右美托咪定调控miR-126对高糖诱导的心肌细胞氧化应激及凋亡的影响

Dexmedetomidine affects high glucose-induced oxidative stress and apoptosis by regulating the expression of miR-126

目的探讨右美托咪定(DEX)对高糖诱导的心肌细胞氧化应激及凋亡的影响及作用机制。方法体外培养心肌细胞H9C2,分别用葡萄糖浓度为5.5、35 mmol/L的DMEM培养基培养,分别作为对照组和高糖损伤模型组。分别使用不同浓度(5、10、20μg/L)DEX处理心肌细胞记为实验1组、实验2组、实验3组。检测各组乳酸脱氢酶(LDH)、丙二醛(MDA)、超氧化物歧化酶(SOD)的含量。采用实时荧光定量聚合酶链反应(qRT-PCR)检测微小RNA-126(miR-126)的表达量。分别将miR-NC、miR-126 mimicis转染至心肌细胞,使用含有葡萄糖浓度为35 mmol/L的DMEM培养基培养24 h,分别记为miR-NC组、miR-126组。分别将miR-NC、miR-126 mimcis、anti-miR-NC、anti-miR-126转染至心肌细胞,采用含有葡萄糖浓度为35 mmol/L与DEX浓度为20μg/L的DMEM培养基培养,分别记为实验3+miR-NC组、实验3+miR-126组、实验3+anti-miR-NC组、实验3+anti-miR-126组。流式细胞术检测各组细胞凋亡率,蛋白免疫印迹法(Western blot)检测各组半胱氨酰天冬氨酸特异性蛋白酶3的前体蛋白(pro-caspase-3)、活化的含半胱氨酸的天冬氨酸蛋白水解酶3(cl-caspase-3)水平。结果与对照组比较,高糖损伤模型组LDH、MDA含量显著升高(P<0.05),SOD含量显著降低(P<0.05),miR-126的表达水平显著降低(P<0.05),细胞凋亡率显著升高(P<0.05),cl-caspase-3蛋白水平显著升高(P<0.05),pro-caspase-3蛋白水平显著降低(P<0.05)。与高糖损伤模型组比较,实验1组、实验2组、实验3组LDH、MDA含量显著降低(P<0.05),SOD含量显著升高(P<0.05),miR-126的表达水平升高(P<0.05),细胞凋亡率显著降低(P<0.05),cl-caspase-3蛋白水平显著降低(P<0.05),pro-caspase-3蛋白水平显著升高(P<0.05),实验1组、实验2组、实验3组间各指标比较差异有统计学意义(P<0.05)。与miR-NC组比较,miR-126组LDH、MDA含量显著降低(P<0.05),SOD含量显著升高(P<0.05),细胞凋亡率显著降低(P<0.05),cl-caspase-3蛋白水平显著降低(P<0.05),pro-caspase-3蛋白水平显著升高(P<0.05)。miR-126过表达能增强DEX对高糖诱导心肌细胞氧化应激及凋亡,抑制miR-126表达能逆减弱DEX对高糖诱导心肌细胞氧化应激及凋亡。结论DEX可通过上调miR-126的表达从而抑制高糖诱导的心肌细胞氧化应激及细胞凋亡。

Objective To investigate the effect and mechanism of DEX on oxidative stress and apoptosis induced by high glucose in cardiomyocytes.Methods The cardiomyocyte H9C2 was cultured in vitro,and a high glucose injury model was established.The cardiomyocytes were treated with DEX with different concentrations(5μg/L,10μg/L,20μg/L),respectively,which were recorded as experiment 1 group,experiment 2 group,experiment 3 group.The contents of LDH,MDA and SOD were detected.qRT-PCR was used to detect the expression of miR-126.miR-NC,miR-126 mimcis,anti-miR-NC,and anti-miR-126 were transfected into cardiomyocytes,respectively,with DMEM medium containing 35 mmol/L glucose and 20μg/L DEX to cultivate.Flow cytometry was used to detect the apoptosis rate.Western blot was used to detect the expression of pro-caspase-3 and cl-caspase-3.Results Compared with the control group,the levels of LDH and MDA in the high glucose injury model group were significantly increased(P<0.05),the content of SOD was significantly reduced(P<0.05),the expression level of miR-126 was significantly reduced(P<0.05),and the apoptosis rate was significantly increased(P<0.05),the level of cl-caspase-3 protein was significantly increased(P<0.05),and the level of pro-caspase-3 protein was significantly decreased(P<0.05).Compared with the high glucose injury model group,the contents of LDH and MDA in experimental group 1,experimental group 2 and experimental group 3 were significantly reduced(P<0.05),the content of SOD was significantly increased(P<0.05),and the expression level of miR-126 was increased(P<0.05),the apoptosis rate was significantly reduced(P<0.05),the level of cl-caspase-3 protein was significantly reduced(P<0.05),and the level of pro-caspase-3 protein was significantly increased(P<0.05).There was a statistically significant difference in each index among the experimental group 1,the experimental group 2,and the experimental group 3(P<0.05).Compared with miR-NC group,the content of LDH and MDA in miR-126 group was significantly reduced(P<0.05),the content of SOD was significantly increased(P<0.05),and the apoptosis rate was significantly reduced(P<0.05),the level of caspase-3 protein was significantly reduced(P<0.05),and the level of pro-caspase-3 protein was significantly increased(P<0.05).Overexpression of miR-126 could enhance the effect of DEX on high glucose-induced oxidative stress and apoptosis in myocardial cells,and inhibition of miR-126 expression could attenuate the effect of DEX on high glucose-induced oxidative stress and apoptosis in cardiac muscle cells.Conclusion DEX could inhibit the oxidative stress and apoptosis of cardiomyocytes induced by high glucose by up-regulating the expression of miR-126.