological diagnosis and research applications, sirius red staining is mainly used for analyzing the degree and characteristics of fibrosis.
The method comprises the following steps: 1) Baking slices; 2) Dewaxing and water placing; 3) Dyeing sirius red; 4) Dehydrating and transparentizing; 5) And (5) sealing the piece.
1.4 liver immunohistochemical staining
Immunohistochemistry is to determine the intracellular antigens (polypeptides and proteins) of tissues by using the basic principle of immunology, namely the principle of antigen-antibody reaction, namely the specific binding of antigen and antibody, and developing the color developing agent (fluorescein, enzyme, metal ion and isotope) of the labeled antibody through chemical reaction, and to carry out the research of localization, qualitative and quantitative analysis.
The method comprises the following steps: 1) Baking slices; 2) Dewaxing and water placing; 3) Antigen retrieval; 4) Removing peroxidase; 5) Dripping first antibody. The method comprises the steps of carrying out a first treatment on the surface of the 6) Incubating the secondary antibody; 7) Developing DAB; 8) Counterstaining with hematoxylin; 9) Hydrochloric acid differentiation; 10 Transparent for dehydration; 11 A sealing piece.
Antibodies used include: YAP (1:100), thy1 (1:100), HRP-sheep anti-mouse (1:500), HRP-sheep anti-rabbit (1:500).
1.5 RT-PCR
1.5.1 Total RNA extraction from animal tissue samples
1) Respectively weighing 10mg of liver tissue samples of each rat, adding 1ml of trizol, homogenizing by a homogenizer, and standing for 5min;
2) 200 μl of chloroform was added, the mixture was vigorously shaken for 15sec, left for 2min, and then centrifuged at 12000g for 15min. 3) Carefully transferring the upper water phase into a new tube, adding 500 μl isopropanol, mixing, standing for 10min after inversion, centrifuging for 10min with 12000g, and discarding the supernatant; 4) RNA pellet was washed with 1.0ml of pre-chilled 75% ethanol, centrifuged at 7500g for 5min, the supernatant was discarded to give total RNA, and reconstituted with 50. Mu.l DEPC water, and diluted to 100 ng/. Mu.l after quantification.
1.5.2 reverse transcription
RNA solution and kit components were prepared in a total volume of 20. Mu.l according to the system ratio required by Takara Bio reverse transcription instructions and reverse transcription was performed by setting the program temperature.
1.5.3 PCR
The primer sequences used for PCR were as follows:
PCR was performed using the system and conditions according to the Monad RT-PCR reaction kit.
1.6 western blot
1) Liver tissue samples of each rat were weighed 20mg separately, 1%ProteaseInhibitor Cocktail (Sigma), 1% phosphatase inhibitor was added, homogenized; 2) Centrifuging 12000g for 10min, transferring the obtained supernatant as extracted total protein sample into a new centrifuge tube, measuring protein content by BCA method, adding 5×loading buffer, mixing, boiling for 10min, and loading; 3) Preparing SDS-PAGE of 3% -5% concentration gel and 6% -12% separation gel, and separating protein samples by electrophoresis; 4) Protein transfer printing is carried out by using wet transfer conditions, constant current is set to be 200mA, and the protein transfer printing is carried out for 90min; 5) The PVDF film after transfer was blocked in a 5% nonfat dry milk blocking solution formulated with TBST solution. A proper amount of primary antibody is prepared and incubated overnight at 4 ℃; 6) Incubating the membrane with HRP-conjugated secondary antibody dilution at 37 ℃ for 60min; 8) ECL color development was exposed.
Antibodies used include: YAP (1:1000), alpha-SMA (1:1000), GPADH (1:1000), HRP-sheep anti-mouse (1:5000), HRP-sheep anti-rabbit (1:5000). GAPDH is an internal reference protein.
1.7 statistical method
Conventional one-way analysis of variance test in GraphPad Prism 8 software, P <0.05, P <0.001.
