Application of unsaturated fat dicarboxylic acid in preparation of medicine for treating liver injury

Abstract

The invention discloses application of unsaturated fat dicarboxylic acid in preparation of a medicine for treating liver injury. The unsaturated fat dicarboxylic acid is selected from a compound shown in a formula I or pharmaceutically acceptable salts thereof and is used for preparing the pharmaceutical composition for relieving and / or treating liver injury.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medical use, and particularly relates to the use of unsaturated aliphatic dicarboxylic acid in the preparation of a drug for treating liver injury. BACKGROUND

[0002] Chronic hepatitis is a kind of chronic inflammatory disease of the liver caused by various causes, and the course usually lasts more than 6 months. Hepatitis B virus (HBV) and hepatitis C virus (HCV) are common causes of chronic hepatitis. In the body of a patient with chronic hepatitis, liver cells are often damaged. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) mainly exist in liver cells, and when liver cells are damaged, the cell membrane permeability increases, and these two enzymes are released into the blood, causing the activity of ALT and AST in the blood to increase. For example, in the mild stage of chronic hepatitis B (mild chronic hepatitis B), the increase of serum ALT and AST levels is of great value for judging the condition.

[0003] Hepatic fibrosis is a reparative response of the liver to chronic injury, characterized by excessive deposition of extracellular matrix (ECM), which can further develop into cirrhosis, liver failure and hepatocellular carcinoma (HCC), and is a result of chronic liver injury. According to statistics, about 2 million people die from liver fibrosis-related complications worldwide each year, among which chronic viral hepatitis (hepatitis B and hepatitis C), alcoholic liver disease (ALD), non-alcoholic steatohepatitis (NASH) and autoimmune hepatitis are the main causes. With the prevalence of obesity and metabolic syndrome, the incidence of NASH-related liver fibrosis is showing a significant upward trend, and it is expected to become the primary indication for liver transplantation by 2030. Liver fibrosis is a reparative response of the liver to various chronic injuries. Under normal circumstances, the extracellular matrix (ECM) of the liver is in a dynamic balance state, and its synthesis and degradation processes are coordinated. When the liver is chronically injured, such as viral hepatitis, alcoholism, drug or toxic injury, autoimmune liver disease, etc., the hepatic stellate cells (HSCs) in the liver are activated. Under normal conditions, HSCs are in a quiescent state, and their main function is to store vitamin A. However, when the liver is injured, HSCs are activated and transformed into cells with the ability to synthesize collagen, and a large amount of extracellular matrix, especially collagen I and III, is synthesized. These extracellular matrices are excessively deposited in the liver, leading to changes in the structure of the liver and the formation of liver fibrosis.

[0004] Although it is generally believed that the degree of liver tissue fibrosis is not significantly correlated with serum ALT levels, during the progression of the disease, repeated liver cell inflammatory damage reflected by persistent ALT elevation is an important basis for the occurrence and development of liver fibrosis. Long-term abnormal elevation of ALT indicates repeated liver damage, which promotes the activation of hepatic stellate cells, and then synthesizes and secretes a large amount of extracellular matrix, leading to gradual aggravation of liver fibrosis.

[0005] AST is associated with the degree of liver fibrosis and cirrhosis. A study of 177 patients with various non-alcoholic chronic liver diseases found that in patients with chronic hepatitis B, the AST / ALT ratio of patients with cirrhosis is often greater than 1.0, while the ratio of patients without cirrhosis is mostly less than 1.0. In addition, the AST level itself is also related to the degree of fibrosis, the higher the degree of fibrosis, the higher the AST level. This is because as liver fibrosis progresses, liver cell damage worsens, mitochondria are damaged, and AST release increases. (A L, Williams, Ratio of serum aspartate to alanine aminotransferase in chronic hepatitis. Relationship to cirrhosis)

