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目的:研究OFSS对小鼠MC3T3成骨细胞TNF-α反应性的影响,为骨力学负荷的抗炎性骨丢失作用提供实验室证据。方法:利用特制的细胞OFSS加载实验装置,在应用TNF-α+CHX诱导MC3T3细胞凋亡之前或过程中,向细胞施加OFSS(10~12 dynes/cm2,1 Hz),观察细胞形态学变化、以及应用SDS-PAGE结合Western blot方法分析凋亡标志性蛋白和IκBα信号的变化,分析OFSS对MC3T3细胞TNF-α反应性的影响。结果:2 h OFSS预处理几乎完全抑制TNF-α诱导的Caspase-3激活和Parp降解,而且Caspase-3的上游关键分子Caspase-8的激活几乎完全抑制;细胞凋亡形态学变化被抑制;TNF-α诱导的IκBα磷酸化也被显著抑制,表明OFSS可作用于IκBα信号上游。此外,在TNF-α/CHX诱导凋亡2 h后开始施加OFSS仍可有效抑制caspase-8的激活、从而抑制细胞凋亡,提示OFSS还可能直接作用于凋亡诱导通路而发挥细胞保护作用。结论:OFSS抑制MC3T3成骨细胞的TNF-α反应性,可能减轻炎症性骨丢失,其作用机制可能涉及多个环节。
Objective: To investigate the effect of OFSS on the TNF-α responsiveness of mouse MC3T3 osteoblasts and to provide laboratory evidence for the anti-inflammatory bone loss effect of osteoblastic load. METHODS: OFSS (10-12 dynes / cm2, 1 Hz) was applied to the cells before and during the induction of MC3T3 cell apoptosis by TNF-α + CHX by using OFSS loading device. The morphological changes of cells were observed, The changes of apoptotic marker protein and IκBα signal were analyzed by SDS-PAGE and Western blot. The effect of OFSS on TNF-α reactivity in MC3T3 cells was analyzed. Results: 2 h OFSS treatment almost completely inhibited TNF-α-induced Caspase-3 activation and Parp degradation, and Caspase-3 upstream key molecule Caspase-8 activation almost completely inhibited; apoptosis morphological changes were inhibited; TNF α-induced IκBα phosphorylation was also significantly inhibited, indicating that OFSS can act on the IκBα signal upstream. In addition, OFSS was also inhibited after 2-h TNF-α / CHX-induced apoptosis and inhibited caspase-8 activation, which indicated that OFSS may play a direct role in the apoptosis-inducing pathway. CONCLUSION: OFSS inhibits the TNF-αreactivity of MC3T3 osteoblasts and may reduce inflammatory bone loss. Its mechanism may involve in several aspects.