阅读说明：本技术 益母草碱用于预防急性肝损伤以及促进肝组织修复与再生 (Leonurine for preventing acute liver injury and promoting liver tissue repair and regeneration ) 是由 王瑞 王余果 余捷妮娜 于 2020-02-13 设计创作，主要内容包括：本发明公开了益母草碱用于制备预防急性肝损伤药物和促进肝组织复与再生的药物中的应用。所述益母草碱对肝组织具有保护作用,能够预防急性肝损伤并能促进肝组织修复与再生。(The invention discloses application of leonurine in preparing a medicament for preventing acute liver injury and promoting liver tissue regeneration. The leonurine has protective effect on liver tissue, can prevent acute liver injury and can promote liver tissue repair and regeneration.)

1. Application of leonurine in preparing medicine for preventing acute liver injury and relevant diseases.

2. Application of leonurine in preparation of medicine for promoting liver tissue repair and regeneration is provided.

3. Application of leonurine in preparing medicine for preventing hepatic fibrosis and related diseases is provided.

4. Use of leonurine in preparing medicine for treating hepatic fibrosis and related diseases is provided.

5. Use according to claim 4, wherein leonurine is used to block mild liver fibrosis.

6. The use according to claim 4, wherein leonurine is used to inhibit moderate liver fibrosis progression.

7. The use according to claim 4, wherein the leonurine is used to reduce the severity of severe liver fibrosis.

8. According toThe use as claimed in any one of claims 1 to 7 wherein the leonurine is leonurine

Technical Field

The invention relates to a new application of leonurine, in particular to an application of leonurine in preparing a medicine for preventing acute liver injury and a medicine for promoting liver tissue repair and regeneration.

Background

With the evolution of human living environment and mode, the incidence of acute and chronic liver diseases worldwide is increasing year by year. According to the literature report, SARS (SARS) -Cov and SARS-CoV-2 can directly combine with ACE2 positive bile duct cell to damage liver tissue. Acute liver injury caused by virus (SARS-Cov, SARS-Cov-2) infection, systemic inflammatory factor storm, and drug (such as aristolochic acid, acetaminophen, etc.) induction occurs continuously. Liver Fibrosis (LF) is a pathological process that is inevitable for all chronic Liver diseases, and is also a pathological state with different branches of chronic Liver injury caused by various reasons (such as viral infection, metabolism or autoimmune diseases). Hepatic fibrosis refers to the diffuse deposition and abnormal distribution of extracellular matrices such as collagen and glycoprotein in the liver after liver cells are repeatedly destroyed. Hepatic fibrosis is the pathological reaction of liver to chronic injury, and is also the key step in the process of the development of various chronic liver diseases to cirrhosis and the important link influencing the prognosis of liver diseases. The further development of hepatic fibrosis can cause the structural disorder of liver tissues, the nodular regeneration of hepatic cells and the formation of false lobules, namely cirrhosis; some patients even cause liver cancer. Once liver fibrosis develops into cirrhosis, the disease is irreversible, the only effective treatment is liver transplantation, and the shortage of liver donors and high medical cost limit clinical application. Friedman, Hepatology, International publication on liver disease Authority, noted that liver fibrosis in animals could be reversed and was also confirmed in humans. Therefore, how to effectively resist hepatic fibrosis becomes the key for treating chronic liver diseases.

Leonurine (SCM-198) is the main bioactive monomer component in Leonurus japonicus of Labiatae family recorded in the pharmacopoeia of the people's republic of China (2000 edition). The current applications are limited to uterine blood, myocardial ischemia, atherosclerosis and diabetes.

Disclosure of Invention

The invention aims to provide a new application of leonurine.

According to one aspect of the invention, the application of leonurine in preparing a medicament for preventing acute liver injury and related diseases thereof is provided.

According to another aspect of the present invention, there is provided a use of leonurine in the preparation of a medicament for promoting liver tissue repair and regeneration.

According to another aspect of the present invention, there is provided a use of leonurine in preparing a medicament for preventing liver fibrosis and related diseases.

According to another aspect of the present invention, there is provided a use of leonurine in the preparation of a medicament for treating liver fibrosis and related diseases.