[0006] Liver fibrosis is a precancerous lesion of cirrhosis and liver cancer. If not treated and controlled, liver fibrosis will gradually progress to cirrhosis. In cirrhosis, the structure of the liver is severely changed, the liver lobule is divided by fibrous tissue to form pseudolobules, leading to increased vascular resistance of the liver and elevated portal vein pressure, thus causing a series of serious complications. For example, portal hypertension can cause esophageal varices, and patients may have symptoms such as hematemesis and melena, which is one of the most common causes of death in cirrhosis. In addition, cirrhosis can also cause ascites, and patients may have symptoms such as abdominal distension and abdominal distension, which seriously affect the quality of life of patients. Further development of liver fibrosis can also increase the risk of liver cancer. Liver cells are prone to genetic mutation and canceration in a long-term inflammatory and fibrotic environment. Studies have shown that the incidence of hepatocellular carcinoma (HCC) in patients with cirrhosis is significantly higher than that in the general population. Therefore, early diagnosis and treatment of liver fibrosis are of great significance for preventing the occurrence of cirrhosis and liver cancer.

[0007] However, the treatment of chronic hepatitis and liver fibrosis still faces some challenges. For example, during antiviral therapy, viral resistance may occur, leading to treatment failure. Some patients have poor tolerance to immunosuppressive agents, and are prone to complications such as infection. In addition, for some patients with end-stage hepatitis, liver transplantation is the only treatment option, but liver transplantation has problems such as donor shortage, high surgical risk, and postoperative immune rejection.

[0008] Despite the progress in basic research and clinical translation, the reversal of liver fibrosis still faces the following bottlenecks: 1) lack of precise non-invasive diagnostic markers to dynamically assess the degree of fibrosis; 2) individualized treatment strategies for different etiologies have not been established; 3) the reprogramming mechanism of the microenvironment of advanced liver cirrhosis needs to be elucidated. Future research needs to integrate multi-omics technologies (single-cell sequencing, spatial transcriptome), organoid models and artificial intelligence prediction systems to develop multifunctional combination therapy. SUMMARY

[0009] The present application provides the use of a compound of Formula I or a pharmaceutically acceptable salt thereof in the manufacture of a pharmaceutical composition for alleviating and / or treating liver injury, such as chronic hepatitis, liver fibrosis,

[0010]

[0011]

[0012] R1, R2 are the same or different, independently of each other selected from H, unsubstituted or with one or more R a substituted C 1-12 alkyl; wherein each R a is the same or different, independently of each other selected from halogen, C 1-12 alkyl, C 1-12 alkyloxy.

[0013] According to an embodiment of the present application, the R1, R2 are the same or different, independently of each other selected from H, unsubstituted or with one or more R a substituted C 1-6 alkyl; wherein each R a is the same or different, independently of each other selected from halogen, C 1-6 alkyl, C 1-6 alkyloxy.

[0014] According to an embodiment of the present application, the R1, R2 are the same or different, independently of each other selected from H, unsubstituted or with one or more R a substituted C 1-4 alkyl; wherein each R a is the same or different, independently of each other selected from halogen, C 1-4 alkyl, C 1-4 alkyloxy.

[0015] According to an embodiment of the present application, the C 1-4 alkyl comprises methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl or t-butyl.

[0016] According to an embodiment of the present application, the R1, R2 are different, independently of each other selected from H or unsubstituted C 1-6alkyl, preferably C 1-4 alkyl (e.g., methyl); for example, R1is H and R2is methyl.

[0017] According to an embodiment of the present application, the compound of Formula I has a structure as shown in Formula I-1 (i.e., mesoxalic acid):

[0018]

[0019] According to an embodiment of the present application, the pharmaceutically acceptable salt is a metal salt of the compound of Formula I, for example, the metal salt is selected from one or more of an alkali metal salt and an alkaline earth metal salt, exemplarily selected from one or more of a sodium salt, a potassium salt, and a calcium salt.

[0020] According to an embodiment of the present application, the pharmaceutical composition comprises a therapeutically effective amount of the compound of Formula I or a pharmaceutically acceptable salt thereof.

[0021] According to an embodiment of the present application, the pharmaceutical composition can further comprise a pharmaceutically acceptable excipient.

[0022] According to an embodiment of the present application, the pharmaceutically acceptable excipient includes, but is not limited to, a pharmaceutically acceptable carrier or excipient. For example, the pharmaceutically acceptable excipient is selected from at least one of a filler, a disintegrant, a binder, a lubricant, a surfactant, a flavoring agent, a humectant, a pH adjusting agent, a solubilizer or co-solubilizer, an osmotic pressure adjusting agent, and the like, but is not limited thereto. In some embodiments, the pharmaceutically acceptable excipient is starch and / or a nanoparticle for facilitating absorption or sustained release of the active ingredient.

