Application of oleanolic acid in the preparation of drugs for treating liver fibrosis

Abstract

This invention provides the application of oleanolic acid in the preparation of a drug for treating liver fibrosis. The drug comprises oleanolic acid as the active ingredient and sodium carboxymethyl cellulose as the excipient. Oleanolic acid enters hepatocytes, enhances the binding of SIRT7 protein to ASC protein, and promotes the deacetylation of ASC protein. The deacetylation of ASC protein blocks the assembly and activation of the NLRP3 inflammasome, leading to a decrease in the expression of activated aspartate protease-1 and a reduction in the production of mature interleukin-1β, thereby blocking hepatocyte pyroptosis. The protective effect of oleanolic acid in alleviating liver fibrosis depends on the presence of SIRT7 protein. The drug is formulated as a suspension, and after application, it can reduce serum alanine aminotransferase activity, reduce the degree of inflammatory cell infiltration in the liver parenchyma, and reduce collagen fiber deposition in the portal area and peri-central venous region, thereby alleviating liver pathological changes and providing pharmacological target support.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the application of oleanolic acid in the preparation of drugs for treating liver fibrosis. Background Technology

[0002] Liver fibrosis is a tissue repair response that occurs after chronic liver damage, characterized by the proliferation and deposition of extracellular matrix within the liver tissue. Many chronic liver diseases can induce fibrosis in liver tissue. If left uncontrolled, liver fibrosis will gradually progress to cirrhosis, leading to destruction of the liver parenchyma and liver failure.

[0003] Conventional clinical interventions for liver fibrosis primarily focus on treating the underlying etiology, aiming to slow disease progression by eliminating primary pathogenic factors. In terms of clinical drug application, remission mainly relies on conventional drugs with hepatoprotective, anti-inflammatory, or antioxidant effects. Developing targeted drugs that can directly intervene in and block the pathological signaling pathways of fibrosis is a key research direction in the treatment of this disease.

[0004] In the development and progression of liver fibrosis, persistent damage and death of hepatocytes are driving factors. Abnormal activation of the NLRP3 inflammasome within hepatocytes mediates pyroptosis, which in turn activates hepatic stellate cells and promotes collagen deposition. However, the specific regulatory mechanisms of the NLRP3 inflammasome in hepatocytes remain unclear, particularly regarding the lack of drugs targeting post-translational modification regulation of ASCs. The current lack of effective drugs to inhibit inflammasome assembly and activation at the post-translational modification level makes it difficult for existing interventions to suppress the expression of activated aspartate protease-1 and the production of mature interleukin-1β, thus failing to block the pyroptosis process at the pathogenesis stage. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides the application of oleanolic acid in the preparation of drugs for treating liver fibrosis, solving the technical problem that existing liver fibrosis intervention methods are unable to block the pathological pathogenesis at the microscopic mechanism level of protein post-translational modification.

[0006] To achieve the above objectives, the present invention provides the application of oleanolic acid in the preparation of drugs for treating liver fibrosis.

[0007] The innovative principle of this invention lies in the following: Oleanolic acid enters hepatocytes, enhances the binding of SIRT7 protein and ASC protein, and promotes the deacetylation of ASC protein; ASC protein deacetylation blocks the assembly and activation of NLRP3 inflammasome, leading to a decrease in the expression of activated aspartate protease-1 and a reduction in the production of mature interleukin-1β, thereby blocking hepatocyte pyroptosis; the protective effect of oleanolic acid in reducing liver fibrosis depends on the presence of SIRT7 protein. Preferably, the drug comprises oleanolic acid as an active ingredient and pharmaceutically acceptable excipients. Preferably, the pharmaceutically acceptable excipient includes sodium carboxymethyl cellulose.

[0008] Preferably, the drug is a suspension, wherein the oleanolic acid is suspended in a 0.5% sodium carboxymethyl cellulose aqueous solution.

[0009] Preferably, the concentration of oleanolic acid in the suspension is prepared to be 2 mg / ml.

[0010] Preferably, the drug is formulated as a dosage form for oral administration by gavage.

[0011] Preferably, the drug is used to treat liver fibrosis accompanied by elevated serum alanine aminotransferase activity.

[0012] Preferably, the treatment of liver fibrosis includes: reducing the degree of inflammatory cell infiltration in the liver parenchyma and reducing collagen fiber deposition in the liver.

