lncRNA OIP5-AS1在脑缺血再灌注大鼠模型中的表达及对缺氧缺糖复供诱导的细胞凋亡的影响

Expression of lncRNA OIP5-AS1 in a Rat Model of Cerebral Ischemia Reperfusion and Its Effect on PC12 Cell Apoptosis Induced by Oxygen-glucose Deprivation/Reperfusion

目的探讨长链非编码RNA(lncRNA)Opa相互作用蛋白5反义转录本1(OIP5-AS1)在脑缺血再灌注大鼠模型中的表达及其对缺氧缺糖复供(OGD/R)诱导的PC12细胞凋亡的影响。方法构建脑缺血再灌注大鼠模型,实时定量聚合酶链反应(qRT-PCR)方法检测脑组织中OIP5-AS1水平。PC12细胞分成Control组(空白对照)、OGD/R组(OGD/R处理)、Vector+OGD/R组(转染阴性对照载体,OGD/R处理)、OIP5-AS1+OGD/R组(转染OIP5-AS1过表达载体后以OGD/R处理)、OIP5-AS1+OGD/R+LY294002组[转染OIP5-AS1过表达载体,并以磷脂酰肌醇3-激酶/蛋白激酶B(PI3K/Akt)信号抑制剂LY294002处理,之后进行OGD/R处理]。CCK-8法检测细胞增殖活性,流式细胞术检测细胞凋亡变化,Western blot法检测细胞中Bax、Bcl-2、磷酸化的磷脂酰肌醇-3激酶(p-PI3K)、PI3K、磷酸化蛋白激酶B(p-Akt)、Akt蛋白水平,硫代巴比妥酸法检测丙二醛(MDA)水平,黄嘌呤氧化法检测超氧化物歧化酶(SOD)水平,DCFH-DA法检测活性氧(ROS)水平。结果脑缺血再灌注大鼠模型脑组织中OIP5-AS1表达水平降低。与Control组比较,OGD/R组PC12细胞存活率降低,细胞凋亡率升高,细胞中Bax蛋白表达水平升高,Bcl-2蛋白表达水平降低,p-PI3K/PI3K、p-Akt/Akt降低,SOD活性降低,MDA和ROS水平升高(均P<0.05)。与Vector+OGD/R组比较,OIP5-AS1+OGD/R组PC12细胞存活率升高,细胞凋亡率降低,细胞中Bax蛋白表达水平降低,Bcl-2蛋白表达水平升高,p-PI3K/PI3K、p-Akt/Akt升高,SOD活性升高,MDA和ROS水平降低(均P<0.05)。与OIP5-AS1+OGD/R组比较,OIP5-AS1+OGD/R+LY294002组PC12细胞中p-PI3K/PI3K、p-Akt/Akt降低,细胞存活率降低,细胞凋亡率升高,细胞中Bax蛋白水平升高,Bcl-2蛋白水平降低,SOD活性降低,MDA和ROS水平升高(均P<0.05)。结论OIP5-AS1在脑缺血再灌注大鼠模型中表达下调,上调OIP5-AS1可抑制OGD/R诱导的PC12细胞凋亡和氧化损伤,其机制与激活PI3K/Akt信号通路有关。

Objective To investigate the expression of long non-coding RNA(lncRNA)Opa-interacting protein 5 antisense transcript 1(OIP5-AS1)in a rat model of cerebral ischemia and reperfusion and its effect on the apoptosis of PC12 cells induced by oxygen-glucose deprivation/reperfusion(OGD/R).Methods A rat model of cerebral ischemia-reperfusion was established.The level of OIP5-AS1 in brain tissue was detected by real-time quantitative polymerase chain reaction(qRT-PCR).PC12 cells were divided into Control group(blank control),OGD/R group(OGD/R treatment),Vector+OGD/R group(transfected with negative control vector+OGD/R treatment),OIP5-AS1+OGD/R group(transfected with OIP5-AS1 overexpression vector+OGD/R treatment),OIP5-AS1+OGD/R+LY294002 group(transfected with OIP5-AS1 overexpression vector+PI3 K/Akt signal inhibitor LY294002 treatment+OGD/R treatment).Cell counting kit(CCK-8)method was used to detect cell proliferation activity,flow cytometry to detect cell apoptosis,Western blotting to test Bcl-2 related X protein(Bax),B-cell lymphoma/leukemia-2(Bcl-2),phosphorylated phosphatidylinositol-3 kinase(p-PI3 K)in cells.And thiobarbituric acid method was applied to detect malondialdehyde(MDA)level,Xanthine oxidation method to detect uperoxide dismutase(SOD)level,and DCFH-DA method to detect reactive oxygen species(ROS)level.Results The expression level of OIP5-AS1 in the brain tissue of the cerebral ischemia-reperfusion rat model decreased.Compared with the Control group,the survival rate of PC12 cells in the OGD/R group decreased,the rate of apoptosis increased,the expression level of Bax protein in the cells increased,the expression level of Bcl-2 protein decreased,and the p-PI3 K/PI3 K,p-Akt/Akt protein levels decreased,SOD activity decreased,MDA level and ROS level increased(P<0.05).Compared with the Vector+OGD/R group,the PC12 cell survival rate in the OIP5-AS1+OGD/R group increased,the apoptosis rate decreased,the Bax protein expression level in the cells decreased,the Bcl-2 protein expression level increased,and p-PI3 K/PI3 K,p-Akt/Akt protein levels increased,SOD activity increased,MDA level and ROS level decreased(P<0.05).Compared with the OIP5-AS1+OGD/R group,the p-PI3 K/PI3 K and p-Akt/Akt protein levels in the PC12 cells of the OIP5-AS1+OGD/R+LY294002 group decreased,the cell survival rate decreased,and the apoptosis rate increased,Bax protein level in cells increased,Bcl-2 protein level decreased,SOD activity decreased,MDA and ROS levels increased(P<0.05).Conclusion The expression of OIP5-AS1 was down-regulated in the rat model of cerebral ischemia-reperfusion,and the up-regulation of OIP5-AS1 inhibited OGD/R-induced apoptosis and oxidative damage of PC12 cells,the mechanism was related to the activation of PI3 K/Akt signaling pathway.