let-7d抑制剂对海马神经元离体氧糖剥夺/复氧细胞损伤的凋亡抑制作用

Inhibitory effect of let-7d inhibitor on apoptosis of hippocampal neurons after oxygen-glucose deprivation/reoxygenation injury

目的探讨let-7d microRNAs(miRNAs)抑制剂对海马神经元离体氧糖剥夺/复氧(OGD/R)细胞损伤中的凋亡抑制作用及机制。方法培养小鼠海马神经元HT22细胞系,利用三气缺氧细胞培养箱及培养基缺糖方式建立OGD/R细胞损伤模型,实时荧光定量PCR检测海马神经元OGD/R细胞损伤后6,12,24 h let-7d mRNA表达。将细胞分为正常组、OGD/R+miRNA-NC组、OGD/R+let-7d mimics组和OGD/R+let-7d inhibitor组,实时荧光定量PCR检测各组细胞let-7d和NGF mRNA表达,CCK-8法检测各组细胞活性,Hoechst33342/PI双染法检测各组细胞凋亡率。结果与正常组比较,海马神经元OGD/R细胞损伤后6,12,24 h let-7d表达显著降低(P<0.001)。与正常组比较,OGD/R+miRNA-NC组NGF mRNA表达和细胞凋亡率显著增高(P<0.001),let-7d mRNA表达和细胞活性显著降低(P<0.001)。与OGD/R+miRNA-NC组比较,OGD/R+let-7d inhibitor组let-7d mRNA表达和细胞凋亡率显著降低,NGF mRNA表达和细胞活性显著增高。而OGD/R+let-7d mimics组作用与之相反(P<0.001)。结论let-7d inhibitor能够抑制OGD/R诱导的海马神经元细胞凋亡,其机制可能与上调内源性保护因子NGF表达有关。

Objective To investigate the inhibitory effect and mechanism of let-7d microRNAs(miRNAs)inhibitors on apoptosis in hippocampal neurons with oxygen-glucose deprivation/reoxygenation(OGD/R)cell injury in vitro.Methods HT22 hippocampal neurons in mice were cultured.OGD/R cell injury model was established by three gas anoxic cell incubator and glucose-free medium.Real-time fluorescence quantitative PCR was used to detect let-7d expression in hippocampal neurons 6,12 and 24 h after OGD/R injury.The cells were divided into normal group,OGD/R+miRNA-NC group,OGD/R+let-7d mimics group and OGD/R+let-7d inhibitor group.The expression of let-7d and NGF mRNA was detected in each group.The cell viability was detected by CCK8 method,and the apoptotic rate was detected by Hoechst 33342/PI double staining method.Results The expression of let-7d in hippocampal neurons was significantly decreased after OGD/R injury(P<0.001).Compared with normal group,NGF expression and apoptotic rate were significantly increased in OGD/R+miRNA-NC group,while let-7d expression and cell activity were significantly decreased(P<0.001).Compared with OGD/R+miRNA-NC group,the expression of let-7d and apoptotic rate of cells decreased significantly in OGD/R+let-7d inhibitor group(P<0.001),while the expression of NGF and cell activity increased significantly(P<0.001).Whereas OGD/R+let-7d mimics significantly increased the expression of let-7d and apoptotic rate of cells,and decreased the expression of NGF and cell activity(P<0.001).Conclusion The let-7d inhibitor may inhibit the apoptosis of hippocampal neuron ischemia/reperfusion cells induced by OGD/R,which may be related to the up-regulation of endogenous protective factor NGF expression.