[0023] According to an embodiment of the present application, the pharmaceutical composition can further comprise one or more other drugs for treating liver injury (e.g., chronic hepatitis, liver fibrosis), for example, comprising one or more of an antiviral drug, an antioxidant drug, an anti-inflammatory drug, an immunomodulatory drug, an anti-fibrotic drug, and the like.

[0024] According to an embodiment of the present application, the administration route of the pharmaceutical composition includes, but is not limited to, intravenous infusion, intravenous drip, subcutaneous administration, intradermal administration, intramuscular injection, oral spray, oral administration, in situ administration to a tumor, and the like.

[0025] According to an embodiment of the present application, the pharmaceutical composition is preferably a pharmaceutical preparation, for example, a tablet, a capsule, a pill, a granule, a solution, a suspension, a syrup, an injection (including an injection solution, a sterile powder for injection, or a concentrated solution for injection), a suppository, an inhalant, or a spray.

[0026] According to an embodiment of the present application, the pharmaceutical preparation is a single-dose preparation or a multi-dose preparation. The multi-dose preparation comprises more than one unit package, for example consists of 2 to 10 unit packages, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 unit packages. Among them, the content of the compound of formula I, pharmaceutically acceptable salt of the compound of formula I contained in each unit package is the same, or at least the content of the compound of formula I, pharmaceutically acceptable salt of the compound of formula I contained in two unit packages is not the same.

[0027] The present application also provides a method for alleviating and / or treating liver injury (for example chronic hepatitis, liver fibrosis), comprising administering to a patient a therapeutically effective amount of the compound of formula I, pharmaceutically acceptable salt thereof or the pharmaceutical composition.

[0028] According to an embodiment of the present application, the chronic hepatitis includes chronic hepatitis B, chronic hepatitis C, alcoholic hepatitis, autoimmune hepatitis, chronic persistent hepatitis and chronic active hepatitis.

[0029] According to an embodiment of the present application, the liver fibrosis of the present application includes F1 stage, fibrosis of the portal area but no fibrous septum formation according to the Metavir staging system; F2 stage, fibrosis of the portal area with a small number of fibrous septum formation; F3 stage, more fibrous septum formation but no cirrhosis; F4 stage, cirrhosis.

[0030] According to an embodiment of the present application, the compound of formula I, pharmaceutically acceptable salt thereof can effectively reduce the activity of related enzymes (for example ALT and / or AST) in liver fibrosis serum.

[0031] According to an embodiment of the present application, the compound of formula I, pharmaceutically acceptable salt thereof can reduce the expression level of fibrosis index α-SMA protein in the liver, and the degree of fibrosis is reduced.

[0032] According to an embodiment of the present application, the compound of formula I, pharmaceutically acceptable salt thereof can improve the degree of liver tissue inflammation and the degree of lipid vacuolization, and reduce the area of collagen and fibrous deposition.

[0033] According to an embodiment of the present application, when liver cells are damaged, the activities of serum glutamic oxalacetic transaminase (AST) and glutamic pyruvic transaminase (ALT) will increase. The compound of formula I, pharmaceutically acceptable salt thereof can treat liver injury, for example treat chronic hepatitis and liver fibrosis by inhibiting the activities of glutamic oxalacetic transaminase (AST) and glutamic pyruvic transaminase (ALT).

[0034] According to an embodiment of the present application, the compound of formula I, pharmaceutically acceptable salt thereof can alleviate and / or treat liver fibrosis by inhibiting the expression of α-SMA protein.

[0035] The application also provides use of the compound of formula I or a pharmaceutically acceptable salt thereof in the preparation of a pharmaceutical composition capable of effectively reducing the activity of related enzymes (such as ALT and / or AST) in serum of liver fibrosis.

[0036] The application also provides use of the compound of formula I or a pharmaceutically acceptable salt thereof in the preparation of a pharmaceutical composition capable of reducing the expression level of fibrosis index α-SMA protein in the liver, and reducing the degree of fibrosis.