[0013] Preferably, the reduction of intrahepatic collagen fiber deposition includes: reducing collagen fiber deposition in the portal area and the perivenous region of the central vein.

[0014] Preferably, the drug is used to prepare a drug that reduces liver histopathological changes by blocking NLRP3 inflammasome activation.

[0015] This invention provides the application of oleanolic acid in the preparation of drugs for treating liver fibrosis. It has the following beneficial effects: 1. This invention uses oleanolic acid as an active ingredient to intervene in hepatocytes, enhancing the binding of SIRT7 protein and ASC protein in hepatocytes and promoting the deacetylation of ASC protein, blocking the assembly and activation of NLRP3 inflammasome, resulting in a decrease in the expression of activated aspartate protease-1 and a reduction in the production of mature interleukin-1β, thereby blocking hepatocyte pyroptosis. This achieves the drug intervention effect of inhibiting the liver inflammatory response and blocking the pathogenesis of liver fibrosis from the level of protein post-translational modification.

[0016] 2. This invention prepares a drug suspension containing oleanolic acid and sodium carboxymethyl cellulose excipients into a specific concentration and dosage form for oral gavage administration. This drug is then administered to animal models of liver fibrosis, reducing the infiltration of inflammatory cells in the liver parenchyma, decreasing collagen fiber deposition in the portal area and peri-central vein region, and lowering abnormally elevated serum alanine aminotransferase (ALT) activity. This achieves the therapeutic effect of improving liver histopathological signs and treating liver fibrosis accompanied by elevated serum ALT activity.

[0017] 3. This invention uses a hepatocyte-specific SIRT7 gene knockout model for comparative verification analysis to confirm that the protective effects of oleanolic acid in reducing hepatic parenchymal inflammatory cell infiltration, collagen fiber deposition, and serum alanine aminotransferase against liver fibrosis depend on the presence of SIRT7 protein. This verifies the direct target of oleanolic acid's pharmacological intervention and provides a technical effect with clear target specificity and a well-defined biological mechanism to support the development of anti-liver fibrosis drugs. Attached Figure Description

[0018] Figure 1 This is a graph showing the results of immunoprecipitation of the effect of oleanolic acid on the binding of SIRT7 and ASC in hepatocytes according to the present invention. Figure 2 This is a Western blot result showing the effect of oleanolic acid on the activation of inflammasomes in hepatocytes according to the present invention. Figure 3 This figure shows the effect of oleanolic acid on the pathological changes of liver tissue in mice with CCl4-induced liver fibrosis. Detailed Implementation

[0019] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] To achieve the goal of preparing drugs for treating liver fibrosis using oleanolic acid, the pharmacodynamic evaluation and mechanism verification process of this invention follows the logic of microscopic molecular target identification, cell phenotype observation, and macroscopic animal pharmacodynamic verification. Oleanolic acid is used as the active ingredient in the preparation of drugs for treating liver fibrosis. The major steps of pharmacodynamic and mechanism verification are as follows: S1, Molecular Mechanism Confirmation Steps. Oleanolic acid acts on hepatocytes, enhancing the interaction between SIRT7 and ASC proteins and promoting ASC protein deacetylation. ASC deacetylation blocks the over-assembly and aberrant activation of the NLRP3 inflammasome, leading to a decrease in the expression of cleaved caspase-1 and a reduction in the production of mature interleukin-1β (mature IL-1β). The inhibition of the NLRP3 inflammasome activation pathway thus blocks hepatocyte pyroptosis mediated by aberrant NLRP3 inflammasome activation.

[0021] S2, In vivo efficacy evaluation procedure. Oleanolic acid was prepared as a drug delivery formulation by suspending it in a solvent. Liver fibrosis model animals were administered the drug via gavage using specific dosages and suspension volume parameters corresponding to the animal's body weight. After drug intervention, serum samples were collected from the animals to detect alanine aminotransferase (ALT) activity levels, confirming that oleanolic acid significantly reduced abnormally elevated serum ALT levels. Pathological section staining of liver tissue from the animals confirmed that oleanolic acid reduced the degree of inflammatory cell infiltration in the liver parenchyma and decreased abnormal collagen fiber deposition around the portal areas and central veins.