Wherein the leonurine is used for blocking mild hepatic fibrosis.

Wherein the leonurine is used for inhibiting moderate liver fibrosis progression.

Wherein the leonurine is used for reducing the degree of severe liver fibrosis.

In the invention, the chemical structure of the leonurine is

The leonurine has protective effect on liver tissue, can prevent acute liver injury and can promote liver tissue repair and regeneration.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the specification.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present specification and together with the description, serve to explain the principles of the specification.

FIG. 1a is a diagram showing the chemical structure of SCM-198;

FIG. 1b is a graph showing the results of measuring the lactate dehydrogenase content of L02 cells;

FIG. 1c is a graph showing the results of CCK-8 assay of L02 cell activity;

FIG. 1d is a graph showing the results of detection of Caspase-3 protein activity in L02 cells;

FIG. 1e is a graph showing the results of TUNEL staining experiments with L02 apoptosis;

FIG. 2a is a graph showing survival of acute liver injury within 1.5 hours after intraperitoneal injection of TAA;

FIG. 2b is a graph showing survival of acute liver injury within 2.5 hours after intraperitoneal injection of TAA;

FIG. 2c is a graph showing body weight in mice with acute liver injury;

FIG. 2d is a diagram showing a pathological section of acute liver injury;

FIG. 2e is a graph showing serum biochemical marker levels in mice with acute liver injury;

FIG. 3a is a graph showing the results of an LX-2 cell migration assay;

FIG. 3b is a graph showing the result of LX-2 cell Edu staining;

FIG. 3c is a graph showing the results of an assay for alpha-SMA protein content in LX-2 cells;

FIG. 4a is a diagram showing ultrasonic digital image biopsy of mild liver fibrosis mice;

FIG. 4b is a diagram showing pathological sections of mild liver fibrosis;

FIG. 4c is a graph showing the levels of serum biochemical indicators in mild liver fibrosis mice;

FIG. 4d is a graph showing body weight of a mouse with mild liver fibrosis;

FIG. 5a is a graph showing digital ultrasound image biopsy of a mouse with moderate liver fibrosis;

FIG. 5b is a graph showing pathological sections of a mouse with moderate liver fibrosis;

FIG. 5c is a graph showing the levels of serum biochemical markers in mice with moderate liver fibrosis;

FIG. 5d is a graph showing moderate liver fibrosis mouse body weight;

FIG. 6a is a diagram showing ultrasound digital image biopsy of severe liver fibrosis mice;

FIG. 6b is a diagram showing pathological sections of a mouse with severe liver fibrosis;

FIG. 6c is a graph showing the levels of serum biochemical markers in mice with severe liver fibrosis;

FIG. 6d is a graph showing body weight of a mouse with severe liver fibrosis;

FIG. 7a is a diagram showing a pathological section for liver tissue regeneration in a mouse with severe liver fibrosis;

FIG. 7b is a graph showing the result of staining of liver tissue regeneration Edu in a mouse with severe liver fibrosis;

FIG. 7c is a graph showing the results of the measurement of serum albumin content in mice with severe liver fibrosis;

FIG. 8a is a diagram showing in vivo ultrasound digital image examination of an unmolded mouse;

FIG. 8b is a diagram showing pathological sections of unmolded mice;

FIG. 8c is a graph showing the levels of serum biochemical markers in unmolded mice.

Detailed Description

Acute liver injury caused by virus (SARS-Cov, SARS-Cov-2) infection, systemic inflammatory factor storm, and drug (such as aristolochic acid, acetaminophen, etc.), and there is no specific liver protection drug in clinic. The treatment of chronic liver disease is mainly divided into two aspects of primary pathogenesis and anti-fibrosis. From the viewpoint of treating primary diseases, not only are the causes of diseases (virus, metabolism, immunity, etc.) diverse, but also, a great deal of basic and clinical practices prove that even after the causes of diseases are removed, hepatic fibrosis still progresses, and even liver cirrhosis or liver cancer progresses. The reason is that when the liver is damaged, hepatic stellate cells are stimulated by paracrine, transformed into myofibroblasts, and synthesized extracellular mechanisms increase. Even if paracrine action is gradually lost after removing the cause, hepatic stellate cells can still be self-activated by autocrine, and paracrine of neighboring hepatic stellate cells, promoting the persistence and progression of hepatic fibrosis. From the viewpoint of anti-fibrosis, no specific medicine for treating hepatic fibrosis exists. Once liver fibrosis progresses to cirrhosis, the disease is irreversible. There are no effective therapeutic drugs and therapeutic strategies other than liver transplantation, and the shortage of liver donors and high medical expenses limit the clinical application of liver transplantation.

