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中华关节外科杂志(电子版)

中华关节外科杂志(电子版) ›› 2024, Vol. 18 ›› Issue (01) : 60 -68. doi: 10.3877/cma.j.issn.1674-134X.2024.01.009

基础论著

线粒体动力学相关蛋白影响破骨细胞分化机制探讨
周月惠1, 江梦钰1, 薛宇轩1, 卫杨文祥1, 凡一诺2, 万子艺1, 刘予豪3, 陈镇秋3, 周驰4,†()   
  1. 1. 510405　广州中医药大学第一临床医学院；510388　广州中医药大学岭南医学研究中心
    2. 510378　广州中医药大学第三附属医院
    3. 510405　广州中医药大学第一附属医院
    4. 510405　广州中医药大学第一附属医院；525022　广州中医药大学茂名医院（茂名市中医院）
  • 收稿日期:2024-01-08 出版日期:2024-02-01
  • 通信作者: 周驰
  • 基金资助:
    国家自然科学基金(82374475); 广东省中医药局中医药科研项目(20233001); 广州市科技计划项目(202201020314)

Study on mechanism of mitochondrial dynamics related protein affects osteoclast differentiation

Yuehui Zhou1, Mengyu Jiang1, Yuxuan Xue1, Wenxiang Weriyang1, Yinuo Fan2, Ziyi Wan1, Yuhao Liu3, Zhenqiu Chen3, Chi Zhou4,†()   

  1. 1. TheFirst Clinical Medical College of Guangzhou University of Chinese Medicine, Guangzhou 510405, China; Lingnan Medical Research Center, Guangzhou University of Chinese Medicine, Guangzhou 510388, China
    2. The Third Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510378, China
    3. The First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510405, China
    4. The First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510405, China; Maoming Hospital of Guangzhou University of Traditional Chinese Medicine (Maoming Hospital of Traditional Chinese Medicine), Maoming 525022, China
  • Received:2024-01-08 Published:2024-02-01
  • Corresponding author: Chi Zhou
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引用本文：

周月惠, 江梦钰, 薛宇轩, 卫杨文祥, 凡一诺, 万子艺, 刘予豪, 陈镇秋, 周驰. 线粒体动力学相关蛋白影响破骨细胞分化机制探讨[J]. 中华关节外科杂志(电子版), 2024, 18(01): 60-68.

Yuehui Zhou, Mengyu Jiang, Yuxuan Xue, Wenxiang Weriyang, Yinuo Fan, Ziyi Wan, Yuhao Liu, Zhenqiu Chen, Chi Zhou. Study on mechanism of mitochondrial dynamics related protein affects osteoclast differentiation[J]. Chinese Journal of Joint Surgery(Electronic Edition), 2024, 18(01): 60-68.

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。

关键词: 线粒体, 线粒体动力学, 破骨细胞
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.

Key words: Mitochondria, Mitochondrial dynamics, Dynamins, Osteoclasts
图1 线粒体分裂参与调节破骨分化。图A为抗TRAP(酒石酸酸性磷酸酶)染色(放大图标＝50 μm)，示Mdivi-1浓度在10 μmol/L时对RAW264.7细胞破骨分化呈抑制作用；图B为图A中破骨细胞数量；图C为细胞毒性CCK-8实验结果；图D为Mdivi-1干预RAW264.7细胞的NFATc1和CTSK表达的蛋白电泳结果；图E为破骨细胞分化相关蛋白NFATc1表达水平；图F为CTSK的表达水平注：****-P<0.001；Mdivi-1-线粒体分裂抑制剂1；CCK-细胞计数试剂盒；RANKL-核因子受体配体激活因子；NFATc1-活化T细胞核因子c1；CTSK-组织蛋白酶K
Figure 1 Mitochondrial fission participates in the regulation of osteoclast differentiation. A is the anti-tartrate acid phosphatase staining(enlarged icon＝50 μm), showing that Mdivi-1 of 10 μμmol/L was inhibitory to osteoclastic differentiation of RAW264.7 cells; B shows the number of osteoclasts in A; C is the results of cytotoxicity CCK-8 assay; D is the western blot results of NFATc1 and CTSK expressions after intervention of Mdivi-1 on RAW264.7 cells; E is the expression of NFATc1; F is the expression level of CTSK Note: ****-P<0.001；Mdivi-1- mitochondrial division inhibitor 1；CCK-cell count kit；RANKL- nuclear factor receptor ligand activating factor；NFATc1- nuclear factor of activated T cells c1；CTSK- cathepsin K protein
图2 线粒体分裂参与影响破骨分化的表面肌动蛋白环形成。图A为破骨细胞表面肌动蛋白环染色(标尺：200 μm)；图B为破骨细胞肌动蛋白环面积值；图C为破骨细胞数量注：****-P<0.001；RANKL-核因子受体配体激活因子；DAPI-4，6-二脒基-2-苯基吲哚；F-actin-纤维肌动蛋白
Figure 2 Mitochondrial division participating in surface actin ring formation affects osteoclast differentiation. A shows actin ring staining on the surface of osteoclasts(scale bar：200 μm); B is actin ring area values of osteoclasts; C is the number of osteoclasts Note: ****-P<0.001；RANKL- nuclear factor receptor ligand activating factor；DAPI-4，6-diamidino-2-phenylindole
图3 DRP1(发动蛋白相关蛋白1)调节破骨分化中的线粒体形态变化。图A为透射电镜结果(第一行放大区域间距＝5 μm，第二行放大区域间距＝1 μm)；图B为破骨分化过程中DRP1蛋白的表达；图C为破骨细胞DRP1蛋白表达水平注：****-P<0.001；RANKL-核因子受体配体激活因子
Figure 3 DRP1 regulates Mitochondrial morphologyl changes during osteoclast differentiation. A is the results of transmission electron microscopy (the first row magnification＝5 μm, the second row magnification＝1 μm); B is the expression of DRP1 protein during osteoclastic differentiation; C is the expression level of DRP1 protein in osteoclasts Note: ****-P<0.001；RANKL- nuclear factor receptor ligand activating factor
图4 DRP1(发动蛋白相关蛋白1)调节破骨分化中的线粒体动力学改变。图A为流式细胞仪器检测的细胞ROS含量；图B为流式细胞仪检测的细胞ROS的平均荧光强度；图C为细胞的线粒体膜电位水平荧光图；图D、E为图C中红、绿色荧光强度注：****-P<0.001；RANKL-核因子受体配体激活因子；ROS-活性氧
Figure 4 DRP1 regulates mitochondrial dynamics during osteoclast differentiation. A shows the cellular ROS content detected by the flow cytometry instrument; B is the average fluorescence intensity of cellular ROS detected by the flow cytometry; C shows the horizontal fluorescence of the mitochondrial membrane potential of the cell; D and E are the intensities of red and green fluorescence in C Note: ****-P<0.001；RANKL- nuclear factor receptor ligand activating factor；ROS -reactive oxygen species
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