International Research in Chinese Medicine

Objective To investigate whether 3-methyladenine (3-MA) exacerbates CCl₄-induced liver fibrosis in mice by inhibiting autophagy, thereby antagonizing the anti-fibrotic effect of betulinic acid (BA). Methods Fifty SPF-grade male C57BL/6 mice were randomly divided into a control group, a model group, a betulinic acid group, a 3-MA treatment group, and a 3-MA+BA group, with 10 mice in each group. Except for the control group, which received intraperitoneal injections of 1.5 ml/kg•d of pure olive oil, all other groups underwent intraperitoneal injections of a 25% carbon tetrachloride (CCl₄) and olive oil solution (CCl₄: olive oil = 1:3) at 2 ml/kg•d to establish a liver fibrosis model. Interventional drugs were administered simultaneously from the first week of model establishment. The 3-MA treatment group and the 3-MA+BA group received intraperitoneal injections of 3-MA (10 mg/kg) once daily 2 hours before the intraperitoneal injection of the CCl₄ and olive oil solution. The BA group and the 3-MA+BA group received gavage with a 30 mg/kg suspension of betulinic acid half an hour after the intraperitoneal injection of the CCl₄ and olive oil solution, with continuous intervention for 6 weeks. Liver histopathological damage was assessed using HE staining, collagen fiber deposition was detected using Sirius red-picric acid staining, and the expression level of α-smooth muscle actin (α-SMA), a marker of hepatic stellate cell activation, was detected by Western blot. Results Compared with the control group, the serum levels of ALT and AST in the 3-MA group were significantly increased (P<0.05). The liver tissue of the 3-MA group exhibited more pronounced inflammatory responses in hepatocytes and increased deposition of red collagen fibers in the liver compared with the model group, with upregulation of α-SMA protein expression; The levels of serum ALT and AST in the 3-MA+BA group were significantly lower than those in the 3-MA group (t=12.62, P<0.05; t=12.42, P<0.05). Only a small number of inflammatory cells accumulated in the portal area, and cell edema and vacuolar degeneration were less severe than in the 3-MA group. Collagen deposition was reduced, and α-SMA protein expression was between that of the 3-MA group and the BA group. The levels of serum ALT and AST in the BA group were significantly lower than those in the 3-MA+BA group, and the pathological changes were significantly alleviated. The cell structure was relatively intact, with no obvious cell edema or vacuolar degeneration. Only a small amount of collagen fibers were present in the portal area, and α-SMA protein expression was significantly reduced compared to the model group. Conclusion 3-MA significantly exacerbates CCl₄-induced liver fibrosis in mice by inhibiting autophagy, antagonizing the anti-fibrotic effect of betulinic acid.

3-methyladenine; Autophagy; Liver fibrosis; Betulinic acid; α-smooth muscle actin