[0037] The application also provides use of the compound of formula I or a pharmaceutically acceptable salt thereof in the preparation of a pharmaceutical composition capable of improving the degree of inflammation and the degree of lipid vacuolization of liver tissue, and reducing the area of collagen and fibrous deposition.

[0038] According to an embodiment of the application, the pharmaceutical composition has the limitations as shown above.

[0039] Advantages of the application:

[0040] The application provides use of the compound of formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating liver damage, especially liver fibrosis and chronic hepatitis.

[0041] The application first discovers and confirms through animal experiments that mesaconic acid can effectively reduce the activity of ALT and AST related enzymes in serum of liver fibrosis mice, and reduce the expression level of fibrosis index α-SMA protein in the liver. Moreover, through HE staining of the liver, it is found that mesaconic acid can reduce the fibrosis level of liver tissue, reduce inflammatory cell infiltration, and improve the abnormal liver histology. Therefore, mesaconic acid has strong liver protection potential, can effectively delay the disease progression of liver fibrosis and chronic hepatitis, has good application prospects in preventing and / or treating liver fibrosis and chronic hepatitis, and can lay a theoretical research and material basis for the application of mesaconic acid in preventing and / or treating liver fibrosis and chronic hepatitis.

[0042] Definitions and explanations of terms

[0043] Unless otherwise specified, the definitions of the terms recorded in the specification and claims of the present application, including the definitions of examples, exemplary definitions, preferred definitions, definitions recorded in tables, definitions of specific compounds in examples, etc., can be combined and integrated with each other in any manner. Such combinations and integrations shall belong to the scope recorded in the specification of the present application.

[0044] Unless otherwise indicated, the numerical values in the description and the claims herein are to be rounded to the nearest whole number. For example, the numerical range "1-12" is equivalent to reciting each of the integer values 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 in the numerical range "1-12."

[0045] It is to be understood that the description and claims herein refer to "a" or "an" element or "the" element by attention drawn to a singular form, which also covers the plural form unless otherwise indicated. For example, a reference to "one" element is a reference to one or more elements unless otherwise indicated.

[0046] The term "liver injury" refers to damage to liver cells caused by factors such as viral infection, drugs, alcohol, autoimmunity, etc. Such damage can be mild or severe, and can even lead to liver failure.

[0047] The term "C 1-12 "alkyl" is to be understood as meaning a straight-chain and branched-chain alkyl radical having 1 to 12 carbon atoms, "C 1-6 "alkyl" means a straight-chain and branched-chain alkyl radical having 1, 2, 3, 4, 5 or 6 carbon atoms. Said alkyl radical is, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1 -methylbutyl, 1 -ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1 -dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1 -methylpentyl, 2-ethylbutyl, 1 -ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1 -dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl or 1,2-dimethylbutyl, and the like or their isomers.

[0048] The term "alkoxy" means -O-(alkyl), wherein alkyl is defined as above. Non-limiting examples of alkoxy groups include methoxy, ethoxy, propoxy, butoxy.

[0049] The term "halogen" includes F, CI, Br and I.

[0050] The term "pharmaceutically acceptable excipient" means an excipient that is not biologically or otherwise undesirable, i.e., the excipient can be administered to an organism without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained.

[0051] Unless otherwise indicated, the compounds of Formula I described in the present specification and / or pharmaceutically acceptable salts thereof can include tautomers, amorphous forms, polymorphic forms, solvates, isotopically-labeled forms (such as deuterated derivatives), and the like.

[0052] The term "tautomer" refers to isomers of a functional group that result from the rapid movement of an atom in a molecule between two positions. The compounds of the present disclosure can exhibit tautomerism. Compounds that tautomerize can exist in two or more interconvertible species. Proton-shift tautomers result from the migration of a hydrogen atom covalently bonded between two atoms. Tautomers generally exist in equilibrium, and attempts to isolate a single tautomer usually result in a mixture whose physical and chemical properties are consistent with a mixture of compounds. The position of equilibrium depends on the chemical properties within the molecule. For example, in many aliphatic aldehydes and ketones such as acetaldehyde, the keto form predominates; in phenols, the enol form predominates. The present disclosure includes all tautomeric forms of the compounds.

