Autophagy attenuates the oxidative stress-induced apoptosis of Mc3T3-E1 osteoblasts

D.-Y. Li, J.-C. Yu, L. Xiao, W. Miao, K. Ji, S.-C. Wang, Y.-X. Geng

Department of Orthopedics, Zhangjiagang Hospital of Traditional Chinese Medicine Affiliated to Nanjing University of Chinese Medicine, China. yixiao1981@163.com

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• Orthopedics

OBJECTIVE: The oxidative stress-induced osteoblast apoptosis plays an important role in the pathological process of osteoporosis, but the roles of autophagy in oxidative stress and apoptosis of osteoblasts remain unclear. This study aimed to observe the role of autophagy in oxidative stress injury of osteoblasts and the relationship between autophagy and apoptosis.

MATERIALS AND METHODS: Mc3T3-E1 cells were stimulated with different concentrations (0.1, 0.5, and 1 mM) of hydrogen peroxide. The cell viability was detected via cell counting kit 8 (CCK8) at different time points (0, 2, 6, 8, and 12 h), the apoptosis was detected via Western blotting and flow cytometry, and the autophagy was detected via macrophage-derived chemokine (MDC) and transmission electron microscope. The changes in expression of autophagy-associated protein, Beclin1, and LC3II/I ratio, were detected via Western blotting. Moreover, the intracellular reactive oxygen species (ROS) level and extracellular superoxide dismutase (SOD) level were observed using the autophagy regulators, rapamycin (Rap) and 3-methyladenine (3-MA), so as to clarify the interaction between autophagy and cellular oxidation.

RESULTS: Hydrogen peroxide-induced apoptosis and autophagy of osteoblasts were in dose- and time-dependent manners; the hydrogen peroxide inhibitors could inhibit the autophagy level, and autophagy inhibitor (3-MA) could significantly enhance the hydrogen peroxide-induced ROS level and apoptosis rate in cells. Besides, Western blotting confirmed that the cleaved caspase-3 and cleaved poly adenosine diphosphate ribose polymerase (PARP) proteins were increased. The autophagy inducer (Rap) partially inhibited the hydrogen peroxide-induced oxidative stress and apoptosis.

CONCLUSIONS: Autophagy inhibits the oxidative stress-mediated apoptosis of osteoblasts, which is a potential target for the osteoporosis treatment.

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