Kurarinol A from Sophora flavescens suppresses hepatic stellate cell activation via the TLR4/MyD88/NF-κB pathway

Abstract

Sophora flavescens is traditionally used in TCM and Miao/Mongolian/Yi ethnomedicines for hepatobiliary disorders, yet its bioactive flavonoid constituents and mechanisms remain incompletely understood. This study investigated the anti-hepatic fibrosis efficacy, mechanism, and target of the ethyl acetate fraction (EAF) from S. flavescens and its key lavandulyl flavonoid kurarinol A (KA). A CCl4-induced mouse hepatic fibrosis model was established to evaluate liver injury and fibrosis by H&E/Masson staining and detection of serum ALT/AST and hepatic α-SMA/fibronectin/Hyp. A TGF-β1-activated LX-2 cell model was established, with cell viability, migration, and apoptosis evaluated by MTS, wound-healing, and Annexin V-FITC/PI assays. Transcriptomics, KEGG analysis, molecular docking, Molecular dynamics (MD) simulation, CETSA, and MyD88 knockdown/overexpression assays were applied to explore the underlying mechanism. In vivo , EAF (50 and 100 mg/kg) alleviated CCl4-induced liver injury and fibrosis and decreased the levels of fibrotic serum biomarkers. In vitro , KA significantly inhibited LX-2 cell activation with an IC50 of 12.65 μM (n = 3), suppressing cell proliferation and migration while inducing apoptosis. KA’s anti-fibrotic activity was validated only in vitro , and its contribution to the in vivo efficacy of the EAF remains inferred rather than directly proven. Mechanistically, KA downregulated key signaling molecules of the TLR4/NF- κ B pathway, thereby blocking pathway activation. Molecular docking, MD simulations and CETSA supported a direct interaction between KA and MyD88. MyD88 knockdown and overexpression assays further supported that MyD88 serves as a candidate molecular target mediating KA’s anti-fibrotic activity. The EAF exerts anti-fibrotic effects in vivo, and KA exhibits consistent anti-fibrotic activity in vitro . These findings support MyD88 as a potential molecular target mediating KA’s in vitro anti-fibrotic activity via TLR4/NF- κ B signaling. These findings not only validate the traditional use of S. flavescens in hepatobiliary disorders, but also offer a promising MyD88-targeting candidate warranting lead optimization for anti-fibrotic drug development.