Glutathione peroxidase 4 plays an important role in oxidative homeostasis and wound repair in corneal epithelial cells

Oxidative stress is involved in the pathologies of corneal epithelial cells. However, the importance of specific antioxidant enzymes in corneal epithelial cells is not fully understood. The purpose of this study is to elucidate the role of glutathione peroxidase 4 (GPx4) in corneal epithelial cells. For in vitro experiments, an immortalized human corneal epithelial cell line was used. Cytotoxicity measured through LDH activity, lipid peroxidation immunostained for 4‐hydroxynonenal, cell viability, and cell death were compared between cells transfected with either GPx4 siRNA or scrambled control siRNA. In addition, the rescue effects of α‐tocopherol and ferrostatin‐1, a ferroptosis inhibitor, were examined in the cells with deficient GPx4 expression. For in vivo experiments, we applied n‐heptanol on the cornea of GPx4+/+ and GPx4+/− mice to create corneal epithelial wound. The epithelial defect area size was measured up to 48 h after epithelial wound creation. Knockdown of GPx4 strongly induced cytotoxicity and cell death in human corneal epithelial cells. Cell death induced by GPx4 knockdown was characterized by positive staining for both annexin V and propidium iodide, nuclear translocation of AIF, and without activation of caspase 3, and was rescued by α‐tocopherol and ferrostatin‐1. The delayed wound healing of GPx4 siRNA‐transfected cells were ameliorated by α‐tocopherol in vitro. In addition, loss of one GPx4 allele was sufficient to significantly delay the healing of experimental corneal epithelial wounds in vivo. Our results suggest that the antioxidant enzyme GPx4 plays an important role in oxidative homeostasis, cell survival, and wound healing in corneal epithelial cells. GPx4‐deficient corneal epithelial cells in vitro undergo significant cytotoxicity and nonapoptotic cell death partially rescued by α‐tocopherol and ferrostatin‐1. In vivo, loss of one GPx4 allele is sufficient to significantly delay the healing of experimental corneal epithelial wounds.