In order to solve the problems, the invention provides a new application of leonurine (SCM-198) in preventing acute liver injury and promoting liver tissue repair and regeneration.

In the invention, pharmacodynamic experiments developed by in vitro cell pharmacodynamic experiments and in vivo animal pharmacodynamic experiments prove that the SCM-198 can effectively prevent acute liver injury and remarkably promote the liver tissue repair and regeneration of experimental animals.

SCM-198 is a compound synthesized based on the chemical structure of leonurine, a main bioactive monomer component of Chinese herbal medicine motherwort, and the molecular formula of the compound is as follows: c₁₄H₂₁N₃O₅(ii) a The molecular weight is: 311, the structural formula is shown in FIG. 1 a.

The invention carries out pharmacodynamic experiments through in vitro cell models and in vivo animal models. First, 3% Hydrogen peroxide (H) was used₂O₂) Preparation of injured human liver parenchymal cell line L02 cellThe results of experiments on the acute liver injury cell model show that SCM-198 can remarkably promote the survival of L02 cells, show that the survival rate of L02 cells is improved, the activity of caspase-3 (cysteine aspartic protease-3, caspase-3) is inhibited, L02 cell apoptosis is inhibited, a hepatic fibrosis cell model is prepared by stimulating human hepatic stellate cell line LX-2 cells with human Transforming growth factor- β 1 (transforminggrowth factor- β 1, TGF- β 1) and shows that the SCM-198 can remarkably inhibit the activation of hepatic liver-2 cells, show that LX-2 cell migration and proliferation are inhibited, and promote the apoptosis of human hepatic stellate cell line LX-2 cells, and down-regulate α -actin (Alacoothromucotactin, α -actin) expression, an SCM-198 tissue regeneration-198 and a liver fibrosis regeneration-tissue regeneration-related drug is prepared by using SCM-198, and particularly has the effects of remarkably preventing liver fibrosis and repairing liver tissue regeneration related to treat liver fibrosis with acute liver injury, chronic liver injury, liver cirrhosis prevention of mice and liver cirrhosis.

The medicine can only contain the SCM-198 as an active ingredient, also can contain the SCM-198 and other medicinal ingredients with the same functions, and also can contain the SCM-198 as the active ingredient and pharmaceutically acceptable auxiliary agents.

The medicament can be administered orally or intravenously. The person skilled in the art can determine the dosage to be administered according to the actual circumstances.

The medicine can be made into injection, tablet, pill, dispersant, soft capsule, dripping pill, granule, solution, suspension, emulsion, etc.

For the purpose of facilitating understanding, the present invention will be described in detail below with reference to specific embodiments thereof, which are illustrated in the accompanying drawings. It is specifically intended that the detailed description and drawings be regarded as illustrative only.

Primary reagent

SCM-198 Anhui Bomei Biochemical company; thioacetamide Biopsis (Shanghai) Inc.; EDTA antigen retrieval liquid google bio, wuhan; citric acid antigen retrieval liquid wuhan google bio; HRP-labeled anti-IgGPerkin Elmer; DAPI Invitrogen corporation; chemical reagent company, paraformaldehyde, national drug group; antibody dilutions Perkinelmer; chemical reagent company, national ethanol Water free; xylene national chemical group chemical agents corporation; cell culture grade PBS beijing solibao science and technology; perkinelmer Corp for confining liquids; anti-fluorescence quenching mounting medium, vectorelabs, usa; human TGF-. beta.1 MEC; 0.5% TritonX-100, Beijing Soilebao; TBST, Beijing Soilebao.