[0022] S3, Target-Dependent Evidence Step. An animal model with hepatocyte-specific SIRT7 knockout was constructed, and the aforementioned formulation administration by gavage and efficacy evaluation process were repeated. The differences in histopathological changes and serum biochemical results between the wild-type and knockout animal models were compared to confirm that the anti-hepatic fibrosis efficacy of oleanolic acid in reducing inflammatory cell infiltration, decreasing collagen deposition, and lowering serum alanine aminotransferase is dependent on the SIRT7 protein.

[0023] Regarding the molecular mechanism confirmation steps described in S1, the specific material preparation and testing implementation methods include: S101, Preparation of materials for cellular level experiments. The human hepatocellular carcinoma cell line Huh7 (Procell, China) was selected as the cell model for in vitro mechanism studies. The active pharmaceutical ingredients used were oleanolic acid, ursolic acid, and tetrandrine (Yuanye, China) with a purity greater than or equal to 98%. Huh7 cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. Oleanolic acid, ursolic acid, and tetrandrine were diluted to specific concentrations using complete culture medium, ensuring that the final concentration of dimethyl sulfoxide in the final system was less than 0.1%.

[0024] S102, Construction and treatment of the in vitro intervention model. Huh7 cells were seeded in six-well plates and cultured at 37°C with 5% CO2. When the cells reached 70%-80% confluence, they were simultaneously treated with 1 μg / ml lipopolysaccharide (LPS) and 20 μM oleanolic acid for 6 hours. This was followed by treatment with 20 μM nigrain for 2 hours. The control group was treated with an equal volume of dimethyl sulfoxide (DMSO). In the validation of the immunoprecipitation deacetylation detection system, 5 nM TSA was used as a positive control to treat cells for 2 hours to confirm the effectiveness of the deacetylation detection system.

[0025] S103, Protein Binding and Deacetylation Detection. 400 μg of total protein lysis buffer was incubated overnight at 4°C with Protein A / G PLUS-Agarose (BioLinkedin, China) conjugated with anti-ASC antibody (CST, USA). After incubation, the agarose beads conjugated with the immunoprecipitation complex were washed three times with TBST buffer. Elution buffer containing 1% sodium dodecyl sulfate and 0.01 M sodium bicarbonate was added, and the mixture was eluted at 75°C for 10 minutes. The nuclear extract and immunoprecipitated proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

[0026] S104, Immunoblot assay of inflammasome-related proteins. Cell and tissue samples were lysed using RIPA lysis buffer (Millipore, MA, USA). Lysis buffer was centrifuged at 12,000 rpm for 10 minutes at 4°C, and the supernatant was collected. Protein concentration was determined using a diquinoline formate assay kit (Thermo Fisher Scientific). 20 μg of total protein from each lysis buffer was separated by 10% or 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then electrotransferred to a polyvinylidene fluoride membrane (Millipore, MA, USA). The membrane was blocked for 1 hour at room temperature with 5% skim milk blocking buffer containing 0.1% Tween-20 and Tris buffer. The membrane was then incubated overnight at 4°C with primary antibody, followed by incubation with horseradish peroxidase (HRP)-labeled secondary antibody. Developing was performed using chemiluminescent substrate (ECL) in an imaging system.

[0027] Regarding the in vivo efficacy evaluation steps described in S2, the specific material preparation and testing implementation methods include: S201, Preparation and Dosage Conversion of In Vivo Gavage Suspension. Sodium carboxymethyl cellulose (CMC-Na, Shanghai Experimental, China) was selected as an excipient. Oleanolic acid was suspended in a 0.5% sodium carboxymethyl cellulose aqueous solution to prepare a suspension with an oleanolic acid concentration of 2 mg / ml. Gavage was administered to mice based on their body weight, with a standard gavage volume of 5 μl / g according to the actual body weight of the experimental animals. Based on the aforementioned concentration and volume standards, the final dosage of oleanolic acid was calculated to be 10 mg / kg. For example, if 0.2 mg of the drug was administered to a 20g mouse, the corresponding suspension volume would be 100 μl.

