Abstract
Purpose:
Purpose: The purpose of the present study was to investigate whether glutathione peroxidase 4 (GPx4) could be an essential antioxidative enzyme in retinal ganglion cells.
Methods:
Methods: In vitro experiments, an immortalized rat retinal precursor cell line R28 was used. Cells were transfected with siRNA specifically silencing GPx4 or control siRNA. Knockdown was confirmed by real-time RT-PCR and Western blot. Lipid peroxidation was evaluated by immunostaining of 4-hydroxy-2-nonenal (4-HNE). The cytotoxicity was assessed by LDH activity assay and Annexin V/propidium iodide (PI) staining. Cells transfected with GPx4 siRNA or control siRNA were treated with glutamate (final concentration: 2 mM), and the cytotoxicity was evaluated by LDH activity assay. In vivo study, retinal ganglion cell damage was induced by intravitreal injection of 25 mM N-methyl-D-aspartate (NMDA, 2 mL/eye) in GPx4+/+ and GPx4+/− mice. Evaluation of lipid peroxidation (immunostaining of 4-HNE), apoptosis (TUNEL staining) and cell density in the ganglion cell layer (GCL) were performed at 12 hr, 1 day and 7 days after NMDA injection, respectively.
Results:
Results: Knockdown of GPx4 significantly induced cytotoxicity and increased the level of lipid oxidation by 3.2 fold in R28 cells (p<0.01). The level of LDH activity was increased by GPx4 knockdown (13.9 fold, P<0.01). Moreover, knockdown of GPx4 enhanced glutamate-induced cytotoxicity determined by LDH activity (1.52 fold, P<0.01), though glutamate did not influence LDH activity of cells treated with control siRNA. In vivo study, GPx4+/− mice had higher levels of lipid peroxidation than GPx4+/+ mice in the retina treated with NMDA (1.26 fold, P<0.05). GPx4+/− mice had more TUNEL-positive cells induced by NMDA in GCL (1.45 fold, P<0.05). In addition, the cell density in GCL of GPx4+/− mice was significantly lower than that of GPx4+/+ mice after treatment with NMDA (19%, P<0.05).
Conclusions:
Conclusion: These results suggested that GPx4 was essential for maintaining oxidative homeostasis and protecting from glutamate-induced cytotoxicity in retinal ganglion cells.