3 results of experiments
3.1 The liver injury treatment effect of TCDCA Sodium on rat biliary obstruction is superior to UDCA
Total Bile Acid (TBA) is a sensitive and effective liver function test, and the content of serum bile acid is trace under physiological conditions, and when liver cell damage or intrahepatic and extrahepatic obstruction occurs, the metabolism of bile acid is abnormal, and the level of total bile acid is increased. Clinically, it is mainly used for diagnosing liver lesions and cholestasis syndrome. As can be seen from fig. 1, the degree of liver injury significantly increased after bile duct obstruction (BA group); the liver injury degree is improved after UDCA treatment (BA+UDCA group), and the total bile acid content (TBA) (P < 0.05); the improvement of liver injury after TCDCA Sodium treatment (ba+tcdca group) was more pronounced compared to UDCA treatment, TBA content was significantly reduced (P < 0.05).
3.2 The therapeutic effect of TCDCA Sodium on hepatic fibrosis caused by rat bile duct obstruction is superior to that of UDCA.
Hydroxyproline (HYP) is one of the imino acids, is one of the main components of collagen tissue, and is a unique amino acid in collagen, and is used as a gold index for detecting liver fibrosis. From fig. 2, it can be seen that the HYP content increases significantly after bile duct obstruction (BA group); the HYP content is obviously reduced (P < 0.05) after UDCA treatment (BA+UDCA group); the HYP content was significantly reduced (P < 0.05) after TCDCA treatment (ba+tcdca group) compared to UDCA treatment.
In clinical pathological diagnosis and research applications, sirius red staining is mainly used for analyzing the degree and characteristics of fibrosis. According to the staining results of sirius red (fig. 3), large-area liver fibrosis appears after bile duct ligation (BA group), the area of sirius red positive is significantly reduced (P < 0.05) compared with BA group after UDCA treatment (ba+udca group); and the red positive area of sirius is obviously reduced (P < 0.01) after TCDCA Sodium treatment (BA+TCDCA group) compared with that of BA+UDCA group.
3.3 TCDCA Sodium inhibits α -SMA expression.
Based on the results of real-time RT-PCR (FIG. 4), the α -SMA mRNA levels were significantly reduced (P < 0.01), and WB results showed significant changes in the protein levels of α -SMA.
3.4 Effects of TCDCA Sodium on the Hippo-YAP signaling pathway.
According to the results of real-time RT-PCR (fig. 5), bile duct blockage (BA group) resulted in YAP overactivation, while expression of downstream target genes CTGF, CYR61, ankrd1, birc5 was very significantly upregulated (P < 0.001), and Mst1 gene in Hippo pathway inhibiting YAP expression was also very significantly downregulated (P < 0.001); whereas (ba+tcdca) YAP activation was inhibited following TCDCA Sodium treatment, expression of downstream target genes CTGF, CYR61, ankrd1, birc5 was significantly down-regulated (P < 0.001), while the Mst1 gene was restored to levels near Sham group; while immunohistochemical staining showed that TCDCA Sodium inhibited YAP expression in PFs.
3.5 TCDCA Sodium inhibits YAP activation in PFs.
The affs are normally not expressed, only around Portal Vein (PV) in the early stages of bile duct obstruction. As bile duct obstruction time increases, affs gradually spread around the Central Vein (CV) and activate HSCs, exacerbating the degree of liver fibrosis.
Immunohistochemical co-staining was performed on YAP and Thy1 (affs markers) using the serial section method. The results show (fig. 6) that large areas of co-staining of Thy1 and YAP signals after bile duct obstruction (BA group) occurred in the same field of view and spread around the CV, indicating that overexpression of YAP in affs resulted in its continued activation and spread; however, the TCDCA Sodium treated (ba+tcdca group) Thy1 and YAP had only a small portion of co-staining around PV (black arrows), indicating that TCDCA Sodium treated inhibited YAP expression in affs at the early stage of biliary obstruction, and further inhibited continued activation of affs and subsequent liver fibrosis progression.
Claims (4)
1. Application of taurochenodeoxycholic acid or pharmaceutically acceptable salt thereof in preparing anti-hepatic fibrosis medicines.
2. The use according to claim 1, wherein: the liver fibrosis is cholestatic liver fibrosis.
3. The use according to claim 1, wherein: the salt is a soluble salt formed by taurochenodeoxycholic acid and various cations such as sodium ion, potassium ion and ammonia ion.
4. The use according to claim 1, wherein: the medicine is an oral medicine.
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