Use of ursodeoxycholic acid in the preparation of a medicament for the treatment of liver fibrosis

Abstract

The application of ursolic acid in the preparation of drugs for treating liver fibrosis can effectively solve the problem of medication for treating liver fibrosis. By regulating the differentiation of immune cells (Th17 cells) and reducing inflammatory damage, ursolic acid intervenes in the liver's immune microenvironment, effectively reversing liver fibrosis, and thus enabling its application in the preparation of drugs for treating liver fibrosis. This invention develops a new drug for treating liver fibrosis, opening up a new avenue for medication in the treatment of liver fibrosis. It represents a major innovation in the treatment of liver fibrosis, demonstrating good efficacy, safety, and practical application value.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of ursolic acid in the preparation of drugs for treating liver fibrosis. Background Technology

[0002] Liver fibrosis is a key pathological step in the progression of many chronic liver diseases to cirrhosis and liver cancer. It can be caused by various factors such as viral hepatitis, alcohol, and aflatoxin. It is a reversible pathophysiological phenomenon. Clinically, ursodeoxycholic acid (UDCA) is the first-line drug for treating fibrosis associated with cholestatic liver disease. However, about 40% of patients with primary biliary cholangitis do not respond well to it. These patients experience continuous progression of liver fibrosis and have a poor prognosis. Second-line drugs such as obeticholic acid often have side effects such as severe itching, which limits their use. Currently, there are no specifically approved drugs to reverse liver fibrosis worldwide. Therefore, there is an urgent clinical need to develop effective treatments for liver fibrosis, especially for patients who do not respond well to UDCA.

[0003] Ursolic acid is a naturally occurring bile acid, a white to light brown solid or powder, soluble in DMSO, ethanol, and methanol (sonic dissolution is required). It is primarily found in mammalian bile and is mainly used as an impurity reference standard for drug quality control (e.g., ursodeoxycholic acid EP impurity D). It is also used in research fields such as bile acid metabolism, hepatocyte function, and gut microbiota-host interactions. While existing technologies disclose the application of ursolic acid in the treatment of cholelithiasis and chronic cholestasis, there is a lack of effective technical solutions for reversing existing liver fibrosis using it. Furthermore, current research on anti-liver fibrosis drugs has limited exploration of immune pathways. This invention aims to solve the technical challenge of effectively reversing liver fibrosis by using ursolic acid to regulate the differentiation of immune cells (Th17 cells), reduce inflammatory damage, and thus intervene in the liver's immune microenvironment.

[0004] Although ursolic acid (URA) is structurally similar to UDCA and belongs to the ursane bile acid derivative class, differences in their side-chain structures may endow them with different pharmacological activities. Currently, there are no research reports on the use of URA in the treatment of liver fibrosis. Based on the structural differences between URA and UDCA and their potential novel pharmacological effects, this invention proposes the application of URA in the preparation of drugs for treating liver fibrosis, thus filling a gap in the existing technology. Summary of the Invention

[0005] In view of the above situation and to overcome the shortcomings of the prior art, the purpose of this invention is to provide an application of ursolic acid in the preparation of drugs for treating liver fibrosis, which can effectively solve the problem of medication for treating liver fibrosis.

[0006] The technical solution provided by this invention is the application of ursolic acid in the preparation of drugs for treating liver fibrosis. By regulating the differentiation of immune cells and reducing inflammatory damage, ursolic acid can effectively reverse liver fibrosis and thus be used in the preparation of drugs for treating liver fibrosis.

[0007] This invention can effectively inhibit the secretion of IL-17A cytokine, thereby improving the liver's immune microenvironment, reducing inflammatory damage, and playing a role in the treatment of liver fibrosis. It has developed a new drug for the treatment of liver fibrosis, opened up a new approach to the treatment of liver fibrosis, and is a major innovation in the treatment of liver fibrosis. It has good effects and has practical value for promotion and application. Attached Figure Description

[0008] Figure 1 This is the chemical structural diagram of ursolic acid; Figure 2 Comparison of gross morphology of the liver; Figure 3 A comparison chart of liver function coefficients; Figure 4 A comparison chart of serum liver function indicators; Figure 5 A comparison chart of serum liver fibrosis markers; Figure 6 A comparison chart of H&E; Figure 7 Comparison image of MASSON staining (blue); Figure 8 This is a comparison image of immunoblot data; Figure 9 A comparison of expression levels of Collagen I and α-SMA; Figure 10 Comparison of flow cytometry results for proportional Th17 cells in the liver; Figure 11 A comparison chart of liver IL-17A concentrations; Figure 12 Comparison of in vitro Th17 cell polarization results; Figure 13 A comparison of IL-17A concentrations in the supernatant of polarized cell cultures. Detailed Implementation

[0009] The specific embodiments of the present invention will be described in detail below with reference to specific circumstances.