[0053] In the present application, the term "solvate" refers to a compound of the present disclosure or a salt thereof, including stoichiometric or non-stoichiometric amounts of solvent, when the solvent is water, then the solvate is a hydrate.

[0054] "Isotopes" are all isotopes of atoms occurring in the compounds of the present application. Isotopes include those atoms having the same atomic number but different mass numbers. Examples of isotopes suitable for inclusion in the compounds of the present application are of hydrogen, carbon, oxygen, fluorine, and chlorine, such as but not limited to 2 H, 3 H, 13 C, 14 C, 18 O, 18 F and 36 C1. Isotopically-labeled compounds of the present application can generally be prepared by conventional techniques known to those skilled in the art, or by processes analogous to those described in the accompanying Examples using an appropriate isotopically-labeled reagent in place of the non- isotopically labeled reagent employed. Such compounds are useful in, e.g., determination of biological activity. In the case of stable isotopes, such compounds are useful in, e.g., providing a more active metabolite.

[0055] The term "patient" means any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, swine; cattle, sheep, horses, or primates, and most preferably humans.

[0056] The term “therapeutic effective dose” refers to the amount of an active compound or drug that researchers, veterinarians, physicians, or other clinicians are searching for in a tissue, system, animal, individual, or human to elicit a biological or medical response. It includes one or more of the following: (1) prevention of disease: e.g., prevention of disease, disorder, or condition in an individual who is susceptible to disease, disorder, or symptom but has not yet experienced or developed the pathology or symptoms of the disease. (2) inhibition of disease: e.g., inhibition of disease, disorder, or symptom in an individual experiencing or developing the pathology or symptoms of the disease (i.e., prevention of further development of the pathology and / or symptoms). (3) relief of disease: e.g., relief of disease, disorder, or symptom in an individual experiencing or developing the pathology or symptoms of the disease (i.e., reversal of the pathology and / or symptom). The specific dosage will vary depending on factors such as the specific compound chosen, the dosing regimen, whether it is administered in combination with other compounds, the timing of administration, the tissue to which the drug is administered, and the physical delivery system used. Attached Figure Description

[0057] Figure 1 The graph shows the results of weight changes in mice in the Control and MA groups in a mouse model of liver fibrosis.

[0058] Figure 2 The results of H&E staining in the livers of mice in the Control and MA groups in a mouse model of liver fibrosis.

[0059] Figure 3 Masson staining results of livers in mice with liver fibrosis in the Control and MA groups in a mouse model of liver fibrosis;

[0060] Figure 4 Sirius red staining results of liver fibrosis mice in the Control and MA groups in a liver fibrosis mouse model.

[0061] Figure 5 Immunohistochemical staining results of liver fibrosis-related indicators in Control and MA mice in a liver fibrosis mouse model.

[0062] Figure 6 and Figure 7 The results of α-SMA protein expression, a liver fibrosis-related marker, in mice of the Control and MA groups in a liver fibrosis mouse model.

[0063] Figure 8 The results of serum ALT and AST-related enzyme activity expression in the Control and MA groups of a mouse model of liver fibrosis.

[0064] in, Figures 1-7In the experiment, the Control group represents the liver fibrosis mouse group which is gavaged with the corresponding dose of sterile ultrapure water by body weight; the MA group represents the liver fibrosis mouse group which is gavaged with the dose of 10 mg / kg / day of mesaconic acid. DETAILED DESCRIPTION

[0065] The technical solutions of the present application will be further described in detail below in combination with specific examples. It should be understood that the following examples are only illustratively described and explained the present application, and should not be interpreted as limiting the scope of protection of the present application. Any technology realized based on the above description of the present application is covered within the scope of the present application.

[0066] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0067] In the following examples, the C57BL / 6J mice used are purchased from Liaoning Changsheng Biotechnology Co., Ltd. The mesaconic acid is purchased from Shanghai Maikelin Biotechnology Co., Ltd. The preparation method of the mesaconic acid solution is as follows: the mesaconic acid powder is dissolved in sterile ultrapure water to obtain a mesaconic acid solution with a final concentration. The CCl4 required for modeling is purchased from Tianjin Fuyu Fine Chemical Co., Ltd., and the olive oil is purchased from Shanghai Maikelin Biotechnology Co., Ltd.

