AIM: To investigate the effect of growth arrest - specific protein 6( Gas 6 ) on H9c2 cell apoptosis induced by anoxia - reoxygenation ( A/R ) and its possible relationship with PI3K/Akt pathway.METHODS: Cultured H9c2 cell line of cardiomyocytes was randomly divided into 4 groups: normal control group, anoxia -reoxygenation group ( A/R ), anoxia - reoxygenation + Gas6 group ( A/R + Gas6 ) and anoxia/reoxygenation + Gas6 + LY294002 group ( A/R + Gas6 + LY294002 ).The procedure of A/R was performed in cultured H9c2 cells by 3 h of anoxia and then 3 h of reoxygenation.The viability of the cells and the activity of caspase - 3 were detected by automatic biochemistry analytic instrument.Cell apoptotic rates were evaluated by flow cytometry.The protein level of phosphorylated Akt( p - Akt ) was determined by Western blotting.RESULTS: Compared with control group, the cell viability was significantly decreased, and caspase - 3 activity, cell apoptotic rate and the protein level of p - Akt were increased in A/R group.Compared with A/R group, the caspase - 3 activity and cell apoptotic rate reduced markedly, while the cell viability and the protein level of p -Akt were significantly increased in A/R + Gas6 group.The effect of Gas6 was inhibited by LY294002.CONCLUSION: Gas6 may protect the H9c2 cells from anoxia - reoxygenation - induced apoptosis.Its mechanism is possibly involved in the activation of PI3K/Akt survival pathway via increasing the phosphorylation of Akt protein.%目的:观察外源性重组人生长停滞特异性蛋白6(Gas6)对缺氧复氧诱导的H9c2心肌细胞系凋亡的影响,并初步探讨其与磷脂酰肌醇3-激酶/蛋白激酶B(PI3K/Akt)通路的关系.方法:对体外培养的H9c2细胞进行缺氧复氧(3 h/3 h),模拟大鼠心肌缺血再灌注模型,将细胞随机分为4组:正常对照组(control组)、缺氧/复氧组(A/R组)、缺氧/复氧+ Gas6预处理组(A/R+Gas6组)及缺氧/复氧+ Gas6预处理+PI3K/Akt特异性阻断剂LY294002干预组(A/R+Gas6+LY294002组).分别采用MTT法检测心肌细胞活力,生化法检测细胞中caspase-3活性水平,Annexin V/PI 双染法及流式细胞仪检测细胞凋亡率,Western免疫印迹法检测胞内磷酸化Akt(p-Akt)蛋白含量表达情况.结果:A/R组较control组细胞活力下降,caspase-3活性、凋亡率及p-Akt水平均较control组增加(P<0.05).但经Gas6预处理后,细胞活力及p-Akt表达水平较A/R组显著增加,caspase-3活性、凋亡率均减少(P<0.05),而Gas6的这些作用能被PI3K/Akt阻断剂抑制.结论:Gas6能减少缺氧复氧诱导的H9c2细胞凋亡,此作用机制可能是通过提高Akt磷酸化水平而激活PI3K/Akt通路实现的.
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