Department of Orthopedics, Zhangjiagang Hospital of Traditional Chinese Medicine Affiliated to Nanjing University of Chinese Medicine, China.
Eur Rev Med Pharmacol Sci. 2017 Dec;21(24):5548-5556. doi: 10.26355/eurrev_201712_13991.
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.
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.
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.
Autophagy inhibits the oxidative stress-mediated apoptosis of osteoblasts, which is a potential target for the osteoporosis treatment.
氧化应激诱导的成骨细胞凋亡在骨质疏松症的病理过程中起重要作用，但自噬在成骨细胞氧化应激和凋亡中的作用尚不清楚。本研究旨在观察自噬在成骨细胞氧化应激损伤中的作用以及自噬与凋亡的关系。
用不同浓度（0.1、0.5 和 1 mM）的过氧化氢刺激 Mc3T3-E1 细胞。在不同时间点（0、2、6、8 和 12 h）用细胞计数试剂盒 8（CCK8）检测细胞活力，用 Western blot 和流式细胞术检测细胞凋亡，用巨噬细胞来源趋化因子（MDC）和透射电子显微镜检测自噬。用 Western blot 检测自噬相关蛋白 Beclin1 和 LC3II/I 比值的变化。此外，用自噬调节剂雷帕霉素（Rap）和 3-甲基腺嘌呤（3-MA）观察细胞内活性氧（ROS）水平和细胞外超氧化物歧化酶（SOD）水平，以阐明自噬与细胞氧化之间的相互作用。
过氧化氢诱导成骨细胞凋亡和自噬呈剂量和时间依赖性；过氧化氢抑制剂可抑制自噬水平，自噬抑制剂（3-MA）可显著增强细胞内 ROS 水平和过氧化氢诱导的凋亡率。此外，Western blot 证实，caspase-3 剪切和多聚腺苷二磷酸核糖聚合酶（PARP）的剪切蛋白增加。自噬诱导剂（Rap）部分抑制了过氧化氢诱导的氧化应激和凋亡。
自噬抑制氧化应激介导的成骨细胞凋亡，这可能成为骨质疏松症治疗的潜在靶点。