[0068] Example 1 Pharmacodynamic test of mesaconic acid

[0069] The pharmacodynamic test of mesaconic acid was carried out in this test, and the test materials, test grouping and test process are as follows:

[0070] Test materials: ordinary feed C57BL / 6J mice. The establishment of liver fibrosis model is to prepare 25% CCl4 solution by mixing CCl4 and olive oil in a ratio of 1:3, and then inject the prepared solution into the abdominal cavity at a dose of 2.5 ml / kg. The recommended injection dose is 50 μL, twice a week and record the body weight change of the mice.

[0071] Liver fibrosis test grouping: the "CONTROL" group represents the ordinary feed liver fibrosis model mouse group which is gavaged with the same volume of sterile ultrapure water, and the "MA" group represents the liver fibrosis model mouse group which is fed with mesaconic acid at a dose of 10 mg / kg / day.

[0072] Test process:

[0073] Mouse liver fibrosis model grouping: C57BL / 6J mice were grouped according to their pre-modeling body weight. The Control group and MA group received intraperitoneal injections of a prepared 25% CCl4 solution at a dose of 2.5 ml / kg twice weekly. The MA group began receiving the drug after the sixth week of modeling, and also received intraperitoneal injections of 25% CCl4 solution twice weekly during this period. The MA group received intragastric administration of 10 mg / kg / day of methylconazole to the liver fibrosis model mice in the MA group, while the Control group received an appropriate volume of ultrapure water via gavage based on their body weight. The experiment lasted 10 weeks. Mice were weighed weekly, and tissue samples were collected at the end of the experiment for subsequent index analysis.

[0074] I. Weight Change Test

[0075] Weight was measured using standard weighing methods. The weekly weight changes of mice in the Control group and MA group are shown below. Figure 1 As shown.

[0076] according to Figure 1 It was found that there was no significant difference in body weight among the groups of animals, indicating that zhongconic acid has good safety. Furthermore, the biocompatibility of zhongconic acid has been confirmed in Chinese patent application No. 2022112460706.

[0077] II. H&E staining

[0078] Mice administered the drug for 4 weeks and untreated mice were dissected, and their livers were harvested for Hematoxylin and Escherichia coli (H&E) staining. The H&E staining procedure was as follows: adipose tissue was fixed in 4% paraformaldehyde for 48 hours before subsequent experiments. The tissue was first dehydrated and embedded, then cut into 4μm sections. The sections were then dewaxed, stained with hematoxylin and eosin, dehydrated, and mounted with neutral resin. The staining results are shown below. Figure 2 As shown.

[0079] according to Figure 2 The results showed that the degree of liver inflammation and lipid vacuolation in the MA group (after administration of citric acid) was improved compared with that in the Control group, and collagen and fiber deposition were reduced.

[0080] III. Masson staining

[0081] The tissue slices of process two were deparaffinated and rehydrated, Weigert iron hematoxylin staining solution was prepared, and the tissue was stained for 10 minutes; after spinning dry, acid ethanol differentiation solution was added for 20 seconds, and then the tissue was washed with flowing water for 2 minutes; Masson blue solution was added for 4 minutes, and then the tissue was washed with flowing water for 2 minutes, distilled water for 1 minute, and then eosin solution was added for 10 minutes; the eosin solution was discarded, and then weak acid working solution was added for 1 minute; after spinning dry, phosphomolybdic acid solution was added for 2 minutes, and then weak acid working solution was added for 1 minute; the tissue was dehydrated and transparentized, and then neutral quick-drying glue was used for sealing the slices, and the slices were naturally dried in a fume hood. The staining results are shown in Figure 3 .

[0082] According to the results shown in Figure 3 , it can be seen that the area of liver tissue fibrosis deposition in the Control group is significantly increased compared with the MA group.

