A method for constructing an animal model of liver fibrosis or non-alcoholic steatohepatitis and its application

Abstract

The present invention belongs to the field of pharmaceutical technology, and specifically relates to a method for constructing an animal model of liver fibrosis or non-alcoholic steatohepatitis and its application. The present invention discloses a method for constructing an animal model of liver fibrosis, which includes the following steps: S1. Breeding GSDMD-NT mice with Alb-Cre mice to obtain GSDMD+ / -; Alb-Cre+ / - mice; S2. Inducing GSDMD+ / -; Alb-Cre+ / - mice with DOX at a concentration of 15 μg / mL and adding a high-fat diet to obtain an animal model of liver fibrosis. The application of the animal model of liver fibrosis or non-alcoholic steatohepatitis obtained by the construction method in studying the mechanism of liver fibrosis or NASH and / or for the efficacy evaluation of drugs or pharmacological experiments. Through the present invention, it is possible to achieve fibrosis modeling in mice with different degrees and speeds, providing a more reliable model tool for the mechanism research of liver fibrosis and non-alcoholic steatohepatitis (NASH) and the efficacy evaluation of drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine, and particularly relates to a method for constructing an animal model of liver fibrosis or non-alcoholic steatohepatitis and its application. Background Art

[0002] The liver is the largest solid organ in the abdominal cavity and has various important physiological functions such as metabolism, detoxification, and immunity. A variety of physical, chemical, and biological factors can cause liver injury. The results of the injury often lead to liver necrosis, fatty liver, cholestasis, liver fibrosis, cirrhosis, and liver cancer. Among them, fibrosis, that is, the excessive deposition and abnormal distribution of various components of the extracellular matrix of hepatocytes, is a common response of the liver to various chronic injuries. Further progression of liver fibrosis can cause changes in liver lobules, formation of false lobules and nodules, and enter the stage of cirrhosis. Currently, China is a major country with liver diseases in the world. Chronic liver diseases will all develop fibrosis during their disease processes and progress towards cirrhosis and liver cancer. Therefore, preventing liver fibrosis and reversing fibrosis are important topics in clinical and pharmaceutical research.

[0003] To study liver fibrosis, it is necessary to establish an animal model of liver fibrosis. The traditional method for establishing the model is to inject carbon tetrachloride (CCl4) into the abdominal cavity of mice to induce the occurrence of liver fibrosis in mice. The CCl4-induced model has the characteristics of a relatively fast formation rate of fibrosis and a stable model establishment. However, it will not only cause liver fibrosis but also damage other organs of mice and has a high mortality rate. Therefore, it is necessary to develop a mouse model that specifically causes liver fibrosis. In the patent "A Method for Constructing and Applying a Conditional Cell Death Animal Model" (Publication No.: CN112868603B) authorized to the applicant, a conditional cell death animal model mediated by GSDMD protein was constructed. In the examples of this patent, 2 mg / mL of DOX was used for induction to create an acute liver injury model in mice. Since liver fibrosis is a chronic liver injury process, it is necessary to optimize the DOX induction conditions to slow down the death of mouse liver cells and induce the symptoms of liver fibrosis in mice. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for constructing an animal model of liver fibrosis or non-alcoholic steatohepatitis and its application. 1. An animal model of liver fibrosis constructed by this method can achieve different degrees of liver fibrosis and different speeds of liver fibrosis model establishment, providing a more reliable model tool for the drug treatment and mechanism research of liver fibrosis. 2. An animal model of non-alcoholic steatohepatitis (NASH) constructed by this method can achieve NASH model establishment, providing a more reliable model tool for the drug treatment and mechanism research of NASH.

[0005] To achieve the above-mentioned invention objectives, the present invention adopts the following technical solutions:

[0006] A method for constructing a liver fibrosis animal model, comprising the following steps:

[0007] S1. Breed GSDMD-NT mice with Alb-Cre mice to obtain GSDMD+ / -; Alb-Cre+ / - mice;

[0008] S2. Use DOX with a concentration of 15 μg / mL and add a high-fat diet to induce GSDMD+ / -; Alb-Cre+ / - mice to obtain a liver fibrosis animal model.

