[Study on anti- fibrotic mechanism of Fuzheng -Huayu formula to suppress autophagy in mice].

Objective To determine whether the anti-hepatic fibrosis effect of Fuzheng-Huayu formula is related to suppress autophagy in mice. Methods C57 mice were randomly divided into normal group (N group) and model group. The model group was induced by intraperitoneal injection of carbon tetrachloride to induce liver fibrosis in mice, and the normal group was injected with equal volume of olive oil. After 1 week, the model group was randomly divided into model (M) group, rapamycin (Rapa) group, rapamycin plus chloroquine (Rapa+CQ) group, rapamycin plus salvianolic acid B (Rapa+Sal B) group, rapamycin plus Fuzheng -Huayu formula (Rapa+FZ) group. Each drug group was administered corresponding drugs by gavage on a daily basis, and N group and M group were given the equal amount of drinking water by gavage. After 5 weeks, the mice were sacrificed, and HE and Sirius red staining were used to observe the inflammation and collagen deposition on liver tissue in each group. The hydroxyproline content was determined by alkaline hydrolysis method. Western blotting was used to detect changes in the expression of autophagy in liver tissue and microtubule-associated protein 1 light chain 3II/I (LC3II/I), p62, α-smooth muscle actin (ɑ-SMA) and type I collagen expression. Immunofluorescence staining was used to observe the immunofluorescence localization of ɑ-SMA and LC3B in liver tissues of each group. ). A t-test was used to compare the two independent samples. LSD or Dunnett’s T3 test were used to compare the mean of multiple samples. Results There was no significant difference in N and M groups in terms of body weight. The body weight of the mice in each drug group decreased significantly (F = 14.041, P < 0.001). The liver/spleen /body weight ratios of each drug group and M group were significantly higher than the N group (F = 26.992, 6.589, P < 0.001). The expression of p62 protein in the liver tissue of mice in each drug group was lower than M group, and the difference between Rapa group and Rapa+Sal B group (F = 3.085, P = 0.039, 0.003) was statistically significant, while that of Rapa + Sal B group was lower. Compared with group M, the expression of LC3B II in Rapa group was significantly higher (F = 7.514, P = 0.01). Immunofluorescence staining showed that LC3B and α-SMA CO-stained cells were absent in the liver of mice in N group, and co-stained cells were found in the liver of mice in M group. The co-stained cells in the liver of mice in each drug group were significantly higher than M group, and the co-stained cells in Rapa+FZ group were fewer. Compared with the N group, the collagen deposition of M group and each drug group was significantly increased; the collagen deposition of each drug group was lower than that of the M group. There was no statistically significant difference between each drug group. Compared with N group (77.75 + 48.79), hydroxyproline in liver tissue of mice in M group was significantly increased (293.48 + 84.43) (F = 3.015, P = 0.005), and the content of hydroxyproline in liver tissue of mice in each drug group was lower than M group, but the difference was not statistically significant (F = 0.750, P = 0.573). Compared with the N group, the expressions of α-SMA and type I collagen in the M group were significantly increased (F = 27.718, 18.893, P < 0.01). The expression of α-SMA in Rapa group and Rapa+Sal B group was similar to M group, while Rapa + CQ group and Rapa + FZ group were significantly lower than Rapa group and M group (P < 0.01). The expression of type I collagen in Rapa + CQ group was significantly higher than Rapa group (P = 0.017), while the expression of type I collagen in Rapa + FZ group was significantly lower than M group (P = 0.013). Conclusion Autophagy of hepatic stellate cells was observed in carbon tetrachloride-induced liver fibrosis model. Rapamycin can promote autophagy in hepatocytes and hepatic stellate cells. Fuzheng-Huayu formula and Salvianolic Acid B might antagonize the effect of rapamycin on autophagy. Key words: Liver fibrosis; Hepatic stellate cell; Autophagy; Fuzheng Huayu compound; Salvianolic acid B