GPX4 Promoter Hypermethylation Induced by Ischemia/Reperfusion Injury Regulates Hepatocytic Ferroptosis

Background and aims: Glutathione Peroxidase 4 (GPX4) is a key factor in Ferroptosis, which is involved in ischemia-reperfusion injury. However, little is known about its role in hepatic ischemia-reperfusion injury (HIRI). This study aimed to investigate the role of GPX4 methylation in Ferroptosis during HIRI.

Methods: For the in vitro experiments, an oxygen and glucose deprivation cell model was established. For the in vivo experiments, an ischemia-reperfusion model was created by subjecting mice to simulated HIRI. Ferroptosis occurrence, GPX4 promoter methylation, and global methylation levels were then assessed.

Results: Ferroptosis was observed in oxygen and glucose deprivation, characterized by a significant decrease in cellular viability (P < 0.05), an increase in lipid peroxidation (P < 0.01), iron overload (P < 0.05), and down-regulation of GPX4 (P < 0.05). This Ferroptosis was exacerbated by GPX4 knockdown (P < 0.01) and mitigated by exogenous glutathione (P < 0.01). Similarly, Ferroptosis was evident in mice subjected to HIRI, with a down-regulation of GPX4 mRNA and protein expression (all P < 0.01), and an upregulation of acyl-CoA synthetase long-chain family member 4 mRNA and protein (all P < 0.01), as well as prostaglandin-endoperoxide synthase 2 mRNA and protein expression (all P < 0.05). Methylation levels increased, evidenced by upregulation of DNA methylation transferase expression (P < 0.05) and down-regulation of Ten-eleven translocation family demethylases (P < 0.01), along with an upregulation of GPX4 promoter methylation.

Conclusions: Ferroptosis may be the primary mode of cell death in hepatocytes following ischemia-reperfusion injury. The methylation of the GPX4 promoter and elevated levels of global hepatic methylation are involved in the regulation of Ferroptosis.

Ferroptosis; Glutathione peroxidase 4; Hepatic ischemia-reperfusion injury; Iron overload; Lipid peroxidation; Methylation; Oxygen and glucose deprivation cell model; Promoter.