[0009] The GSDMD-NT gene is a gene encoding the N-terminal domain of the Gasdermin protein family, which can mediate pyroptosis. Pyroptosis is a form of programmed cell death, manifested as cells continuously swelling until the cell membrane ruptures, resulting in the release of cell contents and then activating a strong inflammatory response.

[0010] Furthermore, DOX can specifically induce the expression of GSDMD-NT in the hepatocytes of GSDMD+ / -; Alb-Cre+ / - double-positive mice. Using DOX for induction has the ability to specifically express GSDMD-NT in liver cells. The expressed GSDMD-NT can cause pyroptosis of liver cells. Continuous use of DOX for induction can cause continuous death of liver cells, gradually form scars, and lead to the occurrence of fibrosis.

[0011] Furthermore, under the condition of 15 μg / mL DOX, GSDMD+ / -; Alb-Cre+ / - mice will show symptoms of liver fibrosis around 6 weeks. On the basis of 15 μg / mL DOX, adding a high-fat diet condition, GSDMD+ / -; Alb-Cre+ / - mice develop fibrosis faster and to a higher degree.

[0012] A method for constructing a non-alcoholic steatohepatitis animal model, comprising the following steps:

[0013] S1. Breed GSDMD-NT mice with Alb-Cre mice to obtain GSDMD+ / -; Alb-Cre+ / - mice;

[0014] S2. First, feed GSDMD+ / -; Alb-Cre+ / - mice with a high-fat diet, and then add DOX with a concentration of 0.05 - 20 μg / mL for concentration gradient induction of GSDMD+ / -; Alb-Cre+ / - mice to obtain a non-alcoholic steatohepatitis animal model.

[0015] Preferably, liver pathological sections of the induced GSDMD+ / -; Alb-Cre+ / - mice at different time points were taken to observe the levels of liver fibrosis and hepatitis.

[0016] Preferably, in the step S1, the Alb-Cre mouse is a mouse with specific expression of Cre recombinase in liver cells.

[0017] Preferably, the GSDMD+ / -; Alb-Cre+ / - mouse is a double-positive mouse obtained by breeding an expression GSDMD-NT mouse containing the CRE-LOXP system and an Alb-Cre mouse.

[0018] Application of the liver fibrosis animal model obtained by the above method for constructing a liver fibrosis animal model in studying the mechanism of liver fibrosis or for evaluating the efficacy of drugs or pharmacological experiments.

[0019] Application of the above non-alcoholic steatohepatitis animal model in studying the mechanism of non-alcoholic steatohepatitis or for evaluating the efficacy of drugs or pharmacological experiments.

[0020] The beneficial effects of the present invention are as follows: The present invention provides a method for constructing a liver fibrosis or non-alcoholic steatohepatitis animal model and its application. First, a GSDMD+ / -; Alb-Cre+ / - double-positive mouse is obtained by breeding an expression GSDMD-NT mouse containing the CRE-LOXP system and an Alb-Cre mouse; secondly, the GSDMD+ / -; Alb-Cre+ / - double-positive mouse is induced by different concentrations of DOX. DOX has the ability to specifically express GSDMD-NT in liver cells, and the expressed GSDMD-NT can cause pyroptosis of liver cells, leading to the occurrence of fibrosis; finally, a certain concentration of DOX is obtained to achieve different degrees of liver fibrosis modeling in mice, that is, 15 μg / mL; further, on the basis of 15 μg / mL DOX, a high-fat diet is added to induce the GSDMD+ / -; Alb-Cre+ / - double-positive mouse to achieve different speeds of liver fibrosis modeling in mice. In addition, under the condition of a high-fat diet, the GSDMD+ / -; Alb-Cre+ / - double-positive mouse is induced by different concentrations of DOX, which can lead to the occurrence of non-alcoholic steatohepatitis (NASH); finally, a certain concentration of DOX is obtained to achieve non-alcoholic steatohepatitis (NASH) modeling in mice, that is, after feeding a high-fat diet for 8 weeks, DOX with a concentration gradient of 0.05 - 20 μg / mL is administered.