Preparation of test reagent

1. Preparing a paraformaldehyde solution (4%): ddH₂Heating O (900ml) + NaOH (500mg) to 65-70 deg.C under stirring, adding 40g paraformaldehyde, heating and stirring to dissolve, cooling at room temperature, adding 100ml 10 × PBS and HCl to pH 7.3, and storing at room temperature.

2. Thioacetamide (TAA) formulation: the TAA is dissolved by normal saline to ensure that the dosage of the TAA is 200mg/kg, and the TAA is fully and uniformly mixed for later use.

Main experimental apparatus and equipment

A pipettor Eppendorf; common optical microscope Olympus; laser scanning confocal microscope Leica (SP 5); precision electronic balance Sartorius LA310 s; a multifunctional microplate reader Tecan (Infinite M200); a low temperature centrifuge Eppendorf; fully automated intelligent section analysis system perkinelmer (vectra); lecia of Paraffin microtome; electric heating constant temperature air-blast drying oven Shanghai Fuma laboratory appliances Co., Ltd; refrigerator at 4 ℃ China sea corporation; -80 ℃ ultra low temperature freezer refrigerator Thermo company, usa; ultra pure water unit Millex corporation, usa; plate washer BioTek company, USA.

Experimental methods

Firstly, establishing a cell model

1. Cell cultureCulturing: human hepatocyte cell line L02 and human hepatic stellate cell line LX-2 were purchased from Shanghai Life research institute of Chinese academy of sciences. Washing the culture dish with cell culture grade PBS, adding trypsin to digest cells, adding fresh complete culture medium to stop digesting cells, blowing to beat cells, transferring to a new cell culture dish, placing at 37 deg.C and 5% CO₂The cultivation is continued in the incubator.

2. Acute liver injury cell model L02 cells in logarithmic growth phase were collected by trypsinization at 5 × 10⁴Cells/ml were plated in 12-well plates and after 48 hours of culture, cells were grouped as follows:

normal group: cells were cultured normally without any drug addition.

Placebo group: use of 3% H₂O₂Cells were treated for 2 hours to construct a model of acute oxidative damage of L02 cells. When using 3% H₂O₂The cell lethality rate was approximately 50% after 2 hours of treatment. And treated with placebo (solvent for dissolution of SCM-198) for 24 hours.

SCM-198 group: use of 3% H₂O₂Cells were treated for 2 hours to construct a model of acute oxidative damage to L02 cells and then treated with SCM-198 for 24 hours.

3. Liver fibrosis cell model LX-2 cells in logarithmic growth phase are digested with trypsin, collected and expressed as 5 × 10⁴Cells/ml were seeded in 12-well plates and after 48 hours of culture, cells were grouped as follows:

normal group: cells were cultured normally without any drug addition.

Placebo group: TGF-. beta.1 (5ng) and placebo (the solvent used to dissolve SCM-198) were co-treated for 24 hours.

SCM-198 separate group: SCM-198 was treated alone for 24 hours.

SCM-198 group: TGF-. beta.1 (5ng) was co-treated with SCM-198 for 24 hours.

4. Lactate dehydrogenase content detection

(1) Cell collection: suspending the cells to be detected by PBS, homogenizing, centrifuging and collecting supernatant;

(2) adding cells to be detected and reagents: adding reagents (absolute ethyl alcohol with the same volume as the supernate to be detected, 3ml of thiobarbituric acid and 1ml of trimethyne) into the supernate to be detected, mixing uniformly by vortex, fastening the opening of a test tube by a preservative film, pricking a small hole by a needle, and carrying out boiling water bath for 40 minutes;

(3) cooling, 12000rpm, and centrifuging for 5 minutes;

(4) taking the supernatant, and centrifuging at 12000rpm again for 2 minutes;

(5) the supernatant was collected and absorbance was measured at 532nm for each tube.

5. Cell migration assay

(1) Culturing the cells until the cells are fused;

(2) after washing with PBS, starving with 1% serum overnight;

(3) making scratches on the cells, washing the cells once with PBS, and taking pictures under a microscope;

(4) after adding the medicine with the required concentration, continuing to culture for 24 hours;

(5) images taken after 24 hours were compared with images taken of the same field of view 24 hours ago.