[0028] S202, Induction and Drug Administration of a Mouse Liver Fibrosis Model. SPF-grade C57BL / 6 mice aged 4 to 6 weeks were used. Wild-type mice (SIRT7fl / fl) were used as wild-type control mice. Before modeling, wild-type control mice and hepatocyte-specific SIRT7 knockout mice were administered oleanolic acid or an equal volume of 0.5% sodium carboxymethyl cellulose solution by gavage at a dose of 10 mg / kg daily for 3 consecutive days. Subsequently, mice were intraperitoneally injected with carbon tetrachloride, prepared as a 20% (v / v) solution in olive oil, at a dose of 0.25 mL / kg body weight. This was administered twice weekly for 4 consecutive weeks. During the modeling period, oleanolic acid or the solvent control was continued daily until tissue collection.

[0029] S203, Liver histopathological examination. Mouse liver tissue samples were fixed in 4% formalin, embedded in paraffin, and cut into 3μm thin sections. Liver lesions and collagen deposition were observed by hematoxylin-eosin staining (Sevier, China) and Sirius red staining (Sevier, China), respectively.

[0030] S204, Serum ALT Activity Detection and Analysis. Blood was collected from the orbital sinus of mice and placed in clean centrifuge tubes. The blood was allowed to clot naturally at room temperature for 30 minutes. Centrifuged at 3500 rpm for 15 minutes. The supernatant pale yellow serum was transferred to a new EP tube. The alanine aminotransferase (ALT / GPT) test kit (Reiter method) from Nanjing Jiancheng Biotechnology Institute (Nanjing Jiancheng Biotechnology Institute, China) was used for detection. A standard curve was plotted with the concentration of the standard as the X-axis and the OD value of the standard as the Y-axis. The sample OD value was substituted into the standard curve equation to calculate the serum ALT activity units, expressed in U / L. After confirming that the ALT experimental data conformed to a normal distribution and did not exhibit homogeneity of variance, the Welch test using one-way ANOVA was used to analyze and determine the significance of differences between groups.

[0031] Regarding the target-dependent disproving steps described in S3, the specific material preparation and testing implementation methods include: S301, Introduction and Treatment of the SIRT7 Gene Knockout Model. A hepatocyte-specific SIRT7 gene knockout mouse model (SIRT7HKO) was introduced as a control and validation model. The hepatocyte-specific SIRT7 knockout mice underwent the same modeling and drug administration procedures as wild-type mice. For three consecutive days before modeling, mice were administered oleanolic acid suspension or solvent via gavage at a dose of 10 mg / kg as a control. During the modeling period, carbon tetrachloride was injected intraperitoneally twice weekly for four weeks, while oleanolic acid or solvent was administered daily via gavage as a control. After modeling, tissue and serum samples were extracted for pathological and transaminase detection.

[0032] See attached document Figure 1 , Figure 1 This is an immunoprecipitation result diagram of the effect of oleanolic acid on the binding of SIRT7 and ASC in hepatocytes according to an embodiment of the present invention.

[0033] Example 1: Oleanolic acid enhances the binding of SIRT7 and ASC in hepatocytes Cellular experimental grouping and drug administration intervention procedures. Four different intervention conditions were set up for Huh7 cells. In the control group, Huh7 cells were simultaneously treated with LPS at a final concentration of 1 μg / ml and an equal volume of dimethyl sulfoxide for 6 hours, followed by treatment with nigrain at a final concentration of 20 μM for 2 hours after the intervention. In the oleanolic acid group, Huh7 cells were simultaneously treated with LPS at a final concentration of 1 μg / ml and oleanolic acid at a final concentration of 20 μM for 6 hours, followed by treatment with nigrain at a final concentration of 20 μM for 2 hours after the intervention. In the ursolic acid group, Huh7 cells were simultaneously treated with LPS at a final concentration of 1 μg / ml and ursolic acid at a final concentration of 20 μM for 6 hours, followed by treatment with nigrain at a final concentration of 20 μM for 2 hours after the intervention. In the tetrandrine group, Huh7 cells were treated with LPS at a final concentration of 1 μg / ml and tetrandrine at a final concentration of 2.5 μM for 6 hours. After the intervention, they were treated with nigrain at a final concentration of 20 μM for 2 hours.

[0034] Immunoprecipitation and protein separation were performed. Four groups of processed cell lysates were collected and immunoprecipitated using anti-ASC antibody. The immunoprecipitated complexes were separated by SDS-PAGE, and then detected by Western blotting using anti-SIRT7 antibody.