[0021] This construction method has a fast modeling speed, is easy to operate, and is convenient for repetition; after modeling, the model has a long duration and is stable; the drugs used in modeling have high safety and strong accessibility; it provides a more reliable model tool for the mechanism research of liver fibrosis and non-alcoholic steatohepatitis (NASH) and the efficacy evaluation of drugs. Description of the Drawings

[0022] Figure 1 Schematic diagram of the breeding of a liver fibrosis mouse model;

[0023] Figure 2 Results of gene identification of GSDMD-NT, Alb-Cre double-positive mice;

[0024] Figure 3 ALT (alanine aminotransferase, Figure A) and AST (aspartate aminotransferase, Figure B) test values of GSDMD+ / -; Alb-Cre+ / - mice induced by different concentrations of DOX. The data in Figure A and Figure B are presented as mean ± standard error, and the number of mice = 3;

[0025] Figure 4 Schematic diagram of liver pathological sections of mice under different concentrations of DOX, Bar = 200 μm;

[0026] Figure 5 Schematic diagram of liver pathological sections of mice after induction with 15 μg / mL DOX for different times, Bar = 200 μm;

[0027] Figure 6 Schematic diagram of liver pathological sections of mice induced by 15 μg / mL DOX plus high-fat diet and CCl4 respectively, Bar = 200 μm;

[0028] Figure 7 Schematic diagram of the efficacy evaluation of obeticholic acid;

[0029] Figure 8 Schematic diagram of pathological sections for the evaluation of obeticholic acid, Bar = 100μm;

[0030] Figure 9 Immunohistochemical picture for the evaluation of obeticholic acid, Bar = 100 μm;

[0031] Figure 10 Schematic diagram of the NASH phenotype of GSDMD+ / -; Alb-Cre+ / - mice induced by different concentrations of DOX in drinking water, Bar = 100 μm. Detailed Implementation Modes

[0032] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but it should not be construed as a limitation of the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention all fall within the scope of the present invention. The experimental methods without specific conditions noted in the embodiments and the reagents without the formulated recipes are all in accordance with the conventional conditions in the art.

[0033] Currently, most animal models of liver fibrosis are induced by carbon tetrachloride (CCl4) to cause chronic liver injury and then form liver fibrosis. CCl4 is metabolized in the liver by the monooxygenase of the cytochrome P450 superfamily (CYP family) to trichloromethyl radical (CCl3 - ). Subsequently, this free radical reacts with nucleic acids, proteins and lipids, thereby damaging key cellular processes, leading to altered lipid metabolism and decreased protein content. The trichloromethylperoxyl radical (CCl3OO*) generated by the oxidation of CCl3* further initiates lipid peroxidation and the destruction of polyunsaturated fatty acids. Therefore, the membrane permeability of all cell compartments (mitochondria, endoplasmic reticulum and plasma membrane) decreases, resulting in systemic liver injury characterized by inflammation, fibrosis, cirrhosis and liver cancer. CCl4-induced liver fibrosis has the characteristics of fast speed and stable model establishment. However, the mortality rate is relatively high during the model establishment process, and there are certain differences from the liver fibrosis pathological characteristics in clinical diseases, and the fibrosis cannot fully simulate the clinic.

[0034] Due to the above-mentioned defects of the current liver fibrosis model establishment methods, it is necessary to develop a brand-new animal model of liver fibrosis. The model mice should have the following advantages: (1) The pathological characteristics of liver fibrosis are close to those in the clinic, having clinical research significance; (2) The model establishment speed is fast, the operation is convenient, and it is easy to repeat; (3) After model establishment, the model duration is long and stable; (4) The drugs used for model establishment have high safety and strong accessibility.

[0035] The present invention provides a method for constructing an animal model of liver fibrosis. Through the method, a mouse model of liver fibrosis can be constructed, which can achieve different degrees of liver fibrosis and different speeds of liver fibrosis model establishment, providing a more reliable model tool for the drug treatment and mechanism research of liver fibrosis.