6. Cell proliferation (Edu method) assay

(1) Cell culture, taking cells in logarithmic growth phase, and culturing at 5 × 10 per well⁴Inoculating the cells in a 12-hole plate, and culturing to a normal growth stage;

(2) and (3) drug treatment: carrying out drug treatment according to experimental requirements;

(3) edu marking: the reaction mixture was mixed with PBS at a ratio of 1: a ratio of 1000 dilutes Edu solution. The cell or tissue section is incubated for a short time (<2 hours) at a high concentration of 10-50 muM and for a long time (>24 hours) at a low concentration of 1-10 muM;

(4) fixing cell or tissue section;

(5) DAPI staining of cells or tissue sections;

(6) an image is captured.

7. CCK-8 cell activity assay

(1) Seeding a cell suspension in a 96-well plate for cytotoxicity experiments with about 5000 cells per well;

(2) the specific drug stimulation concentration and time are determined according to experimental needs.

(3) After drug treatment of the cells, 10. mu.l of CCK-8 reagent (using the corresponding amounts of cell culture medium and CCK-8 reagent, but wells without cells as blank; using the corresponding amounts of cell culture medium and CCK-8 reagent, but wells without drugs as no-drug controls) was added to each well;

(4) incubation was continued for 4 hours in the cell incubator.

(5) The absorbance at 450nm was measured with a microplate reader.

8. Caspase-3 protein activity assay

(1) Cells were digested with trypsin and cell culture fluid was collected. Centrifuge at 1000rpm for 5 minutes at 4 ℃ and remove the supernatant. Adding 20 mul cell lysate, re-suspending the precipitate, and lysing the cells in ice bath for 15 minutes;

(2) centrifuging at 12000rpm at 4 ℃ for 15 minutes;

(3) transferring the supernatant into a precooled centrifuge tube;

(4) taking out Ac-DEVD-rho NA (2mM) and placing the Ac-DEVD-rho NA on an ice bath for later use;

(5) Ac-DEVD-. rho.NA (2mM) was added, mixed well and incubated at 37 ℃ for 2 hours.

(6) And subtracting the blank control A405 from the A405 (absorbance) of the sample to be detected, namely obtaining the absorbance generated by the rho NA generated by Caspase-3 catalysis in the sample.

9. Cellular immunofluorescence assay

(1) Soaking and washing the cultured cell-carried fragment of the cell to be detected with PBS for 3 times, 3 minutes each time;

(2) fixing the cell-carried fragment with 4% paraformaldehyde for 15 min, and washing the cell-carried fragment with PBS solution for 3 times, each for 3 min;

(3)0.5 percent TritonX-100 is transparent for 20 minutes at room temperature;

(4) soaking and washing the cell-carried fragment with PBS for 3 times, 3 minutes each time, sucking dry PBS, dripping goat serum on the cell-carried fragment, and sealing at room temperature for 30 minutes;

(5) removing the confining liquid, dropwise adding primary antibody (1: 200 for dilution), and incubating in a wet box at 4 deg.C overnight;

(6) TBST immersion washing cell-carried fragment 3 times, each time for 3 minutes, removing excess liquid, dripping fluorescent secondary antibody (diluted 1: 1000), incubating for 1 hour at room temperature, TBST immersion washing 3 times, each time for 3 minutes;

(7) adding DAPI dropwise, incubating for 5 minutes in a dark place, and washing for 5 minutes for 4 times by TBST;

(8) sucking the liquid on the stem cell fragment, sealing the fragment with a sealing liquid containing an anti-fluorescence quencher, observing under a fluorescence microscope, and collecting images.

10. Apoptosis (TUNEL assay) detection

(1) Cells or tissue sections seeded in 24-well plates were rinsed with PBS for 5 minutes. Fixing with pre-cooled 4% paraformaldehyde at 4 deg.C for 30 min, rinsing with PBS for 3 times, 5 min each time, adding PBS containing 0.5% TritonX-100, and ice-cooling for 5 min;

(2) to the cells or paraffin sections 50. mu.l of 3% H are added dropwise₂O₂Standing at room temperature in dark for 10 minutes;

(3) immersing the cell or paraffin section into PBS, and placing for 5 minutes at room temperature in a dark place;