[0010] This invention relates to the application of ursolic acid in the preparation of a drug for treating liver fibrosis, wherein the molecular structural formula of ursolic acid is: C 24 H 40 O5, molecular weight: 408.6 g / mol, chemical structural formula:

[0011] By utilizing ursolic acid to regulate the differentiation of immune cells (Th17 cells) and reduce inflammatory damage, the liver's immune microenvironment can be intervened, effectively reversing liver fibrosis, thus enabling its application in the preparation of drugs for the treatment of liver fibrosis.

[0012] This invention develops a new drug for treating liver fibrosis, opening up a new avenue for medication in the treatment of liver fibrosis. Animal experiments have shown very good technical results, and the relevant experimental data are as follows:

[0013] Eighteen 8-week-old male C57BL / 6J mice, weighing 20-22g, were divided into three groups after one week of acclimatization feeding: Normal group (Group A): After acclimatization, olive oil was administered by gavage at a dose of 4 mL / kg, twice a week for a total of 8 weeks; Model group (Group B): After adaptive feeding, while continuing to feed, 25% CCL4 (carbon tetrachloride, the same below) olive oil was administered by gavage to establish the model, 4 mL / kg, twice a week for a total of 8 weeks; Ursolic acid group (Group C): After acclimatization, the model was established by gavage with 25% CCL4 olive oil twice a week for 8 weeks. From week 5, ursolic acid was administered by gavage at a dose of 50 mg / kg once a day for 4 weeks.

[0014] like Figure 2 As shown, the liver tissue of mice in group A was reddish in color, with sharp edges, soft texture, and smooth surface; the liver tissue of mice in group B was dull in color, with blunt edges, hardened texture, and diffuse fine granular appearance on the surface; the color, edge, texture, and surface of group C were all close to those of the normal group.

[0015] like Figure 3 As shown, the weight of mice and liver tissue were weighed during sampling. The liver tissue weight was divided by the corresponding mouse body weight to obtain the liver coefficient, which reflects the therapeutic effect of ursolic acid to a certain extent. The results showed that the liver coefficient of the model group increased significantly, while the liver coefficient of the ursolic acid group was significantly decreased compared with that of the model group.

[0016] like Figure 4 As shown, serum AST and ALT can reflect liver function in mice. Biochemical test results showed that compared with the control group, the serum AST and ALT levels in the model group mice were significantly increased, indicating that the liver function of the mice was significantly impaired; compared with the model group, the serum AST and ALT levels in the ursolic acid group mice were significantly decreased, indicating that the liver function of the mice was restored.

[0017] like Figure 5As shown, serum HAase and FN1 can reflect the level of liver fibrosis in mice. ELISA results showed that compared with the control group, the serum HAase and FN1 levels in the model group mice were significantly increased, indicating that the degree of liver fibrosis in the mice was aggravated; compared with the model group, the serum HAase and FN1 levels in the ursolic acid group mice were significantly decreased, indicating that the degree of liver fibrosis in the mice was reduced.

[0018] like Figure 6 As shown, the H&E reaction resulted in pathological liver damage. In the control group, the liver tissue structure of mice was intact, with hepatocyte cords arranged neatly radially around the central vein, and the hepatocytes were uniform in morphology, with no obvious inflammatory cell infiltration. In the model group, obvious focal inflammatory cell infiltration was observed in the central vein and portal areas of the liver tissue, accompanied by mild hepatocyte edema, and the hepatocyte cords were arranged in a disordered manner. The pathological changes in the liver tissue of mice in the ursolic acid group were improved compared with the model group, with inflammatory cells scattered but significantly reduced compared with the model group; the lobular structure of the liver was basically preserved, and the degree of hepatocyte damage was significantly reduced compared with the model group.

[0019] 6. MASSON staining (blue) index: like Figure 7 As shown, MASSON staining (blue) reflects liver collagen deposition, indicating the degree of liver fibrosis: In the control group, collagen fibers (blue staining) were only distributed in the vessel walls, without excessive deposition or fibrous septa formation. In the model group, collagen fibers in the portal area and around blood vessels were significantly increased and thickened, with widened and interconnected fibrous septa forming typical pseudolobular structures. In all treatment groups, collagen fiber deposition was significantly reduced compared to the model group, fibrous septa were thinner, and the pseudolobular structure partially disintegrated, with only a small amount of blue collagen fibers remaining around the vessel walls, and no obvious fibrous septa were observed.