[0036] Specifically, taking mice as an example, on the basis of the conditional cell death animal model mediated by GSDMD protein, a low concentration of DOX was used for induction to prepare an experimental animal model of liver fibrosis. In addition, adding a high-fat diet on the basis of low-concentration DOX induction can promote the process of liver fibrosis and shorten the model establishment time.

[0037] The mouse model is a conditional cell death animal model based on GSDMD protein-mediated constructed in the applicant's authorized patent "A Method for Constructing and Applying a Conditional Cell Death Animal Model" (Publication No.: CN112868603B). Specifically, it is a transgenic mouse that can specifically express the GSDMD-NT gene in liver cells when both Cre recombinase and DOX are present. This transgenic mouse is the double-positive offspring obtained by mating a GSDMD-NT-expressing mouse containing the CRE-LOXP system and an Alb-Cre mouse. This mouse uses GSDMD-NT to clear cells. The GSDMD-NT gene is the gene encoding the N-terminal domain of the Gasdermin protein family, which can cause pyroptosis. Pyroptosis is a form of programmed cell death, manifested as cells continuously swelling until the cell membrane ruptures, resulting in the release of cell contents and then activating a strong inflammatory response.

[0038] The DOX is doxycycline hydrochloride, full name: Doxycycline hydrochloride, abbreviated as DOX in the text, which can specifically induce the expression of GSDMD-NT in the liver cells of GSDMD+ / -;Alb-Cre+ / - double-positive mice.

[0039] The high-fat diet is a 60% high-fat diet, named: Rodent Diet With 60% Kcal% Fat, which is commonly used to induce and construct models of mouse obesity, fatty liver, etc.

[0040] Specifically, a method for constructing a liver fibrosis animal model provided by the present invention, taking mice as an example, includes the following steps:

[0041] (1) Obtain double-positive mouse offspring by mating a GSDMD-NT-expressing mouse containing the CRE-LOXP system and an Alb-Cre mouse;

[0042] (2) Study the liver fibrosis phenotypes induced by different concentrations of DOX in GSDMD+ / -;Alb-Cre+ / - double-positive mice, and determine the specific DOX concentration that causes liver fibrosis in GSDMD+ / -;Alb-Cre+ / - double-positive mice;

[0043] (3) Under the condition of determining the specific DOX concentration, add a high-fat diet to the mice to induce liver fibrosis in the mice more quickly;

[0044] (4) Perform liver pathological sections on the induced double-positive mice at different time stages respectively, and observe the liver fibrosis phenotypes and the time nodes of fibrosis appearance.

[0045] Example 1 Construction of GSDMD+ / -;Alb-Cre+ / - Mice

[0046] The offspring generated by mating GSDMD-NT mice with Alb-Cre mice (a tool mouse with liver-specific expression of Cre recombinase) can be induced with DOX and have the ability to specifically express GSDMD-NT in liver cells. The expressed GSDMD-NT can cause pyroptosis of liver cells. Continuous induction with DOX can cause continuous death of liver cells, gradually form scars, and lead to the occurrence of fibrosis. The construction of GSDMD+ / -; Alb-Cre+ / - mice is specifically implemented as follows:

[0047] First, heterozygous or homozygous GSDMD-NT mice and Alb-Cre mice are obtained through breeding. Then, GSDMD-NT positive mice and Alb-Cre positive mice are crossbred, and the breeding process is as Figure 1 shown. GSDMD+ / -;Alb-Cre+ / - double-positive mice are obtained through gene identification. The gene identification primers are shown in Table 1, and the electrophoresis diagram of the identification results is as Figure 2 shown. B6 is C57BL / 6JGpt, the negative control; N is the negative blank control, the control without template; P is the positive control; the numbers are the mouse tail numbers; Result: 13 positive F0 mice are obtained, as Figure 2 shown. Mice numbered 1587#, 1588#, 1589#, 1591#, 1592#, 1594#, 1596#, 1599#, 1602#, 1605#, 1609#, 1610#, 1611# are the positive mice correctly constructed with GSDMD+ / -; Alb-cre+ / -.

