上调Yes相关蛋白对缺氧复氧心肌细胞损伤保护作用的实验研究

Protective effect of up-regulation of Yes-associated protein on cardiomyocyte injury induced by hypoxia/reoxygenation

目的研究Yes相关蛋白(YAP)在缺氧复氧(H/R)心肌细胞损伤中的作用。方法用过表达YAP重组慢病毒感染心肌细胞,给予H/R处理,用real-time PCR和Western blot检测细胞中YAP表达情况。CCK8法测定增殖变化,二硝基苯肼显色法检测乳酸脱氢酶(LDH)漏出率,流式细胞术检测凋亡变化,Western blot检测活化型Caspase-3和Caspase-9蛋白水平,DCFH-DA法检测活性氧(ROS)水平,黄嘌呤氧化法检测超氧化物歧化酶(SOD)活性,JC-1法检测线粒体膜电位,Western blot法检测胞浆和线粒体中细胞色素C(Cytochrome C)蛋白水平。结果过表达YAP重组慢病毒感染可以提高H/R条件下心肌细胞中YAP表达水平。H/R处理后的心肌细胞增殖活性降低,LDH漏出率升高,细胞凋亡率升高,细胞中活化型Caspase-3和Caspase-9蛋白水平升高,ROS水平也升高,SOD活性降低,线粒体膜电位下降,胞浆中Cytochrome C蛋白水平升高,线粒体中Cytochrome C蛋白水平降低。上调YAP可以提高H/R条件下心肌细胞增殖活性,降低LDH漏出率,减少细胞凋亡,降低细胞中活化型Caspase-3和Caspase-9蛋白水平表达,提高SOD活性,减少细胞中ROS水平,提高线粒体膜电位,降低胞浆中Cytochrome C蛋白水平,提高线粒体中Cytochrome C蛋白水平。结论上调YAP减轻缺氧复氧心肌细胞损伤,减少细胞凋亡,作用机制可能与提高抗氧化酶活性,减少细胞内ROS水平,抑制线粒体凋亡途径有关。

Objective To study the effect of Yes-associated protein(YAP) on cardiomyocyte injury induced by hypoxia/reoxygenation(H/R). Methods The cardiomyocytes were infected with over-expressed YAP recombinant lentivirus and treated with H/R. The expression of YAP was detected by using real-time fluorescence quantitative polymerase chain reaction(RT-PCR) and Western blotting assay. The proliferation changes of cardiomyocytes were detected by using CCK8 method, and leakage rate of lactic dehydrogenase(LDH) was detected by using dinitrophenylhydrazine(DNPH) method. The apoptosis changes of cardiomyocytes were detected by using flow cytometry(FCM), and levels of activated Caspase-3 protein and Caspase-9 protein were detected by using Western blotting assay. The level of reactive oxygen species(ROS) was detected by using DCFH-DA method, activity of superoxide dismutase(SOD) was detected by using xanthine oxidation(XTO) method, mitochondrial membrane potential(MMP) was detected by using JC-1 method, and levels of Cytochrome C in cytoplasma and mitochondrion were detected by using Western blotting assay. Results The infection of over-expressed YAP recombinant lentivirus increased YAP expression in cardiomyocytes under H/R condition. The proliferation activity decreased, leakage rate of LDH increased, apoptosis rate increased, levels of activated Caspase-3 protein and Caspase-9 protein increased, and ROS level increased in cardiomyocytes after H/R treatment. At the same time SOD activity decreased, MMP was reduced, and level of Cytochrome C protein increased in cytoplasma and decreased in mitochondrion. After upregulating YAP, the proliferation activity was improved, leakage rate of LDH was decreased, apoptosis was reduced and expressions of Caspase-3 and Caspase-9 were decreased in cardiomyocytes under H/R condition. And SOD activity was increased, ROS level was decreased, MMP was promoted, and level of Cytochrome C protein was decreased in cytoplasma and increased in mitochondrion. Conclusion The up-regulation of YAP can relieve H/R cardiomyocyte injury and reduce apoptosis, and the mechanism may be related to the improvement of antioxidant enzyme activity, decrease of ROS level and inhibition of mitochondrial apoptosis pathway.