Demethylzeylasteral attenuates hepatic stellate cell activation and liver fibrosis by inhibiting AGAP2 mediated signaling

• Published in: Phytomedicine (Stuttgart) 2022-10, Vol.105, p.154349-154349, Article 154349
• Main Authors: Chen, Ke, Guo, Weiran, Li, Rongxin, Han, Yueqing, Gao, Qi, Wang, Shuzhen
• Format: Article
• Language: English
• Subjects: AGAP2; AKT; Demethylzeylasteral; FAK; Hepatic stellate cells; Liver fibrosis
• ISSN: 0944-7113; 1618-095X
• DOI: 10.1016/j.phymed.2022.154349

•T-96 inhibits the proliferation, migration, and activation of hepatic stellate cells.•T-96 alleviates CCl4-induced hepatic fibrosis in mice.•T-96 improves liver fibrosis by suppressing AGAP2 mediated FAK/AKT signaling. Liver fibrosis is a common cause of chronic liver disease. If left untreated, it can ultimately develop into liver cirrhosis or hepatocellular carcinoma. However, a direct antifibrotic therapy is currently unavailable. A re-examination of existing chemicals might be a potential strategy for finding more lead compounds against liver fibrosis. Demethylzeylasteral (T-96), a naturally occurring bioactive compound found in Tripterygium wilfordii Hook. f. (TwHf) possesses multiple pharmacological properties. However, its antifibrotic potential has not yet been fully evaluated. This study aimed to investigate the antifibrotic properties of T-96 and its underlying molecular mechanisms. The antifibrotic properties of T-96 were investigated in three types of hepatic stellate cells (HSCs) and in a CCl4-induced liver fibrosis mouse model. The effect of T-96 on the proliferation, migration, and activation of HSCs was detected using CCK-8 and scratch/wound healing assays. Hepatic inflammation and fibrosis were evaluated by H&E, Masson's trichrome stain, and Sirius Red staining. The expression of inflammatory and fibrogenic genes was detected by quantitative real-time PCR (qRT-PCR) and western blotting. RNA sequencing (RNA-seq) was performed to explore the potential molecular mechanisms mediating the antifibrotic effect of T-96, which was verified by dual-luciferase reporter assay, qRT-PCR, western blotting, immunofluorescence, and immunoprecipitation analysis. The T-96 treatment significantly suppressed the proliferation, migration, and activation of HSCs in vitro. The administration of T-96 attenuated hepatic injury, inflammation, and fibrosis progression in mice with CCl4-induced liver fibrosis. In addition, the RNA-seq of fibrotic liver tissues and subsequent functional verification indicated that the key mechanisms of the antifibrotic effect of T-96 were mediated by suppressing the expression of AGAP2 (Arf GAP with GTPase-like domain, ankyrin repeat and PH domain 2), inhibiting the subsequent phosphorylation of focal adhesion kinase (FAK) and protein kinase B (AKT), and finally reducing the expression of fibrosis-related genes. Our results provide the first insight that T-96 exerts potent antifibrotic effects both in vitro and in vivo by inhibiting t