[0048] Table 1 Primers for identifying GSDMD+ / -; Alb-Cre+ / - mice

[0049]

[0050] Example 2 Liver injury induced by different concentrations of DOX in GSDMD+ / -; Alb-Cre+ / - mice

[0051] 1. Evaluate liver injury in GSDMD+ / -; Alb-Cre+ / - mice with different concentrations of DOX (2000 μg / mL, 20 μg / mL, 18 μg / mL, 15 μg / mL, 10 μg / mL, 5 μg / mL, 0.5 μg / mL, 0.05 μg / mL). The DOX concentrations are 2 mg / mL and 15 μg / mL respectively, and the DOX administration method is intraperitoneal injection.

[0052] 2. Group GSDMD+ / -; Alb-Cre+ / - mice and induce them with DOX as follows:

[0053] Twenty-seven 8-week-old male GSDMD+ / -; Alb-Cre+ / - mice were used. The experiment was divided into 9 groups with 3 mice in each group. The specific grouping and treatment are shown in Table 2. After 3 weeks of DOX induction by drinking water in the mice except those in Group G1, mouse serum was collected to detect liver function indicators ALT and AST.

[0054] Table 2 Grouping of GSDMD+ / -; Alb-cre+ / - mice induced with liver injury at different DOX concentrations

[0055]

[0056] Note: CD indicates normal diet, and the mice were fed with ordinary feed.

[0057] The detection results are as Figure 3 shown. The mice in Group G1 showed a moribund state on the second day after being induced with 2000 μg / mL DOX by drinking water, and the serum total ALT and AST values were extremely high, indicating that the mice in Group G1 had acute liver injury. For the mice in the remaining groups, the ALT and AST values increased with the increase in the DOX concentration.

[0058] Example 3 Induction of liver fibrosis in GSDMD+ / -; Alb-cre+ / - mice by DOX

[0059] Considering the safety of GSDMD+ / -; Alb-cre+ / - mice and the effectiveness of inducing liver fibrosis, if the concentration is too high, the mice are prone to death, and if it is too low, the phenotypic effect of liver fibrosis cannot be induced. Therefore, the preferred DOX concentration for inducing liver fibrosis in GSDMD+ / -; Alb-cre+ / - mice is 15 μg / mL for implementation. The experimental grouping design is shown in Table 3. The mouse strains are C57BL / 6, GSDMD+ / - and GSDMD+ / -; Alb-Cre+ / - respectively. Liver pathological sections were taken from mice at different time points to observe the level of liver fibrosis. After the experiment started, the status of the mice was observed, and the mice were euthanized in batches 2 weeks, 4 weeks, 6 weeks and 9 weeks after drinking DOX water. The liver tissues were collected for fixation and paraffin embedding, and the sections were stained with HE to evaluate the liver fibrosis situation.

[0060] Table 3 Induction of liver fibrosis in GSDMD+ / -; Alb-cre+ / - mice by DOX

[0061]

[0062] Note: CD indicates normal diet, and the mice were fed with ordinary feed.

[0063] Data showed that under the condition of 15 μg / mL DOX drinking water concentration, GSDMD+ / -; Alb-Cre+ / - mice would develop liver fibrosis symptoms around 6 weeks, and the Sirius red pathological sections of the mice were as Figure 4 shown.

[0064] Example 4 High-fat diet accelerates liver fibrosis induction in GSDMD+ / -; Alb-Cre+ / - mice

[0065] The experimental grouping design for the experiment of high-fat diet accelerating liver fibrosis induction in GSDMD+ / -; Alb-Cre+ / - mice is shown in Table 4.

[0066] Table 4 High-fat diet accelerates liver fibrosis induction in GSDMD+ / -; Alb-cre+ / - mice

[0067]

[0068] Note: CD is normal diet, fed with ordinary feed.

[0069] On the basis of 15 μg / mL DOX drinking water concentration, a high-fat diet was added and observed for 6 weeks. The mice in the high-fat diet group developed liver fibrosis phenotypes after 2 weeks, and the pathological results of Sirius red staining of the liver were as Figure 5 shown. GSDMD+ / -; Alb-Cre+ / - mice showed obvious liver fibrosis phenotypes after 6 weeks of DOX induction. GSDMD+ / -; Alb-Cre+ / - mice showed obvious liver fibrosis phenotypes 2 weeks after induction with DOX plus high-fat diet. Compared with induction with only 15 μg / mL DOX drinking water, the induction of fibrosis in mice with DOX combined with high-fat diet was faster and the degree of fibrosis was higher.

