Rougan Jiangmei Formula medicated serum inhibits activation and autophagy of human hepatic stellate cells

doi:10.12307/2023.762

Abstract

Abstract: BACKGROUND: Autophagy can play an important role in liver fibrosis by inducing the progression of hepatic stellate cell activation. Previous studies have shown that Rougan Jiangmei Formula could protect liver function, anti-inflammation, and anti-oxidative stress response. The mechanism of fighting liver fibrosis through autophagy has not been elucidated.
OBJECTIVE: To investigate the effect of Rougan Jiangmei Formula medicated serum on activation and autophagy of human hepatic stellate cells induced by lipopolysaccharide and its underlying mechanism.
METHODS: Human hepatic stellate cell activation models were established using lipopolysaccharide and divided into various treatment groups: (1) Blank group: human hepatic stellate cells+blank serum; (2) model group: human hepatic stellate cells+blank serum+lipopolysaccharide; (3) low-, medium- and high-dose Rougan Jiangmei Formula medicated serum groups: human hepatic stellate cells+low-, medium- and high-dose Rougan Jiangmei Formula medicated serum+lipopolysaccharide; (4) high-dose Rougan Jiangmei Formula medicated serum combined with rapamycin group: based on high-dose group+rapamycin; (5) high-dose Rougan Jiangmei Formula medicated serum combined with PI3K inhibitor group: based on high-dose group+LY294002. Cell proliferation was observed by CCK-8 assay. The autophagy was observed by monodansylcadaverine staining. The effects of Rougan Jiangmei Formula medicated serum on the expression levels of cell pathway-related proteins (PI3K, P-PI3K, AKT, P-AKT, mTOR, P-mTOR), autophagy-related proteins (P62, Beclin-1, LC3-I, LC3-II), the activation marker protein of hepatic stellate cells (α-smooth-muscle actin and collagen I) were determined by western blot assay.
RESULTS AND CONCLUSION: (1) Rougan Jiangmei Formula medicated serum significantly reduced the proliferation of activated human hepatic stellate cells, down-regulated the protein expression of α-smooth-muscle actin and collagen I, up-regulated the expression of autophagy protein P62, and decreased the expression of Beclin-1 and LC3-II in a dose-dependent manner. (2) Monodansylcadaverine staining results exhibited that Rougan Jiangmei Formula medicated serum significantly inhibited the fluorescence intensity of the cell green spots. (3) After the addition of autophagy agonist rapamycin, the expression of autophagy protein P62 was reduced and the expression levels of Beclin-1 and LC3-II were increased. (4) After Rougan Jiangmei Formula medicated serum treatment, the phosphorylation levels of PI3K, Akt, and mTOR proteins were increased in a dose-dependent manner. This effect could be inhibited by the PI3K inhibitor LY294002. (5) It is indicated that Rougan Jiangmei Formula medicated serum may inhibit autophagy via the PI3K/Akt/mTOR signaling pathway and then reduce lipopolysaccharide-induced human hepatic stellate cell activation.

Key words: Rougan Jiangmei Formula, cell autophagy, Akt, mTOR, LC3-II, human hepatic stellate cell

CLC Number:

Tao Bonan, Wang Yonglan, Jiang Lu, Zhang Zongxing, Liu Daozhong, Wan Xing, Huang Debin, Yuan Lin. Rougan Jiangmei Formula medicated serum inhibits activation and autophagy of human hepatic stellate cells[J]. Chinese Journal of Tissue Engineering Research, 2023, 27(33): 5334-5341.

Figures/Tables 10

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