Natural small molecule evodiamine alleviates liver fibrosis by targeting the ANXA2-p110α axis in hepatic stellate cells

BACKGROUND: Liver fibrosis is a common consequence of chronic liver injury, whereas pharmacological interventions that directly impede fibrogenesis remain limited. PURPOSE: This study aimed to screen natural compounds that inhibit hepatic stellate cell (HSC) activation from clinically effective antifibrotic traditional Chinese medicine (TCM) formulas and to elucidate the underlying mechanisms. STUDY DESIGN AND METHODS: Using network pharmacology integrated with a TGF-β1-induced HSC activation model to screen natural small molecules, followed by in vivo validation in CCl₄-, DDC-, and HFD+CCl₄-induced mouse fibrosis models. Direct target identification and mechanistic analyses were conducted by drug affinity responsive target stability (DARTS), biolayer interferometry (BLI) and RNA sequencing. RESULTS: Among 23 prioritized candidates, evodiamine (EVO) significantly suppressed HSC activation and proliferation in human LX-2 and rat HSC-T6 cells. In three mouse models, EVO (50 mg/kg) markedly improved liver function and fibrotic pathology without inducing overt toxicity. Annexin A2 (ANXA2) was identified as a direct binding target of EVO, and was enriched in activated HSCs and upregulated in fibrotic mouse livers and human fibrotic specimens. Mechanistically, EVO bound to ANXA2 at a site involving Ser22 and Val27, and promoted NEDD4L-mediated K48-linked ubiquitination and degradation of ANXA2. Furthermore, EVO disrupted the interaction between ANXA2 and the PI3K catalytic subunit p110α, resulting in the suppression of PI3K-AKT signaling in HSCs. CONCLUSION: These findings identified EVO as an active antifibrotic compound and uncovered a previously unrecognized mechanism whereby EVO targets the ANXA2-p110α axis in HSCs to alleviate liver fibrosis.