表皮生长因子对缺氧缺糖模型大鼠骨骼肌细胞氧化损伤的保护作用

摘要

目的:构建体外骨骼肌 L6细胞缺氧缺糖(OGD)模型,在体外水平上探讨表皮生长因子(EGF)对压疮深部组织损伤(DTI)的保护机制.方法:将对数生长期大鼠骨骼肌 L6成肌细胞分为5组,即正常对照组、OGD组、5 μg·L-1EGF +OGD组、10 μg·L-1EGF +OGD组和20 μg·L-1EGF+OGD组.MTT法检测各组细胞生存率,流式细胞术检测各组细胞凋亡率,DCFH-DA 法检测各组 L6成肌骨骼肌细胞中活性氧(ROS)水平,Rhodamine 123检测线粒体膜电势,Western blotting法检测各组骨骼肌细胞中 Bcl-2和 Bax蛋白表达.结果:与正常对照组比较,OGD组 OGD 24 h时骨骼肌细胞生存率明显降低(P<0.01),细胞凋亡率明显升高(P<0.01),ROS水平升高(P<0.01),线粒体膜电势下降(P<0.01),Bcl-2/Bax比值明显下降(P<0.01).与 OGD组比较,不同浓度 EGF组细胞存活率明显升高,细胞凋亡率降低,其中10和20 μg·L-1EGF组差异有统计学意义(P<0.05或P<0.01);不同浓度 EGF组骨骼肌细胞中 ROS水平呈浓度依赖性降低,线粒体膜电势明显增加,10和20 μg·L-1EGF组差异有统计学意义(P<0.05或P<0.01);Bcl-2/Bax比值明显下降,并具有浓度依赖性,其中10和20 μg·L-1EGF组差异有统计学意义(P<0.05或P<0.01).结论:EGF通过降低细胞内ROS水平保护线粒体功能,改善OGD诱导的大鼠骨骼肌细胞损伤,并具有浓度依赖性.%Objective:To set up the rat skeletal muscle L6 cell models of oxygen-glucose deprivation(OGD)in vitro,and to investigate the protective effect of EGF in deep tissue inj ury(DTI)of pressure sores.Methods:The rat skeletal muscle cells in the logarithmic phase were divided into normal control group,OGD group,5 μg·L-1 EGF+OGD group,10 μg·L-1EGF+ OGD group and 20 μg·L-1EGF+ OGD group.The survival rates of skeletal muscle cells in various groups were measured by MTT assay;the cell apoptotic rates in various groups were detected by flow cytometry;the reactive oxygen species(ROS)levels were detected by DCFH-DA;Rhodamine 123 was used to detect the mitochondrial membrane potential;the expressions of Bax and Bcl-2 proteins were determined by Western blotting method.Results:Compared with normal control group,the survival rates of skeletal muscle cells in OGD group after 24 h OGD was significantly decreased(P<0.05);the apoptotic rate was markedly increased(P<0.01);the ROS level was increased(P<0.01);the mitochondrial membrane potential was decreased(P<0.01);the ratio of Bcl-2/Bax was significantly decreased(P<0.01).Compared with OGD group, the survival rates of skeletal muscle cells in different concentrations of EGF groups were increased and the apoptotic rates were decreased,especially in 10 and 20 μg·L-1EGF groups(P<0.05 or P<0.01);the ROS levels in skeletal muscle cells in different concentrations of EGF groups were decreased and the mitochondrial membrane potential were increased,especially in 10 and 20 μg·L-1EGF groups(P<0.05 or P<0.01);the Bcl-2/Bax ratios were significantly decreased in a concentration-dependent manner,especially in 10 and 20 μg·L-1EGF groups (P<0.05 or P<0.01).Conclusion:EGF can improve the skeletal muscle cell injury induced by OGD in a concentration-dependent manner via decreasing the ROS levels and protecting the cell mitochondrial function.