咪达唑仑调控PI3K/AKT信号通路对高糖诱导心肌细胞氧化应激和凋亡的影响

Effect of midazolam regulation of PI3K/AKT signaling pathway on high glucose-induced cardiomyocyte oxidative stress and apoptosis

目的探讨咪达唑仑是否通过磷酸肌醇3激酶(PI3K)/蛋白激酶B(AKT)信号通路调控高糖诱导心肌细胞氧化应激和凋亡。方法心肌细胞H9C2采用35 mmol/L葡萄糖诱导损伤,并给予低、中、高(5、10、20 ng/mL)剂量的咪达唑仑或10μmol/L PI3K/AKT特异性抑制剂LY294002,细胞分为对照组、高糖组、高糖+低剂量药物组、高糖+中剂量药物组、高糖+高剂量药物组、高糖+LY294002组,高糖+高剂量药物组+LY294002组。CCK-8检测细胞活性,流式细胞术评估细胞凋亡,Western blot分析Caspase3、磷酸化PI3K(p-PI3K)、PI3K、磷酸化AKT(p-AKT)、AKT的表达,比色法测定超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量。结果与对照组相比,高糖组心肌细胞H9C2的活性、SOD活性、p-PI3K和p-AKT蛋白表达量降低,细胞凋亡率、Caspase3蛋白表达量、MDA含量升高,差异均有统计学意义(P均<0.05),PI3K和AKT蛋白表达水平差异无统计学意义(P>0.05)。与高糖组相比,10、20 ng/mL剂量的咪达唑仑增强高糖诱导的心肌细胞H9C2的细胞活性、SOD活性、p-PI3K和p-AKT蛋白表达水平,并减弱凋亡率、Caspase3蛋白表达水平和MDA含量,差异均有统计学意义(P均<0.05),而PI3K和AKT蛋白表达水平差异无统计学意义(P>0.05)。与高糖组相比,LY294002和高糖处理的心肌细胞H9C2的细胞活性、SOD活性减少,细胞凋亡率、Caspase3蛋白表达量、MDA含量增加,差异均有统计学意义(P均<0.05)。咪达唑仑对高糖诱导的心肌细胞活性、细胞凋亡和氧化应激的影响被LY294002所减弱。结论咪达唑仑通过激活PI3K/AKT信号通路,增强高糖诱导的心肌细胞活性,抑制细胞凋亡并减轻氧化应激。

Objective To explore whether midazolam regulates high glucose-induced cardiomyocyte oxidative stress and apoptosis via PI3K/AKT signaling pathway.Methods Cardiomyocytes H9C2 were injured by 35 mmol/L glucose,and low,medium,and high(5,10,and 20 ng/ml)doses of midazolam or 10μardiomyocytes H9C2 were injured by 35 mmol/L glucose,and low,medium,and high(5,10,and 20 ng/ml)d group,high sugar+low dose drug group,high sugar+medium dose drug group,high sugar+high dose drug group,high sugar+LY294002 group,and high sugar+high dose drug+LY294002 group.Cell activity was detected by Cell Counting Kit-8(CCK-8),apoptosis was evaluated by flow cytometry,and apoptosis-related proteins Caspase3,PI3K/AKT signaling pathway proteins p-PI3K,PI3K,p-AKT,AKT expression were analyzed by Western blot.SOD activity and MDA content were determined by colorimetric method.Results Compared with the control group,H9C2 activity,SOD activity,p-PI3K and p-AKT protein expression levels of cardiomyocytes in the high glucose group decreased,and the apoptosis rate,Caspase3 protein expression level,and MDA content increased.The differences were statistically significant(all P<0.05).But there was no statistically significant difference in protein expression levels of PI3K and AKT(P>0.05).Compared with the high glucose group,10 and 20 ng/ml doses of midazolam enhanced the cell activity,SOD activity,p-PI3K and p-AKT protein expression levels in high glucose-induced cardiomyocyte H9C2,and weakened apoptosis rate,Caspase3 protein expression level and MDA content,which were all statistically significant(P<0.05),while PI3K and AKT protein expression levels were not statistically significant(P>0.05).Compared with the high glucose group,the cell activity and SOD activity of LY294002 and high glucose treated cardiomyocytes H9C2 decreased,and the apoptosis rate,Caspase3 protein level,and MDA content increased.The effect of midazolam on cardiomyocyte activity,apoptosis and oxidative stress induced by high glucose was weakened by LY294002.Conclusion Midazolam activates PI3K/AKT signaling pathway,enhances the activity of cardiomyocytes induced by high glucose,inhibits apoptosis and reduces oxidative stress.