O-GlcNAc Signaling Augmentation Protects Human Corneal Endothelial Cells from Oxidative Stress via AKT Pathway Activation

Abstract

Purpose: To investigate the effect of inhibitor of O-glycosylation on human corneal endothelial cells (HCECs) under oxidative stress.Methods: HCECs were cultured and treated with 10 mM tert-butyl hydroperoxide (tBHP) with or without PUGNAc, a known inhibitor of OGA. Cell viability was assessed. Mitochondrial membrane potential (ΔΨ_m) was measured. Intracellular Ca²⁺ levels and mitochondrial Ca²⁺ levels were measured. Intracellular reactive oxygen species formation was measured. Levels of O-linked β-N-acetylglucosamine (O-GlcNAc), AKT, and pAKT were evaluated by Western blotting.Results: O-GlcNAc augmentation by PUGNAc increased cell viability, attenuated the loss of ΔΨ_m, and intracellular ROS against tBHP-induced oxidative stress (p < .05). O-GlcNAc augmentation reduced tBHP-induced mitochondrial calcium overload (p < .05) while it did not have any effect on intracellular calcium overload with tBHP. Furthermore, AKT signaling was activated in the cells with O-GlcNAc augmentation.Conclusions: O-GlcNAc signaling augmentation protects HCECs from oxidative stress via activation of AKT pathways.

Keywords: AKT pathway; Corneal endothelial cells; O-linked β-N-acetylglucosamine; corneal endothelial wound healing; oxidative stress.