miR-21抑制剂对海马神经元氧糖剥夺/复氧损伤的影响

Effects of microR NA-21 inhibitors on oxygen-glucose deprivation/reoxygenation injury of hippocampal neurons

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(q RT-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。q RT-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/R6 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+miRNANC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+miRNANC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+miRNA-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。

Objective To investigate the effect and mechanism of miR-21 inhibitors on oxygen-glucose deprivation/reoxygenation(OGD/R)injury in hippocampal neurons in vitro.Methods HT22 cells of mouse hippocampal neurons were inoculated into Dulbecco’s modified Eagle’s medium and cultured in incubator which contained volume fraction 95%air and 5%CO2 for 2 days.Then the cells were divided into normal group,OGD/R 6 h group,OGD/R 12 h group and OGD/R 24 h group.The cells in normal group were cultured in normoxia incubator;the cells in OGD/R 6 h group,OGD/R 12 h group and OGD/R 24 h group were established the OGD/R model and then cultured for 6,12,24 h respectively.The HT22 cells which cultured for 24 hours were taken and divided into normal group,OGD/R+miRNA-NC group,OGD/R+miR-21 mimics group and OGD/R+miR-21 inhibitor group.The cells in the normal group were cultured in the normoxia incubator,the cells in the OGD/R+miRNA-NC group were transfected with miRNA negative control plasmids,the cells in the OGD/R+miR-21 mimics group were transfected with miR-21 mimics plasmids,the cells in the OGD/R+miR-21 inhibitor group were transfected with miR-21 inhibitor plasmids;then the above cells were cultured in incubator for 2 days.The expression of miR-21 mRNA in HT22 cells in normal group,OGD/R 6 h group,OGD/R 12 h group and OGD/R 24 h group was detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction(q RT-PCR).The expression of miR-21,brain derived neurotrophic factor(BMNF)mRNA in the normal group,OGD/R+miRNA-NC group,OGD/R+miR-21 mimics group and OGD/R+miR-21 inhibitor group were detected by q RT-PCR;the cell activity in above each group was detected by cell count kit-8;the apoptosis in above each group was detected by Hoechst33342/propidium iodide double staining;the levels of tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)in the cells in above each group were detected by enzyme linked immunosorbent assay.Results The relative expression of miR-21 in the OGD/R 6 h group,OGD/R 12 h group and OGD/R 24 h group was significantly lower than that in the normal group(P<0.05).The expression of miR-21 in the OGD/R+miRNA-NC group and OGD/R+miR-21 inhibitor group was significantly lower than that in the normal group(P<0.05),and the expression of miR-21 in the OGD/R+miR-21 mimics group was significantly higher than that in the normal group(P<0.05).The expression of miR-21 in the OGD/R+miR-21 mimics group was significantly higher than that in the OGD/R+miRNA-NC group(P<0.05);the expression of miR-21 in the OGD/R+miR-21 inhibitor group was significantly lower than that in the OGD/R+miRNA-NC group and OGD/R+miR-21 mimics group(P<0.05).The expression of BDNF mRNA in the OGD/R+miRNA-NC group and OGD/R+miR-21 inhibitor group was significantly higher than that in the normal group(P<0.05);there was no significant difference in BDNF mRNA expression between the OGD/R+miR-21 mimics group and normal group(P>0.05).The expression of BDNF mRNA in the OGD/R+miR-21 mimics group was significantly lower than that in the OGD/R+miRNA-NC group(P<0.05);the expression of BDNF mRNA in the OGD/R+miR-21 inhibitor group was significantly higher than that in the OGD/R+miRNA-NC group and OGD/R+miR-21 mimics group(P<0.05).The cell activity in the OGD/R+miRNA-NC group and OGD/R+miR-21 mimics group was significantly lower than that in the normal group(P<0.05);there was no significant difference in the cell activity between the OGD/R+miR-21 inhibitor group and the normal group(P>0.05).The cell activity in the OGD/R+miR-21 mimics group was significantly lowrer than that in the OGD/R+miRNA-NC group(P<0.05);the cell activity of the OGD/R+miR-21 inhibitor group was significantly higher than that in the OGD/R+miRNA-NC group and OGD/R+miR-21 mimics group(P<0.05).The apoptosis rate in the OGD/R+miRNA-NC group,OGD/R+miR-21 mimics group and OGD/R+miR-21 inhibitor group was significantly higher than that in the normal group(P<0.05).The apoptosis rate in the OGD/R+miR-21 mimics group was significantly higher than that in the OGD/R+miRNA-NC group(P<0.05);the apoptosis rate in the OGD/R+miR-21 inhibitor group was significantly lower than that in the OGD/R+miRNA-NC group and OGD/R+miR-21 mimics group(P<0.05).The levels of TNF-αand IL-1βin cells in the OGD/R+miRNA-NC group,OGD/R+miR-21 mimics group and OGD/R+miR-21 inhibitor group were significantly higher than those in the normal group(P<0.05).The levels of TNF-αand IL-1βin cells in the OGD/R+miR-21 mimics group were significantly higher than those in the OGD/R+miRNA-NC group(P<0.05);the levels of TNF-αand IL-1βin cells in the OGD/R+miR-21 inhibitor group were significantly lower than those in the OGD/R+miRNA-NC group and OGD/R+miR-21 mimics group(P<0.05).Conclusion MiR-21 inhibitor can improve OGD/R-induced ischemia/reperfusion injury in hippocampal neurons,which may be related to up-regulation of BDNF signaling pathway and inhibition inflammatory cytokine signaling pathway.