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Osamu Sakai, Takashi Ueta, Shiro Amano; Role of glutathione peroxidase 4 in corneal endothelial cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2031.
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Reactive oxygen species (ROS) occur as a byproduct of cellular energy metabolism, and react with the polyunsaturated fatty acids of lipid membranes and induce lipid peroxidation. Lipid peroxidation is known to be toxic to a lot of 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 corneal endothelial cells.
An immortalized human corneal endothelial cell line (HCE) cells were used. HCE cells were transfected with GPx4 siRNA or control siRNA, and we tested for lipid peroxidation, proliferation, and cytotoxicity. Lipid peroxidation was confirmed by immunostaining of 4-hydroxy-2-nonenal (4-HNE). Cell proliferation was evaluated by WST-8. Evaluation of cell cytotoxicity was conducted by assay of LDH activity and staining of annexin V. In oxidative stress study, cells transfected by siRNA were treated with hydrogen peroxide or ferric sulfide, and their cytotoxicity was evaluated by assay of LDH activity.
GPx4 knockdown caused 1.5-fold increase in the levels of lipid oxidation (P<0.01 vs control). The proliferation of Gpx4 siRNA-treated cells was reduced by 44% as compared with control siRNA-treated cells (P<0.01 vs control). The levels of LDH activity were increased 2.2-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. In oxidation stress study, GPx4 siRNA knockdown enhanced cytotoxicity induced by hydrogen peroxide (3.0 fold, P<0.01 vs control) or ferric sulfide (2.9 fold, P<0.01 vs control).
GPx4 knockdown decreased the proliferation of cells, and caused the cytotoxicity. In addition, GPx4 siRNA knockdown enhanced cytotoxicity induced by oxidative stress. These results suggest that GPx4 is involved in maintaining homeostasis and antioxidant defense of HCE cells.
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