NOX4 mediates ferroptosis through oxidative stress in diabetic keratopathy

Diabetic keratopathy (DK) affects 47-64% of diabetic patients, yet the cell death mechanisms underlying corneal epithelial dysfunction remain unclear. The corneal epithelium, directly exposed to environmental oxidative stressors, exhibits a uniquely vulnerable redox balance. This study investigated whether ferroptosis contributes to high glucose (HG)-induced corneal epithelial damage and elucidated the role of NADPH oxidase 4 (NOX4) in this process. Human corneal epithelial cells (HCECs) were cultured under HG (50 mM) conditions. Cell viability, ferroptosis markers, and reactive oxygen species levels were assessed. Among multiple cell death inhibitors tested, only the ferroptosis inhibitor Ferrostatin-1 significantly rescued HG-induced cell death. HG treatment induced ferroptotic changes including iron accumulation, lipid peroxidation, GPX4 downregulation, and TfR1 upregulation. Transmission electron microscopy confirmed the characteristic ferroptotic ultrastructural changes in HG-treated HCECs. A bidirectional positive feedback loop was identified between oxidative stress and ferroptosis. Both pharmacological inhibition of NOX4 with GLX-351322 and genetic silencing with siRNA effectively attenuated oxidative stress and ferroptosis, providing complementary evidence for a causal role of NOX4. In diabetic db/db mice, topical Fer-1 accelerated corneal wound healing, and GLX-351322 normalized the ferroptosis-related protein signature. These findings identify NOX4 as the molecular linchpin linking oxidative stress and ferroptosis in DK, representing a promising therapeutic target.