Abstract
Purpose:
Antioxidant enzymes are involved in the homeostasis of corneal epithelial cells. Glutathione peroxidase 4 (GPx4) is one of the antioxidant enzymes that can directly reduce lipid peroxidation caused by oxidative stress. The purpose of the present study was to investigate the role of GPx4 in maintaining homeostasis of corneal epithelial cells.
Methods:
Simian virus 40 immortalized human corneal epithelial (HCE) cells were used. HCE cells were transfected with GPx4 siRNA, and we tested for lipid oxidation, wound healing, and cytotoxicity. Lipid oxidation was confirmed by immunostaining of 4-hydroxy-2-nonenal (4-HNE). In the wound healing model, hole defects were formed in confluent HCE cells, and wound areas were evaluated 2 days after defect formation. Evaluation of cell cytotoxicity was conducted by assay of LDH activity, staining of annexin V/PI, immunostaining of apoptosis inducing factor (AIF), and western blot of caspase 3. In oxidative stress study, cultured HCE cells were transfected with GPx4 overexpressing plasmid and GPx4 siRNA, and then were treated with hydrogen peroxide. Cytotoxicity was evaluated by assay of LDH activity 24 hours after the treatment.
Results:
GPx4 knockdown caused 2.3-fold increase in the levels of lipid oxidation (P<0.01 vs control). The mean wound area of GPx4 knockdown group was larger than that of control group (18.9 ± 1.2 % and 9.3 ± 3.2% as a ratio of the initial damaged area, respectively; P<0.01), showing that GPx4 knockdown delayed wound healing. The levels of LDH activity were increased 4.6-fold by GPx4 knockdown (P<0.01 vs control). Moreover, cell death in Gpx4 siRNA treated cells was characterized by positive staining for annexin V, and mediated by AIF, but not caspase. In oxidation stress study, GPx4 overexpression prevented hydrogen peroxide induced-cytotoxicity by 31% (P<0.01 vs control). Conversely, GPx4 siRNA knockdown enhanced cytotoxicity by 6.3 fold (P<0.01 vs control).
Conclusions:
GPx4 knockdown induced delay of wound healing and cytotoxicity in corneal epithelial cells, and GPx4 overexpression prevent cytotoxicity by oxidative stress. These results suggest that GPx4 is involved in migration and survival of HCE cells.
Keywords: 482 cornea: epithelium •
634 oxidation/oxidative or free radical damage •
514 enzymes/enzyme inhibitors