Abstract
Hepatocellular death is a sensitive parameter for detecting acute liver injury (ALI) of toxic, viral, metabolic, and autoimmune origin. Ferroptosis has recently been implicated in carbon tetrachloride (CCl4)-induced ALI. However, the underpinning mechanism and mechanistic basis remain elusive. In this study, bicyclol, a proprietary hepatoprotectant in China, and ferroptosis-specific inhibitor ferrostatin-1 (Fer-1) were administered in CCl4-injured mice. A panel of ferroptosis-related markers, including mitochondria morphology, reactive oxygen species production, protein adducts in response to lipid peroxidation, and key modulators of ferroptotic process, was determined in vivo. Erastin-treated L-O2 hepatocytes were transfected with glutathione peroxidase 4 (GPx4) or nuclear factor erythroid 2-related factor 2 (Nrf2) siRNA to delineate the pathway of bicyclol against ferroptosis in vitro. As a result, CCl4 led to iron accumulation, excessive reactive oxygen species production, enhanced lipid peroxidation, and characteristic morphological changes in mitochondria, along with a decrease in GPx4 and xCT protein levels in ALI mice liver, all of which were generally observed in ferroptosis. The use of Fer-1 further corroborated that ferroptosis is responsible for liver damage. Bicyclol exerted its hepatoprotection by preventing the aforesaid ferroptotic process. Furthermore, bicyclol alleviated erastin-induced cellular inviability, destruction, and lipid peroxidation in vitro. Knockdown of GPx4 diminished these protective activities against perturbations associated with ferroptosis in L-O2 hepatocytes. Additionally, Nrf2 silencing drastically reduced GPx4 levels, and further impeded the medicinal effects of bicyclol. In summary, positively regulating Nrf2-GPx4 axis by bicyclol can prevent ferroptosis in CCl4-induced ALI in mice.