Apigenin-5-O-glucoside alleviates hepatic fibrosis through NOX4-mediated ferroptosis in zebrafish

Hepatic fibrosis (HF) represents a considerable global burden on health, primarily because of the activation of hepatic stellate cells (HSCs). Although the induction of ferroptosis in activated HSCs has been identified as a promising avenue for therapeutic intervention, the potential application of apigenin-5-O-glucoside (AG) derived from Scutellaria baicalensis Georgi has not yet been investigated.

This study aimed to evaluate the therapeutic properties of AG in the treatment of HF and to elucidate its underlying mechanisms, with a particular emphasis on its capability to promote ferroptosis in HSCs and to identify the essential molecular targets.

Zebrafish models induced by thioacetamide (TAA) and JS-1 cell models stimulated by TGF-β1 were used to assess the anti-fibrotic effects of AG. Network pharmacology, molecular docking, and molecular dynamics simulation techniques were employed to predict potential protein targets. The direct target engagement was confirmed using the cellular thermal shift assay (CETSA), and the functional necessity was further validated through pharmacological inhibition and genetic knockdown methodologies, both in vivo and in vitro.

AG exhibited significant anti-HF efficacy, ameliorating hepatic pathology and suppressing the expression of key fibrotic markers including α-SMA, collagen-Ⅰ and collagen-Ⅲ. Mechanistic investigations demonstrated that AG induced ferroptosis, as evidenced by iron overload, accumulation of lipid reactive oxygen species (ROS) and malondialdehyde (MDA), downregulation of glutathione peroxidase 4 (GPX4), and depletion of glutathione (GSH) Network analysis identified NADPH oxidase 4 (NOX4) as a principal target, with molecular docking studies confirming a strong binding affinity (-7.8 ± 0.8 kcal/mol). Both pharmacological inhibition and genetic knockdown of NOX4 negated AG-induced ferroptosis and reversed its anti-fibrotic effects.

This study suggests that AG alleviates HF through NOX4-dependent ferroptosis in HSCs, revealing a novel mechanism of action and establishing a basis for its potential as a therapeutic candidate for HF.