Formononetin Induces Ferroptosis in Activated Hepatic Stellate Cells to Attenuate Liver Fibrosis by Targeting NADPH Oxidase 4

ABSTRACT Ferroptosis is a newly discovered type of cell death that exerts a crucial role in hepatic fibrosis. Formononetin (FMN), a natural isoflavone compound mainly isolated from Spatholobus suberectus Dunn, shows multiple biological activities, including antioxidant, anti‐inflammatory, and hepatoprotection. This research aims to explore the regulatory mechanism of FMN in liver fibrosis and the relationship between NADPH oxidase 4 (NOX4) and ferroptosis. The effects of FMN on HSC ferroptosis were evaluated in rat model of CCl4‐induced hepatic fibrosis. In vitro, N‐acetyl‐L‐cysteine (NAC) and deferoxamine (DFO) were used to block ferroptosis and then explored the anti‐fibrotic effect of FMN. The target protein of FMN was identified by bio‐orthogonal click chemistry reaction as well as drug affinity responsive target stability (DARTS), cellular thermal shift (CETSA), surface plasmon resonance (SPR) assays, and isothermal titration calorimetry (ITC) analysis. Here, we found that FMN exerted anti‐fibrotic effects via inducing ferroptosis in activated HSCs. NAC and DFO prevented FMN‐induced ferroptotic cell death and collagen reduction. Furthermore, FMN bound directly to NOX4 through possible active amino acid residues sites, and increased NOX4‐based NADPH oxidase activity to enhance levels of NADP+/NADPH, thus promoting ferroptosis of activated HSCs and relieving liver fibrosis. These results demonstrate that the direct target and mechanism by which FMN improves liver fibrosis, suggesting that FMN may be a natural candidate for further development of liver fibrosis therapy.