白藜芦醇通过HIF-1α/BNIP3信号通路促进骨骼肌缺氧损伤修复的机制

目的 探讨白藜芦醇（Res）对二氯化钴（CoCl₂）诱导的骨骼肌细胞缺氧损伤的保护机制。方法 将分化的小鼠骨骼肌细胞系C2C12按不同干预方法分为4组,即对照组、 CoCl₂组、 Res组、 CoCl₂+Res组。观察免疫荧光染色后的细胞形态,统计肌管融合指数;采用荧光定量PCR技术检测肌球蛋白重链（MyHC） mRNA水平的变化;采用蛋白免疫印迹法检测低氧诱导因子-1α（HIF-1α）、 Bcl2/腺病毒E1B相互作用蛋白3（BNIP3）、微管相关蛋白1轻链3（LC3）以及p62和Beclin1蛋白水平。结果 CoCl₂诱导的缺氧损伤使肌细胞形态异常、肌管分化减少,与对照组比较,CoCl₂组肌管融合指数降低（P < 0.001）,MyHC mRNA水平和蛋白表达量降低（P均 < 0.05）,HIF-1α、BNIP3、Beclin1蛋白水平和LC3升高（P均 < 0.001）,p62蛋白水平降低（P < 0.001）。加入Res处理后,细胞形态恢复,肌管分化增多;与CoCl₂组比较,CoCl₂+Res组肌管融合指数升高,MyHC亚型（Myh7、Myh2、Myh4）的mRNA和MyHC蛋白水平升高,HIF-1α、BNIP3和Beclin1蛋白水平降低,p62蛋白水平升高（P均 < 0.05）。结论 CoCl₂诱导的缺氧可抑制MyHC表达,导致肌细胞分化和融合能力下降。Res可增强缺氧条件下成肌细胞的分化和融合能力,对肌细胞的损伤修复有保护作用,可能通过抑制HIF-1α/BNIP3信号通路诱导的自噬来促进肌细胞的损伤修复。

Resveratrol promotes the repair of hypoxia-induced skeletal muscle injury through HIF-1α/BNIP3 signaling pathway

Objective To investigate the protective mechanism of resveratrol （Res） against the CoCl₂-induced hypoxia injury in the skeletal muscle cells. Methods According to different intervention methods, the murine skeletal muscle C2C12 cells were divided into the control, CoCl₂, Res and CoCl₂+Res groups, respectively. The cell morphology was observed after myosin heavy chain （MyHC） immunofluorescence staining. The fusion index of the myotubes was calculated. The expression level of MyHC mRNA was detected by quantitative fluorescence PCR. The expression levels of hypoxia-inducible factor-1α （HIF-1α）, Bcl2/ adenovirus E1B interacting protein 3 （BNIP3）, microtubule associated protein 1 light chain 3 （MAP1LC3, LC3）, p62 and Beclin1 proteins were measured by Western blot. Results After CoCl₂-induced hypoxia, the morphology of myotubes was abnormal and the quantity of differentiated myotubes was reduced. Compared with the control group, the fusion index of differentiated myotubes was significantly decreased （P < 0.001）, the expression levels of MyHC mRNA and protein were significantly down-regulated （both P < 0.05）, those of HIF-1α, BNIP3 and Beclin1 proteins and LC3 were significantly up-regulated （all P < 0.001）, and that of p62 protein was significantly down-regulated （P < 0.001） in the CoCl₂ group, respectively. Following the Res intervention, the cellular morphology was recovered and the quantity of differentiated myotubes was increased. Compared with the CoCl₂ group, the fusion index of differentiated myotubes was significantly elevated, the expression levels of mRNA of MyHC subtypes （Myh7, Myh2 and Myh4） and MyHC protein were significantly up-regulated, those of HIF-1α, BNIP3 and Beclin1 proteins were significantly down-regulated, and that of p62 protein was significantly up-regulated （all P < 0.05）. Conclusions CoCl₂-induced hypoxia can down-regulated the expression of MyHC, resulting in decreased muscle differentiation and fusion. Rescan enhance the differentiation and fusion of myoblasts under hypoxia condition, and extert protective effect upon the repair of muscle cell injury probably by suppressing the autophagy induced by the HIF-1α/BNIP3 signaling pathway, thereby accelerating the repair of muscle cell injury.