摘要
目的:探讨发动蛋白相关蛋白1(DRP1)通过调节线粒体动力学对破骨细胞分化的影响。方法:体外培养稳定生长的小鼠单核巨噬细胞白血病细胞(RAW264.7)。将细胞分为正常组、模型组[核因子受体配体激活因子(RANKL)]、实验组[RANKL ＋线粒体分裂抑制剂1(Mdivi-1)]。使用细胞毒性实验对不同浓度的DRP1抑制剂(Mdivi-1)作用于RAW264.7细胞的结果进行分析，得出安全有效浓度。通过抗酒石酸酸性磷酸酶(TRAP)染色、肌动蛋白环染色和Western Blot实验，观察DRP1对体外破骨细胞分化过程的调控关系。通过透射电镜、流式细胞术和线粒体膜电位荧光探针(JC-1)检测DRP1在破骨细胞分化过程中对线粒体形态、动力学改变的重要作用。TRAP阳性细胞数、肌动蛋白环面积值、JC-1荧光强度及Western Blot结构数据均采用方差分析。结果:细胞计数试剂盒(CCK-8)结果显示10 μmol/L及以下浓度的Mdivi-1对RAW264.7细胞无明显毒性作用(F＝319，P<0.001)，所以选取10 μmol/L浓度的Mdivi-1干预RAW264.7破骨分化进程。在TRAP染色中发现10 μmol/L的Mdivi-1会明显抑制破骨细胞分化，且TRAP阳性细胞数目及破骨细胞的面积明显减少(F＝391.7，P<0.0001)。肌动蛋白环染色结果表明，与模型组比，RANKL＋Mdivi-1组的破骨细胞数量及肌动蛋白环的面积明显变小(F＝321.5、1444，均为P<0.001)。扫描透射电镜观察，与对照相比，RANKL＋ Mdivi-1组线粒体分裂明显减少。通过Western Blot实验，10 μmol/L浓度的Mdivi-1会明显抑制线粒体动力学相关蛋白及破骨细胞分化相关蛋白的表达，包括DRP1、T细胞核因子c1(NFATc1)、组织蛋白酶K(CTSK)(F＝317.8、3510、6404，均为P<0.001)。JC-1荧光实验结果发现，与模型组相比，异常的线粒体膜电位表达有所恢复(F＝2917, P<0.001)。结论:线粒体分裂蛋白DRP1通过影响破骨细胞表面肌动蛋白环的形成参与调节破骨分化；DRP1通过改变线粒体形态和线粒体动力学参与调节破骨分化。
Abstract
Objective:To explore the effect of dynamin-related protein 1 (DRP1) on osteoclasts differentiation by regulating mitochondrial dynamics.Methods:Stably growing mouse monocyte macrophage leukemia cells were cultured in vitro (RAW264.7). The cells were divided into normal, model [(RANKL)], and experimental [RANKL+ mitochondrial division inhibitor 1 (Mdivi-1)] groups. Results of different concentrations of DRP1 protein inhibitor (Mdivi-1) in RAW264.7 cells were analyzed by one-way ANOVA to derive safe and effective concentrations using cytotoxicity assay. The regulatory relationship of DRP1 on the differentiation process of osteoclasts in vitro was observed by anti-tartrate acid phosphatase staining (TRAP), actin ring staining and western blot experiments. The important role of DRP1 on mitochondrial morphology and dynamics alteration during osteoclast differentiation was detected by transmission electron microscopy, flow cytometry and mitochondrial membrane potential fluorescence assay(JC-1). The number of TRAP-positive cells, actin ring area values, JC-1 fluorescence intensity and western blot structural data were analyzed by one-way ANOVA.Results:Cell count kit-8(CCK-8) results showed that 10 μmol/L and lower concentrations of Mdivi-1 had no significant toxic effects on RAW264.7 cells (F=319, P<0.001). Mdivi-1 of 10 μmol/L was selected to intervene in the process of RAW264.7 osteoclastic differentiation. In anti-tartrate acid phosphatase staining, it was found that 10 μmol/L Mdivi-1 significantly inhibited osteoclast differentiation, and the number of TRAP-positive cells and the area of osteoclasts were significantly reduced (F=391.7, P<0.001). The results of actin ring staining showed that number of osteoclasts and area of actin rings in the RANKL+ Mdivi-1 group was significantly smaller compared with the model group (F=321.5, 1444, both P<0.001). Scanning transmission electron microscopy observation that mitochondrial division was significantly reduced in the RANKL+ Mdivi-1 group compared with the control group. By western blot assay, 10 μmol/L concentration of Mdivi-1 significantly inhibited the expression of dynamic-related protein and osteoclast differentiation-related protein, including DRP1, nuclear factor of activated T cells c1(NFATc1) and cathepsin K (CTSK)(F=317.8, 3510, 6404, all P<0.001). JC-1 fluorescence assay revealed that the abnormal mitochondrial membrane potential expression was restored compared with that of the positive control group (F=2917, P<0.001).Conclusion:The mitochondrial splitting protein DRP1 involves in the regulation of osteoclastic differentiation by affecting the formation of actin rings on the surface of osteoclasts; DRP1 can affect osteoclastic differentiation by altering mitochondrial morphology and mitochondrial dynamics.
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