Role of Glutathione Peroxidase 4 in Corneal Endothelial Cells

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

Declaration of interests

Takatoshi Uchida and Osamu Sakai are employees of Senju Pharmaceutical Co., Ltd. Hirotaka Imai and Takashi Ueta report no conflicts of interest in this study.

Funding

This work was supported by Japan Society for the Promotion of Science KAKENHI Grant Number 26861437.

Additional information

Funding

This work was supported by Japan Society for the Promotion of Science KAKENHI Grant Number 26861437.