二甲双胍通过调控MSTN抑制小鼠骨骼肌细胞C2C12分化

目的：探讨二甲双胍对小鼠骨骼肌细胞C2C12分化的影响及其分子机制。方法：将小鼠骨骼肌C2C12细胞系分为对照组和二甲双胍（5 mmol/L）组，用含5%马血清分化培养基诱导C2C12细胞分化，观察肌管形成情况。qPCR检测肌球蛋白重链（MyHC）和肌肉生长抑制素（MSTN）mRNA水平。Western印迹检测My HC、Ⅰ型肌球蛋白重链（MyHCⅠ）、Ⅱa型肌球蛋白重链（MyHCⅡa）、Ⅱb型肌球蛋白重链（MyHCⅡb）、Ⅱx型肌球蛋白重链（MyHCⅡx）和MSTN蛋白表达。应用siRNA下调MSTN的表达，并将小鼠骨骼肌C2C12细胞系分为MSTN敲低阴性对照组（siNC组），MSTN敲低阴性对照+二甲双胍组（siNC+metformin组），MSTN敲低组（siMSTN组），MSTN敲低+二甲双胍组（siMSTN+metformin组），检测二甲双胍是否通过升高MSTN抑制C2C12细胞分化。结果：显微镜观察显示，与对照组相比，二甲双胍组肌管数量减少、肌管直径较小。qPCR结果显示，与对照组相比，二甲双胍组My HC mRNA水平降低（t=29.47,P<0.000 1）,...

Metformin inhibits the differentiation of C2C12 murine skeletal muscle cells by regulating MSTN

Objective: To investigate the effect of metformin on differentiation of murine C2C12 skeletal muscle cells and its potential molecular mechanism. Methods:Murine skeletal muscle C2C12 cells were divided into control group and metformin（5 mmol/L） group. The differentiation medium containing 5% horse serum was used to induce the differentiation of C2C12 cells and the formation of myotubes was observed. The mRNA levels of myosin heavy chain（MyHC） and myostatin（MSTN） were detected by qPCR.The protein expression of MyHC I，MyHCⅡa，MyHC Ⅱb，MyHCⅡx and MSTN were detected by Western blotting. Murine skeletal muscle C2C12 cell lines were divided into MSTN knockdown negative control group（siNC group），MSTN knockdown negative control +metformin group（siNC+metformin group），MSTN knockdown group（siMSTN group），MSTN knockdown+metformin group（siMSTN+metformin group），and the expression of MSTN was down-regulated by siRNA to investigate whether metformin inhibited the differentiation of C2C12 cells by regulating MSTN. Results:Compared with the control group，the metformin group had less myotube number and diameter. The qPCR results showed that compared with the control group，the level of MyHC mRNA in metformin group decreased（t=29.47，P<0.000 1），while the level of MSTN mRNA increased（t=14.14，P<0.001）. Western blotting results showed that compared with the control group，the expression of MyHC protein in metformin group decreased（t=12.57，P<0.001），the expression of MSTN protein increased（t=6.555，P<0.01）. The protein expression of MyHC Ⅰ，MyHCⅡa，MyHCⅡb and MyHCⅡx decreased（t=9.288，6.140，8.348，14.75，all P<0.01）. The expression of MSTN was down-regulated by siRNA. Compared with siNC group，the expression of MyHC in siNC+metformin group was significantly decreased（P<0.001）. Compared with siNC+metformin group，the expression of MyHC protein in siMSTN+metformin group was increased（F=12.44，P<0.001）. Conclusion:Metformin inhibits the differentiation of murine C2C12 skeletal muscle cells by regulating MSTN.