(4) positive control (i.e. placebo TUNEL/DAPI): 50. mu.l of positive control buffer (5. mu.l of 10 XDnase I mixed with 45. mu.l of 1 XDnase I buffer) was added dropwise thereto, and the mixture was allowed to stand at room temperature for 10 minutes, immersed in PBS, and left at room temperature for 5 minutes;

(5) negative control (i.e., normal TUNEL/DAPI): tunel reaction buffer (45. mu.l of 1 × Label Substrate mixed with 5. mu.l of deionized water) without 10 × Enzyme Reagent was added dropwise and incubated for 90 min at 37 ℃ in the absence of light;

(7) immersing in PBS, and standing at room temperature for 5 minutes;

(8) and (3) observation by a fluorescence microscope: cells or tissue sections were counterstained for nuclei with DAPI for 5 minutes, mounted with an anti-fluorescence quencher, excited at 488nm and immediately observed.

Second, establishment of mouse acute and chronic liver injury model

The animal model of acute and chronic liver injury plays an important role in researching the potential mechanism and treatment related to the disease. Currently, acute and chronic liver injury models employ hepatotoxins, such as carbon tetrachloride (CCl)₄) Or TAA intraperitoneal injection, or Common Bile Duct Ligation (CBDL) to prepare acute and chronic liver injury models. The mechanism of TAA damage to parenchymal cells of liver tissue is: TA (TA)A is a thiol-sulfur containing compound that undergoes further metabolism to acetamide and TAA-S-oxide. Acetamide has no hepatonecrosis effect, while TAA-S-oxide continues to be further metabolized by cytochrome P450 monooxygenase to produce a polar product (TAA-S-titanium oxide) called "sulfa-methyl pyrimidine (sulfene)", which is chemically very active. This metabolite can bind to macromolecules in hepatocytes, generate large amounts of oxygen Radicals (ROS), interfere with mitochondrial activity, and cause apoptosis, necrosis, and elevated serum cytokine levels in hepatocytes. First, trauma is involved in the procedure of preparing the common bile duct ligation model, and liver fibrosis that may be induced in this model is insignificant or minimal, and the CBDL model is completed in a short period (typically 3-5 days) as a result of chronic liver injury. In contrast, hepatotoxin caused by liver fibrosis is very stable once formed, and adverse effects of circulating hepatotoxin can be minimized by simply stopping intraperitoneal injection of a molding agent for more than four weeks. Second, with CCl₄Compared with the prior art, TAA-induced hepatic fibrosis has lower mortality, more obvious hepatic injury and more prominent regeneration nodules, and the histology of the hepatic fibrosis is closer to that of human chronic hepatic injury. Therefore, in this example, the internationally accepted modeling method, i.e., continuous/every other day intraperitoneal injection of TAA, was used to prepare acute and chronic liver injury models. The dose of TAA used was 200 mg/kg. In the preparation of acute liver injury models, TAA was injected intraperitoneally once a day for a week. In the preparation of the chronic liver injury model, TAA is injected into the abdominal cavity once every other day for four weeks, eight weeks and twelve weeks according to different experimental periods.

Experimental animals: c57BL/6 mouse, 8 weeks old, weight 20-22 g; purchased from Shanghai Ling Biotech, Inc. Feeding conditions are as follows: the food and the drinking water are fed randomly after 5 pieces of the raw materials are respectively put in a cage, the SPF level is realized, the temperature is kept at 25 ℃, the illumination/darkness is cycled for 12 hours. Feeding places: animal experiment center in Shanxi school zone of Tongji university. Animal management and use: the laboratory animal management and use regulations are strictly followed, which are set by the ethical committee of the college of medicine of the university of Tongji.

1. Acute liver injury experimental animal model

Acute liver failure events caused by viral infections, inflammatory factor storms, or drug induction occur continuously worldwide. N-acetyl cysteine (NAC), an antioxidant, is the most commonly used liver detoxification drug in the clinic.

C57BL/6 mice, 8 weeks old, weighing 20-22g, were randomized into 4 groups:

normal group: normally breeding without any drug treatment.

(II) placebo group: intraperitoneal injection of placebo (solvent for dissolving SCM-198) was added within 1.5 hours and within 2.5 hours after intraperitoneal injection of TAA.

