Nec-1对模拟缺氧条件下骨骼肌细胞损伤修复的作用研究

Effect of Nec-1 on repair of skeletal muscle cell injury under simulated hypoxia

目的探讨Necrostatin-1(Nec-1)在二氯化钴(CoCl_2)诱导缺氧的条件下对骨骼肌细胞损伤修复的保护作用。方法采用小鼠骨骼肌C2C12成肌细胞为体外模型,CoCl_2诱导缺氧,Nec-1为缺氧保护剂。将培养分化72 h后的细胞分为4组,分别加入等量培养基(Control组)、200μM CoCl_2(CoCl_2组)、150μM Nec-1(Nec-1组)、200μM CoCl_2+150μM Nec-1(CoCl_2+Nec-1组),于光镜下观察不同处理组细胞的肌管形态,采用蛋白免疫印迹技术检测肌细胞生成素(Myog)、肌细胞增强因子2A(MEF2A)蛋白表达的变化,统计细胞周期S期比例,划痕实验观察细胞的损伤修复能力。结果 CoCl_2可诱导肌细胞形态异常,减少肌管分化,与Control组比较,CoCl_2组Myog、MEF2A蛋白表达量降低(P均<0.01),同时,S期细胞比例下降(P<0.01),划痕面积修复能力下降(P均<0.01)。加入Nec-1干预后,细胞形态恢复,肌管分化增多,与CoCl_2组比较,CoCl_2+Nec-1组Myog、MEF2A蛋白表达量及S期细胞比例上调(P均<0.05),细胞划痕面积恢复(P<0.05)。结论 CoCl_2模拟缺氧可抑制肌细胞增殖、分化功能,导致肌细胞损伤修复能力下降。Nec-1可增强缺氧条件下肌细胞的增殖和分化能力,对肌细胞的损伤修复有保护作用,可能通过抑制程序性坏死来促进肌细胞的损伤修复。

Objective To investigate the protective effect of Necrostatin-1（Nec-1）on the repair of skeletal muscle cell injury under simulated hypoxia induced by Cobaltous chloride （CoCl2 ） . Methods In this study, mouse skeletal muscle C2C12 cells were used as an in vitro model, CoCl2 was utilized to induce hypoxia and Nec-1 was utilized as a hypoxic protective agent. Following culture and isolation for 72 h, the cells were divided into 4 groups, which were supplemented with an equal volume of culture medium （control group）, 200 μM CoCl2 （CoCl2 group）, 150 μM Nec-1 （Nec-1 group）, and 200 μM CoCl2＋150 μM Nec-1（CoCl2＋Nec-1 group）, respectively. The morphology of myotubes was observed under light microscope. The changes of the expression levels of myogenin （Myog）, myocyte enhancer factor 2A （MEF2A） proteins were quantitatively detected by Western blotting. The proportion of cells in the S phase was statistically calculated. Wound healing assay was adopted to evaluate the ability of cell damage repair. Results CoCl2 could induce the myoblast abnormalities and decreased the differentiation of myotubes. Compared with the control group, the expression levels of Myog and MEF2A proteins were significantly down-regulated （both P 〈0.01）, the proportion of S phase cells and the repair ability were significantly decreased in the CoCl2 group （both P 〈0.01 ） . Following the Nec-1 intervention, the cellular morphology was restored and the differentiation of myotubes was enhanced. Compared with the CoCl2 group, the expression levels of Myog and MEF2A proteins and the proportion of S phase cells were significantly up-regulated （both P 〈0.05）, and the area of cell scar was significantly restored （ P 〈0.05） after the supplement of Nec-1. Conclusions CoCl2-simulated hypoxia can inhibit the proliferation and differentiation of myocytes, leading to decreased ability of cell injury repair. Nec-1 can enhance the proliferation and differentiation of myocytes and protect myocytes from injury under hypoxic conditions and promote the injury repair of myocytes by suppressing programmed necrosis.