[0020] like Figure 8-9 As shown, α-SMA is a marker of hepatic stellate cell activation, which is a hallmark event of liver fibrosis. Activated hepatic stellate cells secrete Collagen I, which is deposited in the liver and is a major component of the extracellular matrix during liver fibrosis. Therefore, the expression levels of Collagen I and α-SMA are often used as one of the evaluation criteria for the degree of liver fibrosis. The results showed that Collagen I and α-SMA were almost not expressed in the control group; compared with the control group, Collagen I and α-SMA were significantly highly expressed in the model group, indicating that the degree of liver fibrosis in the model group was aggravated; compared with the model group, the expression levels of Collagen I and α-SMA in the ursolic acid group were significantly reduced, indicating that ursolic acid can significantly improve the degree of liver fibrosis in mice.

[0021] like Figure 10As shown, Th17 cells are a typical pro-inflammatory and pro-fibrotic CD4+ T cell subtype. Flow cytometry analysis of the proportion of Th17 cells in mouse liver tissue revealed that the proportion of Th17 cells in the liver tissue of control mice was low, while the proportion of Th17 cells in the liver tissue of model mice was significantly increased compared to the control group. Furthermore, the proportion of Th17 cells in the liver tissue of mice in the ursolic acid group was significantly decreased compared to the model group. These results indicate that ursolic acid can significantly reduce the proportion of Th17 cells in the liver of fibrotic mice.

[0022] like Figure 11 As shown, IL-17A is a classic pro-inflammatory and pro-fibrotic cytokine secreted by Th17 cells. ELISA results showed that the IL-17A level in the liver tissue of control mice was low; compared with the control group, the IL-17A level in the liver of model mice was significantly increased; and compared with the model group, the IL-17A level in the liver of mice in the ursolic acid group was significantly decreased. These results indicate that ursolic acid can significantly reduce the IL-17A level in the liver of fibrotic mice. Combined with flow cytometry results, these findings suggest that ursolic acid can significantly reduce the proportion of Th17 cells in the liver of fibrotic mice, thereby reducing IL-17A secretion and exerting anti-inflammatory and anti-fibrotic effects.

[0023] like Figure 12 As shown, in order to further investigate how ursolic acid reduces the proportion of Th17 cells in the liver, immature CD4+ T cells were isolated from mouse spleen and polarization experiments were performed in vitro to induce the differentiation of immature CD4+ T cells into Th17 cells. The results showed that ursolic acid could inhibit the differentiation of immature CD4+ T cells into Th17 cells.

[0024] Polarization conditions (model group): Anti-mouse CD3 5μg / ml, Anti-mouse CD28 5μg / ml, Anti-IL-4 10μg / ml, Anti-IFN-γ 10μg / ml, Anti-IL-2 10μg / ml, Mouse IL-6 20 ng / ml, MouseIL-23 10 ng / ml, Mouse IL-1β 10 ng / ml, Human TGF-β1 2 ng / ml.

[0025] Ursolic acid group: 100 µM ursolic acid was added to the model group.

[0026] like Figure 13The results showed that ursolic acid can inhibit the differentiation of naive CD4+ T cells into Th17 cells, thereby reducing the proportion of Th17 cells in the liver, inhibiting the secretion of IL-17A cytokine, improving the liver immune microenvironment, reducing inflammatory damage, and playing a role in the treatment of liver fibrosis.

[0027] In summary, this invention utilizes ursolic acid to regulate the differentiation of immune cells (Th17 cells), reduce inflammatory damage, and thereby intervene in the liver's immune microenvironment, effectively reversing liver fibrosis and enabling its application in the preparation of drugs for treating liver fibrosis. This invention develops a new drug for treating liver fibrosis, opening up a new avenue for its treatment. It represents a significant innovation in liver fibrosis medication, demonstrating good efficacy, safety, and practical value for widespread application.

Claims

1. The application of ursolic acid in the preparation of drugs for treating liver fibrosis, wherein the molecular structural formula of ursolic acid is: C 24 H 40 O5, molecular weight: 408.6 g / mol, chemical structural formula:

2. The application according to claim 1, characterized in that, By utilizing ursolic acid to regulate the differentiation of Th17 immune cells and reduce inflammatory damage, thereby intervening in the liver's immune microenvironment and reversing liver fibrosis, it can be applied in the preparation of drugs for the treatment of liver fibrosis.

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