[0035] Analysis of protein binding and deacetylation results. Under LPS combined with nigrain stimulation, SIRT7 protein and ASC protein only bound in trace amounts in the control group. The SIRT7-ASC binding signal in the ursolic acid group did not change significantly. Tetrandrine enhanced the co-precipitation signal of SIRT7 and ASC, but the acetylation level of ASC did not change significantly compared to the control group. Oleanolic acid treatment significantly enhanced the co-precipitation signal of SIRT7 and ASC, promoting the interaction between SIRT7 and ASC proteins in hepatocytes and increasing the acetylation level of ASC protein.

[0036] Verification of the findings: Oleanolic acid can promote the binding of SIRT7 and ASC proteins within hepatocytes. The SIRT7-ASC protein binding pathway constitutes the target site for oleanolic acid's anti-hepatic fibrosis pharmacological effect, providing a definitive mechanistic basis for its use in the preparation of drugs for treating liver fibrosis.

[0037] See attached document Figure 2 , Figure 2 This is a Western blot diagram showing the effect of oleanolic acid on the activation of inflammasomes in hepatocytes according to an embodiment of the present invention.

[0038] Example 2: Oleanolic acid inhibits the activation of inflammasomes in hepatocytes Cell grouping and sample collection procedures. Four different intervention conditions were set up for Huh7 cells. The cell grouping and drug treatment methods were completely consistent with those in Example 1 above. Huh7 cell lysates were collected after each drug treatment step.

[0039] Detection of inflammasome-related protein expression levels. Western blotting was used to detect the protein expression levels of pro-CASPASE-1, cleaved CASPASE-1, pro-IL-1β, mature IL-1β, GSDMD-FL, and GSDMD-N in cell lysates.

[0040] Comparative analysis of the results of the inflammasome activation inhibition assay. Under LPS combined with nigericin stimulation, treatment with oleanolic acid significantly reduced the levels of cleaved CASPASE-1 and mature IL-1β proteins, indicating successful inhibition of the inflammasome. While both the ursolic acid and tetrandrine groups showed a decrease, the reduction was not as consistent as in the oleanolic acid group. Pro-CASPASE-1 protein levels decreased in the treated groups, while pro-IL-1β protein expression remained unchanged.

[0041] Mechanism verification conclusions. (Summary) Figure 1The results of SIRT7 binding to ASC and ASC deacetylation assays indicate that oleanolic acid inhibits the activation of the NLRP3 inflammasome by promoting ASC deacetylation. Oleanolic acid's inhibition of NLRP3 inflammasome activation manifests as CASPASE-1 activation and impaired IL-1β maturation. These results demonstrate the mechanism of action of oleanolic acid in inhibiting inflammasome activation, providing a solid pharmacological basis for its use as an active ingredient in the preparation of drugs for treating liver fibrosis.

[0042] See attached document Figure 3 , Figure 3 This is a diagram illustrating the effect of oleanolic acid on the pathological changes in liver tissue of mice with CCl4-induced liver fibrosis, according to an embodiment of the present invention.

[0043] Example 3: Oleanolic acid alleviates CCl4-induced liver fibrosis in mice in a SIRT7-dependent manner.

[0044] Animal grouping and modeling drug administration procedures. SPF-grade C57BL / 6 mice were randomly divided into wild-type control group, wild-type drug administration group, knockout control group, and knockout drug administration group, using hepatocyte-specific SIRT7 gene knockout mice (SIRT7HKO), with 4 mice in each group. Wild-type control and wild-type drug administration groups used wild-type control mice (SIRT7fl / fl). Before modeling, wild-type control mice and hepatocyte-specific SIRT7 knockout mice were administered oleanolic acid or an equal volume of 0.5% sodium carboxymethyl cellulose solution by gavage at a dose of 10 mg / kg daily for 3 consecutive days. Afterward, the mice were intraperitoneally injected with carbon tetrachloride, which was prepared as a 20% (v / v) solution with olive oil, at a dose of 0.25 mL / kg body weight, twice a week for 4 consecutive weeks. During the modeling period, oleanolic acid or the solvent control was continued to be administered daily until tissue collection.

