Role of Glutathione Peroxidase 4 in Corneal Endothelial Cells

Curr Eye Res. 2017 Mar;42(3):380-385. doi: 10.1080/02713683.2016.1196707. Epub 2016 Jul 15.

Abstract

Purpose: To investigate the role of glutathione peroxidase 4 (GPx4) in corneal endothelial cells.

Materials and methods: An immortalized human corneal endothelial cell line was used. Cells were transfected with either siRNA specifically silencing GPx4 or scrambled control siRNA. Knockdown was confirmed by Real-time RT-PCR and immunoblotting. Lipid peroxidation was evaluated by 4-hydroxy-2-nonenal immunostaining. Cytotoxicity, cell death, and cell proliferation were evaluated using a lactate dehydrogenase (LDH) activity assay, Annexin V staining, and WST-8, respectively. Furthermore, cells transfected with GPx4 siRNA or control siRNA were treated with hydrogen peroxide or ferrous sulfate, and cytotoxicity was evaluated using the LDH activity assay.

Results: The treatment of siRNA decreased the expression of GPx4 at both mRNA and protein levels. The knockdown of GPx4 significantly increased the levels of lipid oxidation and LDH activity. Annexin V-positive cells increased in GPx4 siRNA-treated cells. The proliferation of GPx4 siRNA-treated cells was downregulated compared with that of control siRNA-treated cells. GPx4 knockdown enhanced hydrogen peroxide- and ferrous sulfate-induced cytotoxicity.

Conclusion: These results suggest that GPx4 is an important antioxidant enzyme for maintaining redox status and protecting corneal endothelial cells from oxidative stress.

Keywords: Antioxidant; corneal endothelial cells; glutathione peroxidase; oxidative stress; reactive oxygen species.

MeSH terms

  • Blotting, Western
  • Cell Death
  • Cell Line
  • Cell Proliferation
  • Corneal Diseases / genetics*
  • Corneal Diseases / metabolism
  • Corneal Diseases / pathology
  • Endothelium, Corneal / enzymology*
  • Endothelium, Corneal / pathology
  • Gene Expression Regulation*
  • Glutathione Peroxidase / biosynthesis
  • Glutathione Peroxidase / genetics*
  • Humans
  • Lipid Peroxidation
  • Oxidative Stress*
  • Phospholipid Hydroperoxide Glutathione Peroxidase
  • RNA / genetics*
  • Real-Time Polymerase Chain Reaction

Substances

  • RNA
  • Phospholipid Hydroperoxide Glutathione Peroxidase
  • Glutathione Peroxidase