[0083] Four, Sirius red staining

[0084] The mice administered for 4 weeks and mice of the same period without administration were dissected, and the livers were taken out, and then H&E staining was performed. The experimental process of H&E staining is as follows: the adipose tissue was fixed in 4% paraformaldehyde, and after 48 hours of fixation, the subsequent experiments were performed. First, the tissue was dehydrated and embedded, and then the embedded tissue was cut into 4 μm sections. Then the sections were deparaffinized, stained with Sirius red, and finally dehydrated and sealed with neutral gum. The staining results are shown in Figure 4 .

[0085] According to the results shown in Figure 4 , it can be seen that the liver fibrosis deposition of the Control group mice is obvious, and the liver fibrosis deposition area of the MA group mice after intervention of mesaconic acid is significantly reduced.

[0086] Five, immunohistochemical staining

[0087] The tissue slices of process four were deparaffinized and rehydrated, sodium citrate was used for heat antigen repair, and then the temperature was cooled to room temperature. Endogenous peroxidase blocking agent was added, and the tissue was incubated at room temperature for 20 minutes. The tissue was washed with 1×PBS solution for 3 times, 5 minutes each time. The primary antibody was prepared according to the proportion provided in the antibody instruction book, and an appropriate amount of the primary antibody was added to evenly distribute the antibody on the tissue. The sections were placed in a humidified box and incubated at 4°C overnight. The next day, the primary antibody was recovered, and the tissue was washed with 1×PBS solution for 3 times, 5 minutes each time. An appropriate amount of enzyme-labeled goat anti-rabbit IgG polymer was added, and the tissue was incubated in a 37°C incubator for 20 minutes. The tissue was washed with 1×PBS solution for 3 times, 5 minutes each time. Fresh DAB developing solution was prepared and added to the tissue, and the tissue was incubated at room temperature in the dark for 8 minutes. Under a microscope, when the tissue turned brownish yellow, the developing was stopped, and the tissue was washed with 1×PBS solution for 5 minutes. The tissue was dehydrated and transparentized, sealed with neutral quick-drying glue, and naturally dried in a fume hood. The staining results are shown in Figure 5 .

[0088] According to the results of Figure 5 It can be seen that compared with the Control group, the fibrosis index a-SMA in the MA group is significantly reduced, and the degree of fibrosis is reduced.

[0089] Six, the expression of liver fibrosis related factor a-SMA protein

[0090] Test process: After 4 weeks of administration, a certain size of liver tissue of liver fibrosis mice was taken into a 2mL grinding tube, and an appropriate amount of lysis solution (RIPA high-efficiency tissue lysis solution: PMSF: phosphatase inhibitor = 100: 1: 2) was added. The tissue homogenate was prepared on the automatic sample freezing grinder. Then centrifugation was carried out at 4℃, 12000rpm, 30min, the supernatant was aspirated, and the protein concentration was detected using BCA detection kit. After protein quantification, Western Blot test was carried out. Among them, the qualitative and quantitative results of a-SMA protein expression of liver fibrosis related factor in Control group and MA group of liver fibrosis mouse model are shown in Figure 6 and Figure 7 Compared with the Control group, the expression of fibrosis index a-SMA protein in the MA group is significantly reduced. "*" represents "p<0.05"; "**" represents "p<0.01"; "***" represents "p<0.001", all indicating significant difference.

[0091] Seven, AST and ALT related enzyme activity expression

[0092] Test process: a certain amount of corresponding tissue sample was taken, and the sample used in this experiment was mouse serum. The blood was coagulated at room temperature for 30min, centrifuged at 3500r / min for 10min, and the supernatant was taken for standby. Add pre-warmed matrix liquid reagent 20μL and sample 5μL in microplate, mix well, react at 37℃ for 30min, add reagent two (2, 4-dinitrophenylhydrazine liquid) 20μL, react at 37℃ for 20min, add reagent three (concentration of 0.4mol / L sodium hydroxide solution) 200μL, react at room temperature for 15min, measure the absorbance value at 510nm, and calculate the activity of alanine aminotransferase and aspartate aminotransferase according to the standard curve. Among them, Figure 8 is the expression of AST and ALT related enzyme activity in the serum of two groups of mice. "*" represents "p<0.05"; "**" represents "p<0.01"; "***" represents "p<0.001", all indicating significant difference.

[0093] From the Figure 8 It can be seen that the expression of AST and ALT related enzyme activity in the serum of liver fibrosis model mice can be reduced by using mesaconic acid.

