ULK1/Beclin-1/ATG12 Mediated Autophagy Defines the Redox Survival Threshold in Human Osteoblasts

Objective: Oxidative stress and autophagy are crucial determinants of osteoblast survival and bone homeostasis. Hydrogen peroxide (H2O2), a reactive oxygen species (ROS), functions as a signaling molecule at physiological levels but activates cytotoxicity and apoptosis when overproduced. Understanding how osteoblasts adaptively respond to subcytotoxic oxidative stress is critical for elucidating bone metabolism under redox imbalance. This study aims to identify redox-mediated survival thresholds and the molecular interaction between autophagy and apoptosis by examining the autophagic and apoptotic responses in hFOB 1.19 human osteoblast cells exposed to nontoxic concentrations of H2O2. Methods: Following cultivation, the hFOB 1.19 cells were treated with increasing concentrations of H2O2 (0-100 µM). The MTT technique was used to measure cell viability. Sub-cytotoxic concentrations (50 and 60 µM) were selected for qRT-PCR analysis of autophagy-related (ULK1, Beclin-1, ATG12), anti-apoptotic (Bcl-2), and pro-apoptotic (Caspase-3) genes. Results: H2O2 at 75-100 µM significantly decreased cell viability, while 50-60 µM did not induce cytotoxicity. At 50 µM, ULK1 and Beclin-1 were up-regulated by 1.98-fold and 1.40-fold, respectively, while ATG12 showed a slight, non-significant increase. Bcl-2 was sharply increased (2.3-fold) and Caspase-3 remained unchanged. At 60 µM, three autophagy genes and Bcl-2 were significantly elevated, confirming a dose-dependent activation of autophagic signaling and a suppression of apoptosis. Conclusion: Sub-cytotoxic oxidative stress triggers a protective autophagic program rather than apoptotic death in hFOB 1.19 cells. Coordinated upregulation of ULK/Beclin-1/ATG12 and Bcl-2, along with suppressed Caspase-3, promotes redox-adaptive survival in osteoblasts. These results show that autophagy is a key mediator of oxidative resistance and suggest that fine-tuning the redox-autophagy axis may offer a novel therapeutic strategy to alleviate bone degeneration and oxidative skeletal damage.