[0045] Liver histopathology and collagen deposition analysis. Liver tissue was collected after euthanasia of mice, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Hematoxylin-eosin and Sirius red staining were used to observe liver lesions and collagen deposition. Hematoxylin-eosin staining results showed that the knockout control group had higher levels of inflammatory cell infiltration and damage than the wild-type control group; the wild-type control group showed a small amount of inflammatory cell infiltration, but no significant liver damage; the knockout control group had significantly higher levels of inflammatory cell infiltration and liver damage compared to the wild-type control group. Sirius red staining results showed mild collagen deposition in the liver tissue of the wild-type control group; the knockout control group showed abundant red collagen fiber deposition, mainly distributed in the portal area and around the central vein. After gavage treatment with oleanolic acid, collagen deposition in the liver tissue of the wild-type control group was significantly reduced; oleanolic acid did not significantly reduce collagen deposition in the knockout control group.

[0046] Serum alanine aminotransferase (ALT) detection and analysis. Blood was collected from the orbital sinus of mice and placed in clean centrifuge tubes. The blood was allowed to clot naturally at room temperature for 30 minutes. Centrifuged at 3500 rpm for 15 minutes, and the supernatant pale yellow serum was transferred to a new centrifuge tube. Serum ALT was detected using an alanine aminotransferase (ALT / GPT) assay kit. A standard curve was plotted with the concentration of the standard as the X-axis and the OD value of the standard as the Y-axis. The sample OD values ​​were substituted into the standard curve equation to calculate the serum ALT activity units. The obtained ALT experimental data were confirmed to conform to a normal distribution and not to be homogeneous in variance. Statistical analysis was performed using a one-way ANOVA Welch test. The specific ALT values ​​(U / L) measured in each group are as follows: SIRT7fl / fl group: 223, 286, 304, 278; SIRT7HKO group: 413, 334, 450, 478; SIRT7fl / fl+OA group: 117, 95, 134, 84; SIRT7HKO+OA group: 163, 437, 326, 531. Statistical analysis showed that the differences were statistically significant (F=9.694, P=0.0016). The analysis results confirmed that oleanolic acid significantly reduced serum ALT levels in mice in the wild-type drug-treated group.

[0047] In vivo efficacy evaluation conclusions. In vivo experimental results confirmed that oleanolic acid effectively reduces CCl4-induced liver fibrosis and the degree of liver damage. The protective effect of oleanolic acid in reducing liver fibrosis depends on the presence of SIRT7 protein; knockout of SIRT7 significantly weakens the anti-fibrotic effect of oleanolic acid. Experimental results demonstrate that oleanolic acid has a clear effect in reducing CCl4-induced liver fibrosis, providing a basis for its application as an active ingredient in the preparation of drugs for treating liver fibrosis.

[0048] For specific operational methods regarding cell culture passage procedures, basic feeding conditions management of laboratory mice, and routine preparation of buffer solutions for protein extraction systems, those skilled in the art can refer to the standard operating procedures of biological laboratories. The aforementioned basic operations are well-known techniques in this field and will not be elaborated upon here.

[0049] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. Application of oleanolic acid in the preparation of drugs for treating liver fibrosis.

2. The application according to claim 1, characterized in that, The drug contains oleanolic acid as the active ingredient and pharmaceutically acceptable excipients.

3. The application according to claim 2, characterized in that, Pharmaceutically acceptable excipients include sodium carboxymethyl cellulose.

4. The application according to claim 3, characterized in that, The drug is a suspension, wherein the oleanolic acid is suspended in a 0.5% sodium carboxymethyl cellulose aqueous solution.

5. The application according to claim 4, characterized in that, In the suspension, the concentration of oleanolic acid is prepared to be 2 mg / ml.

6. The application according to any one of claims 1 to 5, characterized in that, The drug is formulated as a dosage form for oral administration by gavage.

7. The application according to claim 1, characterized in that, The drug is used to treat liver fibrosis accompanied by elevated serum alanine aminotransferase (ALT) activity.

8. The application according to claim 1, characterized in that, The treatment for liver fibrosis includes: reducing the degree of inflammatory cell infiltration in the liver parenchyma and reducing collagen fiber deposition in the liver.

9. The application according to claim 8, characterized in that, The reduction of intrahepatic collagen fiber deposition includes: reducing collagen fiber deposition in the portal areas and the perivenous region of the central vein.

10. The application according to claim 1, characterized in that, The drug is used to prepare a drug that reduces liver histopathological changes by blocking NLRP3 inflammasome activation.

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