咪唑安定通过上调miR-290-5p调节PI3K/AKT信号通路抑制缺氧诱导心肌细胞损伤的机制研究

Midazolam inhibits hypoxia-induced cardiomyocyte damage by up-regulating miR-290-5p to regulate the PI3K/AKT signaling pathway

目的探讨咪唑安定(MID)对缺氧诱导心肌细胞损伤的影响和分子机制。方法采用不同浓度的MID预处理H9C2细胞24 h,缺氧诱导后,细胞计数试剂盒(CCK-8)检测H9C2细胞存活率,确定MID最佳浓度。将H9C2细胞分为正常(NC)组、缺氧(hypoxia)组、MID+hypoxia组、miR-NC+hypoxia组、miR-290-5p+hypoxia组、anti-miR-NC+MID+hypoxia组、anti-miR-290-5p+MID+hypoxia组。CCK-8法检测细胞存活;流式细胞术检测细胞凋亡;实时荧光定量PCR(RT-qPCR)检测miR-290-5p的表达;蛋白质印记(Western blot)检测磷脂酰肌醇-3-羟激酶/蛋白激酶B(PI3K/AKT)信号通路相关蛋白的表达。结果8、16、32μmol/L的MID预处理H9C2细胞缺氧后细胞存活率显著升高(P<0.05),16μmol/L为MID的最佳浓度。与NC组比较,hypoxia组H9C2细胞凋亡率显著升高,miR-290-5p的表达和PI3K通路活性显著降低(P<0.05)。与hypoxia组比较,MID+hypoxia组H9C2细胞凋亡率显著降低,miR-290-5p的表达显著升高,PI3K通路活性显著升高(P<0.05)。与MID+hypoxia组比较,miR-290-5p+hypoxia组H9C2细胞存活率显著升高,凋亡率显著降低(P<0.05)。与anti-miR-NC+MID+hypoxia组比较,anti-miR-290-5p+MID+hypoxia组H9C2细胞存活率显著降低,凋亡率显著升高,PI3K通路活性显著降低(P<0.05)。结论咪唑安定通过上调miR-290-5p激活PI3K/AKT信号通路有关进而对缺氧诱导的心肌细胞损伤具有保护作用。

Objective To investigate the effects and molecular mechanisms of midazolam(MID)on myocardial cell injury induced by hypoxia.Methods To determine the optimal MID concentration,H9 C2 cells were pretreated with different concentrations of MID for 24 h,induced with hypoxia,and the survival rate was assayed using the cell counting kit(CCK-8).H9 C2 cells were divided into seven groups:normal control(NC);hypoxia;MID+hypoxia;microRNA(miRNA)negative control(NC)+hypoxia;miR-290-5 p+hypoxia;anti-miR-NC+MID+hypoxia;and anti-miR-290-5 p+MID+hypoxia.Flow cytometry was used to detect apoptosis.Real-time quantitative PCR(RT-qPCR)was used to detect miR-290-5 p expression.Western blot was used to detect phosphatidylinositol-3-kinase/protein kinase B(PI3 K/AKT)signaling pathway-related protein expression.Results The cell survival rate of H9 C2 cells pretreated with MID at8,16 and 32μmol/L was significantly increased after hypoxia(P<0.05);16μmol/L was the optimal concentration.Compared with the NC group,the apoptosis rate of H9 C2 cells in the hypoxia group was significantly increased,while the expression of miR-290-5 p and the PI3 K pathway activity were significantly reduced(P<0.05).Compared with the hypoxia group,the apoptosis rate of H9 C2 cells in the MID+hypoxia group was significantly reduced,and the expression of miR-290-5 p and the activity of the PI3 K pathway were significantly increased(P<0.05).Compared with the MID+hypoxia group,the survival rate of H9 C2 cells in the miR-290-5 p+hypoxia group was significantly increased,and the apoptosis rate was significantly reduced(P<0.05).Compared with the anti-miR-NC+MID+hypoxia group,the survival rate of H9 C2 cells in the anti-miR-290-5 p+MID+hypoxia group was significantly reduced,the apoptosis rate was significantly increased,and PI3 K pathway activity was significantly reduced(P<0.05).Conclusions Midazolam protected hypoxia-induced cardiomyocyte damage by upregulating miR-290-5 p to activate PI3 K/AKT signaling pathway.