[0094] In summary, the present application first discovers and confirms that in a liver fibrosis mouse model induced by carbon tetrachloride, mesaconic acid can alleviate the weight loss caused by CCl4 induction, and mesaconic acid can significantly reduce the ALT and AST related enzyme activity of the fibrosis model mice and can reduce the expression level of the fibrosis corresponding index α-SMA protein in the liver. And through the kidney H&E and other related pathological staining, it is found that mesaconic acid can reduce the degree of inflammation and lipid vacuolization of liver tissue, and the deposition of collagen and fiber is also reduced, which proves that the liver tissue damage is alleviated from the aspect of histopathology. That is, mesaconic acid can effectively delay the corresponding process of liver fibrosis, and has a certain potential for liver protection.

[0095] Therefore, the present application proves the application potential of mesaconic acid in preventing and treating liver fibrosis, and clarifies the mechanism of action, which can lay a theoretical and material foundation for the application of mesaconic acid in preventing and / or treating liver fibrosis diseases.

[0096] The above describes the embodiments of the present application. However, the present application is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. Use of a compound of Formula I or a pharmaceutically acceptable salt thereof in the manufacture of a pharmaceutical composition for alleviating and / or treating liver injury, R1 and R2 may be the same or different, and are independently selected from H, without substitution, or by one or more R... a Replacement C 1-12 Alkyl groups; wherein each R a They are the same or different, and are independently selected from halogens and C. 1-12 Alkyl, C 1-12 Alkyloxy; Preferably, the liver injury comprises chronic hepatitis, liver fibrosis.

2. Use according to claim 1, wherein, R1, R2are the same or different, independently of one another, selected from the group consisting of H, unsubstituted or substituted C a substituted C 1-6 alkyl; wherein each R a are the same or different, independently of one another, selected from the group consisting of halogen, C 1-6 alkyl, C 1-6 alkyloxy; Preferably, said R1, R2are the same or different, independently from each other, selected from H, unsubstituted or substituted with one or more R a substituted C 1-4 alkyl; wherein each R a are the same or different, independently from each other, selected from halogen, C 1-4 alkyl, C 1-4 alkyloxy; Preferably, said R1, R2are different and independently from each other selected from H or unsubstituted C 1-6 alkyl, preferably C 1-4 alkyl.

3. Use according to claim 1, wherein, The compound of Formula I has a structure as shown in Formula I-1:

4. The use according to claim 1, wherein, The pharmaceutically acceptable salt is a metal salt of the compound of Formula I, which is selected from one or more of an alkali metal salt and an alkaline earth metal salt.

5. The use according to claim 1, wherein, The pharmaceutical composition comprises a therapeutically effective amount of the compound of Formula I or a pharmaceutically acceptable salt thereof.

6. The use according to claim 1, wherein, The pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

7. The use according to claim 1, wherein, The pharmaceutical composition further comprises one or more other drugs for treating liver injury.

8. The use according to claim 1, wherein, The pharmaceutical composition is a pharmaceutical preparation, such as a tablet, a capsule, a pill, a granule, a solution, a suspension, a syrup, an injection, a suppository, an inhalant or a spray.

9. The use according to claim 1, wherein, The chronic hepatitis comprises chronic hepatitis B, chronic hepatitis C, alcoholic hepatitis, autoimmune hepatitis, chronic persistent hepatitis and chronic active hepatitis. The liver fibrosis comprises F1 stage, fibrosis of the portal area but no fibrous septum formation; F2 stage, fibrosis of the portal area with a few fibrous septum formation; F3 stage, more fibrous septum formation but no cirrhosis; F4 stage, cirrhosis according to the Metavir staging system.

10. Use according to any one of claims 1 to 9, wherein, The compound of Formula I, a pharmaceutically acceptable salt thereof can treat liver injury, such as chronic hepatitis, liver fibrosis by inhibiting the activity of aspartate aminotransferase (AST) and alanine aminotransferase (ALT); and / or, the compound of Formula I, a pharmaceutically acceptable salt thereof can alleviate and / or treat liver fibrosis by inhibiting the expression of α-SMA protein.

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