[0070] Example 5 Comparison with the classical CCl4-induced liver fibrosis disease model

[0071] The DOX-induced GSDMD+ / -; Alb-Cre+ / - mouse liver fibrosis model was compared with the classical CCl4-induced liver fibrosis disease model. The specific implementation steps were as follows: GSDMD+ / -; Alb-Cre+ / - mice were induced with 15 μg / mL DOX drinking water. The specific grouping of 15 μg / mL DOX drinking water induction was shown in Table 5:

[0072] Table 5 Comparison between GSDMD+ / -; Alb-Cre+ / - mice and the classical CCl4-induced liver fibrosis disease model

[0073]

[0074] Note: CD is normal diet, fed with ordinary feed.

[0075] Compared with the classical CCl4 liver fibrosis model, the pathological results of the liver fibrosis animal model described in the present invention after Sirius red staining are significantly different from those of the CCl4-induced liver fibrosis. As Figure 6 shown, the collagen in the CCl4-induced liver fibrosis model is centered on blood vessels and shows a divergent state; GSDMD+ / -; Alb-Cre+ / - mice showed obvious liver fibrosis phenotypes after 2 weeks of induction under the condition of 15 μg / mL DOX plus high-fat diet (as shown in Figure 5 Group G2 in). The collagen in the liver fibrosis animal model described in the present invention shows a diffuse state, which is closer to the pathological sections of liver fibrosis patients in this field.

[0076] Application of the liver fibrosis animal model obtained by the method of inducing GSDMD+ / -; Alb-Cre+ / - mice with DOX in the study of liver fibrosis mechanism and / or drugs.

[0077] Based on GSDMD+ / -; Alb-Cre+ / - mice, the animal model of liver fibrosis disease induced by a certain concentration of DOX can be used for the study of the mechanism of liver fibrosis disease and the evaluation of drugs for alleviating or reversing the progression of liver fibrosis. For example, it can be used for the efficacy evaluation of obeticholic acid in alleviating liver fibrosis, as Figure 7 shown. Figure 8 For the pathological sections evaluated by obeticholic acid, Group G1 is the complete control group (blank vector group), Group G2 is the comparative experimental group (adding DOX without obeticholic acid), Group G3 is the obeticholic acid treatment group (adding DOX and obeticholic acid), and Groups G1-G3 are all on normal diet (CD). After 4 weeks of treatment with obeticholic acid, the liver sections of GSDMD+ / -;Alb-Cre+ / - 15 μg / ml + OCA group (Group G3) mice showed improved liver fibrosis (in the comparative experimental group (Group G2) and the treatment group (Group G3), Masson staining showed a decrease in collagen fibers, and SR staining also showed a decrease in collagen fibers), and a reduction in steatosis (oil red staining showed a decrease in hepatocyte fat accumulation and a reduction in steatosis in the treatment group). Figure 9Immunohistochemical pictures were evaluated for obeticholic acid. Group G1 was the complete control group (blank vector group), Group G2 was the comparative experimental group (adding DOX without obeticholic acid), and Group G3 was the obeticholic acid treatment group (adding DOX and obeticholic acid). Groups G1 - G3 were all on a normal diet (CD). Before treatment, in the GSDMD+ / -;Alb-Cre+ / - 15 μg / ml + Vehicle group (Group G2), hepatic stellate cells were activated and collagen synthesis increased; macrophage infiltration in the liver showed chronic inflammation of the tissue. After obeticholic acid treatment, in the GSDMD+ / -; Alb-Cre+ / - 15 μg / ml + OCA group (Group G3), activation of hepatic stellate cells decreased and macrophage infiltration improved.

[0078] In the above liver fibrosis model, liver fibrosis in the mice of the drug treatment group was alleviated by obeticholic acid.

