miR-21抑制剂对海马神经元氧糖剥夺/复氧损伤的影响

目的 探讨miR-21抑制剂对海马神经元离体氧糖剥夺/复氧(OGD/R)损伤的影响及作用机制.方法 将小鼠海马神经元HT22细胞接种于达尔伯克改良伊格尔培养基,置于含体积分数95％空气和5％CO2培养箱中培养2 d,然后将细胞分为正常组、OGD/R 6 h组、OGD/R 12 h组和OGD/R 24 h组.正常组细胞置于常氧培养箱培养,OGD/R 6 h组、OGD/R 12 h组和OGD/R 24 h组细胞建立OGD/R模型,然后分别培养6、12、24 h.取培养24 h的HT22细胞分为正常组、OGD/R+miRNA-NC组、OGD/R+miR-21 mimics组和OGD/R+miR-21 inhibitor组;正常组细胞置于常氧培养箱培养;OGD/R+miRNA-NC组细胞转染miRNA阴性对照质粒,OGD/R+miR-21 mimics组细胞转染miR-21 mimics质粒,OGD/R+miR-21 inhibitor组细胞转染miR-21 inhibitor质粒,转染后置于培养箱孵育2 d.实时荧光定量反转录聚合酶链反应(qRT-PCR)检测正常组、OGD/R 6 h组、OGD/R 12 h组和OGD/R 24 h组细胞中miR-21表达.qRT-PCR检测正常组、OGD/R+miRNA-NC组、OGD/R+miR-21 mimics组和OGD/R+miR-21 inhibitor组细胞中miR-21和脑源性神经营养因子(BDNF)mRNA表达,细胞计数试剂盒-8检测各组细胞活性,Hoechst33342/碘化丙啶双染法检测各组细胞凋亡情况,酶联免疫吸附试验检测各组细胞中肿瘤坏死因子-α(TNF-α)和白细胞介素-1β(IL-1β)的水平.结果 OGD/R 6 h组、OGD/R 12 h组和OGD/R 24 h组细胞中miR-21相对表达量均显著低于正常组(P<0.05).OGD/R+miRNA-NC组和OGD/R+miR-21 inhibitor组细胞中miR-21相对表达量显著低于正常组(P<0.05),OGD/R+miR-21 mimics组细胞中miR-21相对表达量显著高于正常组(P<0.05).OGD/R+miR-21 mimics组细胞中miR-21相对表达量显著高于OGD/R+miRNA-NC组(P<0.05);OGD/R+miR-21 inhibitor组细胞中miR-21相对表达量显著低于OGD/R+miRNA-NC组和OGD/R+miR-21 mimics组(P<0.05).OGD/R+miRNA-NC组和OGD/R+miR-21 inhibitor组细胞中BDNF mRNA相对表达量显著高于正常组(P<0.05);OGD/R+miR-21 mimics组细胞中BDNF mRNA相对表达量与正常组比较差异无统计学意义(P>0.05).OGD/R+miR-21 mimics组细胞中BDNF mRNA相对表达量显著低于OGD/R+miR-NA-NC组(P<0.05);OGD/R+miR-21 inhibitor组细胞中BDNF mRNA相对表达量显著高于OGD/R+miRNA-NC组和OGD/R+miR-21 mimics组(P<0.05).OGD/R+miRNA-NC组和OGD/R+miR-21 mimics组细胞活性显著低于正常组(P<0.05);OGD/R+miR-21 inhibitor组细胞活性与正常组比较差异无统计学意义(P>0.05).OGD/R+miR-21 mimics组细胞活性显著低于OGD/R+miRNA-NC组(P<0.05),OGD/R+miR-21 inhibitor组细胞活性显著高于OGD/R+miRNA-NC组和OGD/R+miR-21 mimics组(P<0.05).OGD/R+miRNA-NC组、OGD/R+miR-21 mimics组和OGD/R+miR-21 inhibitor组细胞凋亡率均显著高于正常组(P<0.05).OGD/R+miR-21 mimics组细胞凋亡率显著高于OGD/R+miRNA-NC组(P<0.05);OGD/R+miR-21 inhibitor组细胞凋亡率显著低于OGD/R+miRNA-NC组和OGD/R+miR-21 mimics组(P<0.05).OGD/R+miRNA-NC组、OGD/R+miR-21 mimics组和OGD/R+miR-21 inhibitor组细胞中炎症因子TNF-α和IL-1β水平均显著高于正常组(P<0.05).OGD/R+miR-21 mimics组细胞中TNF-α、IL-1β水平显著高于OGD/R+miRNA-NC组(P<0.05);OGD/R+miR-21 inhibitor组细胞中TNF-α、IL-1β水平显著低于OGD/R+miRNA-NC组和OGD/R+miR-21 mimics组(P<0.05).结论 miR-21抑制剂可改善OGD/R诱导的海马神经元缺血/再灌注损伤,其机制可能与上调BDNF信号通路、抑制炎症因子信号途径有关.