(III) NAC group: NAC (250mg/Kg) was intraperitoneally injected within 1.5 hours and 2.5 hours after the intraperitoneal injection of TAA.

(IV) SCM-198 group: SCM-198(2mg/Kg) was administered intraperitoneally within 1.5 hours and 2.5 hours after the intraperitoneal administration of TAA.

Acute liver injury was induced by intraperitoneal injection of TAA (200mg/Kg) once a day, and continued until the end of the experiment. The first experiment was a survival experiment for mouse survival analysis. The second experiment was a pathological one and was used for pathological analysis of mice. During the second experiment, 4 days after TAA induction, the remaining mice were all sacrificed and liver tissue from all sacrificed mice was harvested for pathological analysis.

2. Experimental animal model for chronic liver injury

(1) Preparation of hepatic fibrosis experimental animal model

Mild liver fibrosis experimental animal model: mice received every other day intraperitoneal injections of TAA, and after four weeks of molding, developed marked fibroplasia from the central venous or the junction to the peripheral areas, and junction-junction (P-bridge) and junction-central venous (P-C-bridge) were observed. The hepatic fibrosis stage Ishak index is 3, belonging to the S1 stage mild hepatic fibrosis. Therefore, the mild hepatic fibrosis of the mouse is successfully modeled.

Moderate hepatic fibrosis experimental animal model: after the mice receive the intraperitoneal injection of TAA in the next day, the mice are subjected to model building for eight weeks, the mice expand from the central vein or the junction area to the peripheral area, obvious fibrous tissue hyperplasia appears, and P-P bridging and P-C bridging are obvious. The hepatic fibrosis stage Ishak index is 4, belonging to the middle-degree hepatic fibrosis of S2 stage. Therefore, the mouse is successfully modeled after moderate hepatic fibrosis.

Severe liver fibrosis experimental animal model: after the mice receive intraperitoneal injection of TAA in the alternate days, twelve weeks after modeling, a large amount of fiber intervals can be formed in liver tissues, and liver lobules are separated and damaged, so that the lobules are disordered in structure, liver cirrhosis does not appear, and portal hypertension appears in part of the mice. The hepatic fibrosis stage Ishak index is 5, belonging to S3 stage severe hepatic fibrosis. Therefore, the mouse severe hepatic fibrosis model is successfully made.

(2) Grouping of chronic liver injury experimental animal models

According to the hepatic fibrosis experimental animals of each molding period, randomly dividing the experimental animals into 3 groups:

normal group: normally breeding without any drug treatment;

(II) placebo group: according to different experimental periods, after the TAA modeling is successfully injected intraperitoneally, the placebo (solvent for dissolving the SCM-198) is injected intraperitoneally;

(III) SCM-198 group: according to different experimental periods, after the TAA is injected into the abdominal cavity for modeling successfully, SCM-198(2mg/Kg) is injected into the abdominal cavity.

Third, collection of mouse blood and liver tissue specimen

1. Collecting a blood sample: mice were anesthetized with isoflurane and blood was removed by the eye-picking method. After collecting whole blood in a 1.5 ml centrifuge tube, the whole blood was allowed to stand at room temperature for 6 hours. 10000g, centrifuging at 4 ℃ for 10 minutes, collecting upper layer light yellow liquid, and storing in a refrigerator at-80 ℃.

2. Liver tissue collection: the mice were soaked in 75% ethanol for a while. The abdominal cavity was exposed, and the whole liver was removed with scissors and forceps and placed in 4% paraformaldehyde at room temperature.

Fourth, biochemical detection of serum

The serum biochemical indexes of the mice, namely alanine transaminase (ALT), aspartate transaminase (AST), Alkaline phosphatase (ALP) and Albumin (Albumin, ALB) are respectively detected by the following steps: alanine aminotransferase detection kit (Bioassay Systems, Cat #: EALT-100); glutamic-oxaloacetic transaminase detection kit (BioAssay Systems, Cat #: EASTR-100); alkaline phosphatase detection kit (BioAssay Systems, Cat #: DALT-250); albumin detection kit (BioAssay Systems, Cat #: DIAG-250). The procedure was carried out according to the above kit operating manual.

