Targeting activated hepatic stellate cells through ferroptosis: phenethyl isothiocyanate attenuates CCl4-induced liver fibrosis by regulating iron metabolism, redox homeostasis, and autophagy.

Phenethyl isothiocyanate (PEITC), a dietary compound from cruciferous vegetables, is known for its chemopreventive activity, but its effects on liver fibrosis remain largely unexplored. In this study, we show that PEITC administration attenuated carbon tetrachloride (CCl4)-induced hepatic injury and fibrosis in mice, as evidenced by reduced collagen deposition, hydroxyproline content and fibrotic marker expression, and improved liver function. PEITC inhibited cell survival and fibrogenic phenotypes in transforming growth factor β (TGF-β)-stimulated LX-2 human hepatic stellate cells (HSCs) while exhibiting minimal cytotoxicity towards normal hepatocytes. Mechanistically, PEITC depleted glutathione, inhibited glutathione peroxidase 4 (GPX4), and elevated reactive oxygen species (ROS), lipid peroxidation, and malondialdehyde, thereby triggering ferroptosis, as also confirmed by transmission electron microscopy showing mitochondrial shrinkage, increased membrane density, and cristae loss. Additionally, PEITC activated autophagy and promoted nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy, leading to ferritin degradation. The involvement of autophagy was further validated by co-treatment with autophagy inhibitors, which partially reversed PEITC-induced ferritin heavy chain (FTH1) downregulation and NCOA4 upregulation. In vivo immunofluorescence staining revealed that PEITC suppressed antioxidant defense and promoted ferritin degradation in activated HSCs, as shown by reduced GPX4 and FTH1 fluorescence signals within α-smooth muscle actin (α-SMA)-positive areas in mouse liver sections. Collectively, these findings elucidate that PEITC alleviates liver fibrosis by promoting ferroptosis-related processes in activated HSCs, mainly through the NCOA4/FTH1 axis, positioning it as a promising nutraceutical candidate for antifibrotic therapy.