Loureirin B ameliorates cholestatic liver fibrosis via AKT/mTOR/ATG7-mediated autophagy of hepatic stellate cells

Chronic cholestasis leads to liver fibrosis, which lacks effective treatment. In this study, we investigated the role and mechanisms of action of loureirin B (LB) in cholestatic liver fibrosis. Bile duct ligation (BDL)-induced hepatic fibrosis mice were used as in vivo models. Transforming growth factor-β1 (TGF-β1)-pretreated HSC-T6 cells were used to explore the mechanism by which LB attenuates liver fibrosis in vitro. RNA sequencing, quantitative PCR (qPCR), western blotting, immunohistochemistry and immunofluorescence were performed to detect the fibrosis markers and measure autophagy levels. Flow cytometry, cell counting kit-8 (CCK-8) assay, and 5′-ethynyl-2′-deoxyuridine (EdU) assay were conducted to detect cell proliferation and viability. GFP-RFP-LC3 adenovirus, autophagy-related protein 7 (ATG7) siRNA, and bafilomycin A1 (BafA1) were used to verify autophagic flux. Our results showed that LB ameliorates liver injury, inhibits collagen deposition, and decreases the expressions of fibrosis-related markers in BDL-induced mouse livers. In vitro, we found that LB inhibited proliferation and migration, promoted apoptosis, and inhibited the activation of HSC-T6 cells pretreated with TGF-β1. RNA sequencing analysis of HSC-T6 cells showed that LB treatment predominantly targeted autophagy-related pathways. Further protein analysis indicated that LB downregulated the expression of phosphorylated AKT (p-AKT) and phosphorylated mTOR (p-mTOR), and upregulated LC3-II, p62, and ATG7 both in vivo and in vitro. Intriguingly, ATG7 inactivation reversed the antifibrotic effects of LB on HSC-T6 cells. LB can improve BDL-induced liver fibrosis by inhibiting the activation and proliferation of HSCs and is expected to be a promising antifibrotic drug. [Display omitted] •Loureirin B (LB) exerts an anti-hepatic fibrosis effect on bile duct ligation (BDL)-induced liver fibrosis.•LB inhibits TGF-β1-induced proliferation and activation of HSC-T6 cells.•LB inhibits cell proliferation and activation by activating autophagy and inhibiting autophagy flux.•LB inhibits hepatic fibrosis and HSC-T6 activation via AKT/mTOR signaling pathway.