Pathological analysis of liver tissue specimen

1. Pathological section:

(1) fixing a sample: after being fixed for 24 hours at room temperature by 4 percent paraformaldehyde, the mixture is placed in a refrigerator at 4 ℃ for standby.

(2) And (3) dehydrating: after being cleaned, the tissue sample is placed in gradient ethanol for dehydration, namely: 75%, 85%, 90%, 95% ethanol, each soaking for 30 min, 10 min, 5 min, 30 min, and repeating 3 times. And finally, soaking in pure ethanol for 60 minutes.

(3) And (3) transparency: the samples were soaked in xylene for 20 minutes to allow the tissue to become transparent.

(4) Paraffin-embedded tissue samples: the samples were sequentially soaked in paraffin for 30 min, 45 min, 60 min and finally embedded in paraffin with a melting point of 58 ℃. After the embedding, the sections were cut to a thickness of about 4nm with a microtome, attached to a slide glass, incubated at 60 ℃ for 2 hours at room temperature overnight, and stored at 4 ℃.

2. Immunohistochemical staining: the cut sections were oven dried at 75 ℃ for 150 minutes to wax drop. Dewaxing treatment is sequentially carried out. Dewaxing was done with xylene for 10 minutes × 2 times, followed by treatment with 100%, 90%, 70% ethanol for 5 minutes each. And soaking in deionized water for 1 minute to remove peroxidase interference.

3. HE staining: paraffin section is dewaxed, hematoxylin is stained for 10 minutes, 1% hydrochloric acid ethanol is differentiated for 5 seconds, the section is washed by deionized water until turning blue, and then ethanol and xylene are sequentially used for dehydration. And finally, sealing the neutral gum.

4. Dyeing with picric acid sirius red: the dyeing is carried out for 1 hour by using a pre-dyeing solution and then the dyeing is rinsed by tap water. Then soaking the mixture in 95% and 100% ethanol for 3 min, and then, transparent xylene for 30 min. Finally, sealing the tablet by using gum.

5. PAS dyeing: soaking with periodic acid at 37 deg.C for 60 min, and washing. Then soaking the mixture in Schiff reagent at 37 ℃ for 60 minutes. Then the mixture is dehydrated by ethanol gradient, and the xylene is transparent. And finally, sealing the neutral gum.

6. Dyeing the masson pine:

(1) hematoxylin staining of cell nucleus: firstly, staining the fabric with hematoxylin for 5 minutes, and washing the fabric with warm water for 10 minutes; the cells were differentiated for 5 seconds with 1% HCl and rinsed with deionized water until the cells turned blue.

(2) Ponceau red staining: stain with ponceau solution for 10 minutes and rinse with deionized water several times for 3 times.

(3) Phosphomolybdic acid differentiation: the phosphomolybdic acid solution differentiated for 15 minutes, leaving the collagen staining no longer red.

(4) And (3) aniline blue dyeing: sections were directly transferred to aniline blue solution for counterstaining for 5 minutes and after rinsing in distilled water for 5 seconds, treated with 1% glacial acetic acid for 1 minute.

(5) Dewatering and sealing: and putting the slices into 95% ethanol, absolute ethanol and xylene in sequence, and removing the slices to be transparent after 5 minutes of each time. Finally, the neutral gum is encapsulated.

7. And (4) analyzing results: liver fibrosis was assessed according to the Ishak system scoring rule (table 1.1). Further analysis and refinement are carried out according to the Beijing standard, wherein the fiber interval Ishak is more than or equal to 3 periods (Table 1.2).

TABLE 1.1 Ishak System Scoring rules

TABLE 1.2 hepatic fibrosis staging criteria (1995, Beijing)

Sixth, statistical analysis

Quantification of immunochemical stained positive cells and positive stained areas of paraffin sections was analyzed using ImagG software. The survival analysis was performed by a time-scale test (log-rank test) and a Wilcoxon test. Experimental results using the two-tailed T-test, P <0.05 indicated statistically significant differences, and experimental data indicated mean ± standard deviation. The statistical plots were plotted by GraphPad Prism 7.0.