运动上调GPR48-RANKL通路影响 2型糖尿病小鼠破骨细胞分化

陈祥和; 孙 朋; 杨念恩; 李世昌; 徐会金; 张娜娜

doi:10.15942/j.jcsu.2017.03.014

运动上调GPR48-RANKL通路影响 2型糖尿病小鼠破骨细胞分化

Effects of Exercise on Osteoclast Differentiation through the Up-regulation GPR48-RANKL Pathway in Type 2 Diabetic Mice

摘要

摘要: 目的: 探究T2DM小鼠OC分化变化及不同力学刺激通过GPR48-RANKL通路对T2DM小鼠OC分化的分子调控作用。 方法: 40只4周龄雄性C57BL/6小鼠,适应性喂养1周后随机分为T2DM造模组(30只)和正常对照组(ZC, 10只)。T2DM造模组小鼠6周高脂膳食结束空腹12 h后注射STZ,2周后检测小鼠血糖,有27只造模成功并将其随机分为T2DM对照组(TC, 9只)、T2DM游泳组(TS, 9只)和T2DM游泳组(TD, 9只),TS和TD组小鼠分别进行8周游泳和下坡跑训练。结束后,取右侧股骨并利用RT-PCR法检测相关因子mRNA表达;取左侧胫骨并利用West-blotting法检测相关因子蛋白表达;取小鼠BMM并诱导其向OC分化,利用TRAP染液对OC染色;取右侧胫骨并利用游标卡尺检测其大小。 结果: 与ZC组相比,TC组GPR48、OPG、RANKL、RANK、NFATc2、CTSK的mRNA和GPR48、RANKL、RANK蛋白表达均显著变化(P<0.05或P<0.01),OC数量显著增多,远端冠状面宽度和近端冠状面宽度显著变小(P<0.05)。与TC组相比,TS组GPR48、OPG、RANKL、NFATc2、CTSKmRNA和GPR48、RANKL蛋白表达均显著变化(P<0.05或P<0.01),OC数量显著减少;TD组GPR48、OPG、RANKL、RANK、NFATc2、CTSKmRNA和GPR48、RANKL、RANK蛋白表达均显著上调(P<0.05或P<0.01),OC数量显著减少,胫骨长度和中间矢状轴宽度显著减少(P<0.05或P<0.01)。与TS组相比,TD组OPG和CTSKmRNA及GPR48、RANK、RANKL蛋白表达均显著变化(P<0.05或P<0.01),OC数量显著减少。 结论: T2DM小鼠OC分化显著增强;直接作用力激活T2DM小鼠骨中GPR48-RANKL通路,进而抑制RANK及其下游靶基因表达,抑制OC分化,且其作用效果优于间接作用力。

Abstract: Objective: :To investigate the change of OC differentiation in T2DM mice and effect of different mechanical stimulation on OC differentiation by GPR48- RANKL pathway in T2DM mice. Methods: Forty four-week old C57BL/6 male mice were randomly divided into normal control group (ZC) and T2DM group. Use the method of high-fat diet and STZ to build the model of T2DM mice, and the T2DM mice were randomly divided into T2DM control group (TC), T2DM swimming group (TS) and T2DM downhill run group (TD). And use the swimming and downhill running to train the T2DM mice for eight weeks. After this, use the RT-PCR to test the mRNA expression in the left tibia. Use the WB to test the protein expression in right femur. Take the BMM and induced it differentiation into osteoclast, use the TRAP solution to dye the osteoclasts. Use the vernier caliper to test the shape size of right tibia. Results: Compared to ZC group, the mRNA expression of GPR48、OPG、RANKL、RANK, NFATc2, CTSK and protein expression of GPR48、RANKL、RANK of TC group were all significantly changed (P<0.05 or P<0.01), the number of OC was significantly increased, the width of distal coronary surface and the surface of the proximal coronary was significantly reduced(P<0.05). Compared to TC group, the mRNA expression of GPR48, OPG, RANKL, NFATc2, CTSK and the protein expression of GPR48 and RANKLof TS group were significantly changed(P<0.05 or P<0.01). The number of osteoclast was significantly reduced. The mRNA expression of GPR48, OPG, RANKL, RANK, NFATc2, CTSK and the protein expression of GPR48, RANKL, RANK were all significantly changed(P<0.05 or P<0.01). The number of osteoclast was significantly reduced, the length of tibia and width of the middle sagittal axis were significantly increased(P<0.05). Compared to TS group, the mRNA expression of OPG, CTSK and the protein expression of GPR48, RANK, RANKL of TD group were significantly changed(P<0.05 or P<0.01), OC number were drastically reduced. Conclusion: The osteoclast differentiation of type 2 diabetic mice was significantly increased. The direct mechanical stimulation activited the GPR48-RANKL pathway, which inhibited the RANK and their target gene expression, and then inhibited the osteoclast differentiation of type 2 diabetic mice. The effect of direct mechanical stimulation was better than that of indirect one.

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