Melatonin Attenuates H2O2-Induced Oxidative Stress by Restoring Redox Balance, Mitochondrial Integrity and Reducing Apoptosis in Buffalo Fibroblasts.

Abstract

Oxidative stress critically affects cellular viability and function under in vitro culture conditions, often compromising physiological integrity of somatic cells used in livestock biotechnology. This study aimed to investigate hydrogen peroxide (H₂O₂)-induced oxidative stress in buffalo fibroblasts and evaluated the cytoprotective effects of melatonin, focusing on redox homeostasis, mitochondrial function, apoptosis, and antioxidant defence. Fibroblasts were exposed to graded concentrations of H₂O₂ (100-1000 µM) for 2 h, followed by treatment for 72 h in culture media with and without melatonin (10^-9 M). Oxidative stress markers, including GSSG/GSH ratio, ROS generation, mitochondrial membrane potential (MMP), and apoptosis, were assessed using flow cytometry and biochemical assays, while antioxidant (GPx, SOD, CAT) and apoptotic (BAX, Caspase 9) gene expression was analyzed by qPCR. H₂O₂ exposure induced a dose-dependent increase in oxidative stress, evidenced by elevated ROS, redox imbalance, mitochondrial depolarization, and enhanced apoptosis. Severe oxidative damage was observed at higher H₂O₂ (500-1000 µM) concentrations. Melatonin (MT) significantly (p ≤ 0.05) alleviated oxidative stress under mild to moderate conditions (100-200 µM H₂O₂) by restoring redox homeostasis, preserving mitochondrial integrity, suppressing ROS accumulation, enhancing antioxidant defence, and reducing apoptosis. However, its protective efficacy was lost under severe oxidative stress, indicating a defined redox threshold beyond which cellular damage becomes irreversible. These findings suggest that melatonin exerts cytoprotective effect against oxidative stress within a limited oxidative window and provide mechanistic insights for improving fibroblasts culture systems in livestock biotechnology and regenerative applications.