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

Background: 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.

Purpose: This study aimed to investigate the antifibrotic properties of T-96 and its underlying molecular mechanisms.

Methods: 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.

Results: 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.

Conclusion: Our results provide the first insight that T-96 exerts potent antifibrotic effects both in vitro and in vivo by inhibiting the AGAP2 mediated FAK/Akt signaling axis, and that T-96 may serve as a potential therapeutic candidate for the treatment of liver fibrosis.