[0079] The above data indicate that the present invention expresses GSDMD-NT specifically in hepatocytes of GSDMD+ / -; Alb-Cre+ / - double-positive mice induced by DOX. Induced by DOX, it has the ability to specifically express GSDMD-NT in liver cells. The expressed GSDMD-NT can cause pyroptosis of liver cells. Continuous induction with DOX can cause continuous death of liver cells, gradually form scars, and lead to the occurrence of fibrosis. Furthermore, on the basis of a DOX concentration of 15 μg / mL, adding a high-fat diet can further promote the process of liver fibrosis, shorten the modeling time, and achieve liver fibrosis of different degrees and liver fibrosis modeling at different speeds, which is an ideal animal model of liver fibrosis.

[0080] Example 7 Induction of non-alcoholic steatohepatitis (NASH) in GSDMD+ / -; Alb-cre+ / - mice by DOX

[0081] On GSDMD+ / -; Alb-cre+ / - mice, different concentrations of DOX and high-fat diet were added at different times for induction to explore the NASH modeling conditions. The data show that after feeding a 60% high-fat diet for 8 weeks and then adding DOX to the drinking water, with a DOX concentration of 0.05 - 20 μg / mL and changing the concentration every three days, GSDMD+ / -; Alb-Cre+ / - mice will show NASH symptoms. SR pathological staining is as Figure 10 shown. Group G1 was the control group, that is, B6 mice on a normal diet (CD) without adding DOX. Group G2 was fed a 60% high-fat diet and showed obvious NASH phenotypes 21 days after DOX treatment, and the DOX concentration was 15 μg / mL at this time.

[0082] The above are only the preferred embodiments of the present invention, and do not impose any form of limitation on the present invention. Based on the technical essence of the present invention, any simple modifications, equivalent replacements, and improvements made to the above embodiments within the spirit and principles of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A method for constructing an animal model of liver fibrosis, characterized in that, It includes the following steps: S1. Breed GSDMD-NT mice with Alb-Cre mice to obtain GSDMD+ / -; Alb-Cre+ / - mice; S2. Induce GSDMD+ / -; Alb-Cre+ / - mice with DOX at a concentration of 15 μg / mL and add a high-fat diet to obtain a liver fibrosis animal model; In the step S1, the Alb-Cre mice are mice with specific expression of Cre recombinase in liver cells; The GSDMD+ / -; Alb-Cre+ / - mice are double-positive mice obtained by breeding GSDMD-NT mice containing the CRE-LOXP system with Alb-Cre mice.

2. The construction method of a liver fibrosis animal model according to claim 1, characterized in that, Take liver tissues of the induced GSDMD+ / -; Alb-Cre+ / - mice at different time points for pathological sectioning to observe the level of liver fibrosis.

3. A method for constructing an animal model of non-alcoholic steatohepatitis, characterized in that, It includes the following steps: S1. Breed GSDMD-NT mice with Alb-Cre mice to obtain GSDMD+ / -; Alb-Cre+ / - mice; S2. First, feed GSDMD+ / -; Alb-Cre+ / - mice with a high-fat diet, and then add DOX at a concentration gradient of 0.05 - 20 μg / mL to induce GSDMD+ / -; Alb-Cre+ / - mice. Change the concentration every three days. Take liver tissues of the induced GSDMD+ / -; Alb-Cre+ / - mice at different time points for pathological sectioning to observe the level of hepatitis and obtain a non-alcoholic steatohepatitis animal model.

4. The method for constructing a non-alcoholic steatohepatitis animal model according to claim 3, characterized in that, In the step S1, the Alb-Cre mice are mice with specific expression of Cre recombinase in liver cells; the GSDMD+ / -; Alb-Cre+ / - mice are double-positive mice obtained by breeding GSDMD-NT mice containing the CRE-LOXP system with Alb-Cre mice.

5. Application of the liver fibrosis animal model obtained by the method for constructing a liver fibrosis animal model described in claim 1 in studying the mechanism of liver fibrosis or for evaluating the efficacy of drugs or pharmacological experiments.

6. Application of the non-alcoholic steatohepatitis animal model described in claim 3 in studying the mechanism of non-alcoholic steatohepatitis or for evaluating the efficacy of drugs or pharmacological experiments.

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