氯吡格雷上调长链非编码RNA-4231减轻缺氧/复氧诱导的心肌细胞损伤

Clopidogrel alleviates hypoxia/reoxygenation induced cardiomyocyte injury by up-regulating lncRNA-4231

目的:探讨氯吡格雷对缺氧/复氧(H/R)后心肌细胞的保护作用及机制。方法:体外培养大鼠H9c2心肌细胞,对细胞进行H/R处理,建立细胞H/R损伤模型,分别使用不同浓度(H/R组0、低剂量药物组12.5μg/mL、中剂量药物组25μg/mL、高剂量药物组50μg/mL)的氯吡格雷处理细胞,正常培养的细胞设为对照组。用pcDNA-长链非编码RNA-4231(lncRNA-4231)转染细胞,设为pcDNA-lncRNA-4231组,pcDNA组为仅转染pcDNA,两组均进行H/R处理。采用甲基噻唑基四唑(MTT)法检测细胞增殖情况,流式细胞术检测细胞凋亡率,试剂盒检测细胞丙二醛(MDA)水平、超氧化物歧化酶(SOD)活性,实时荧光定量聚合酶链反应检测细胞lncRNA-4231表达水平,Western blot法检测活化胱天蛋白酶-3(cleaved caspase-3)的蛋白表达水平。结果:与对照组比较,H/R组细胞存活率、SOD活性及lncRNA-4231的表达水平降低,细胞凋亡率、cleaved caspase-3蛋白表达水平及MDA水平升高(P均<0.05);与H/R组比较,中剂量药物组、高剂量药物组细胞存活率、SOD活性及lncRNA-4231的表达水平升高,细胞凋亡率、cleaved caspase-3蛋白表达水平及MDA水平降低(P均<0.05);低剂量药物组与中剂量药物组、中剂量药物组与高剂量药物组各指标的差异均具有统计学意义(P均<0.05)。与pcDNA组比较,pcDNA-lncRNA-4231组细胞存活率和SOD活性升高,细胞凋亡率、cleaved caspase-3蛋白表达水平及MDA水平降低(P均<0.05)。结论:氯吡格雷可能通过上调lncRNA-4231的表达水平减轻H/R诱导的心肌细胞损伤。

Objective:To investigate the protective effects and mechanism of clopidogrel on myocardial cell after hypoxia/reoxygenation(H/R).Methods:Rat cardiomyocytes H9c2 were cultured in vitro,and the damage models were established by treating the cells with H/R.Cells were divided into H/R group,low-dose drug group,medium-dose drug group and high-dose drug group(exposed to clopidogrel at 0,12.5μg/mL,25μg/mL and 50μg/mL,respectively).Normally cultured cells were used as the control group.MTT method was used to detect cell proliferation.Flow cytometry was used to detect the apoptosis rate.The level of malondialdehyde(MDA)and the activity of superoxide dismutase(SOD)in cells were detected by kit.qRT-PCR was used to detect the mRNA expression of long non-coding RNA-4231(lncRNA-4231).H/R-induced cardiomyocytes were transfected with pcDNA(pcDNA group)and pcDNA-lncRNA-4231(pcDNA-lncRNA-4231 group),and cell survival rate and apoptosis rate were detected by the above method.Western blot was used to detect the expression of cleaved caspase-3 protein.Results:Compared with the control group,the cell survival rate,the activity of SOD and the expression level of lncRNA-4231 were decreased in the H/R group;while the apoptosis rate,the expression level of cleaved caspase-3 and the level of MDA were increased(all P<0.05).Compared with the H/R group,the cell survival rates,the activity of SOD and the expression levels of lncRNA-4231 were increased in the medium-dose drug group and the high-dose drug group;while the apoptosis rates,the expression levels of cleaved caspase-3 and the levels of MDA were decreased(all P<0.05).These statistical differences were also found between the low-dose group and medium-dose group,as well as between the medium-dose drug group and the high-dose drug group(all P<0.05).Compared with the pcDNA group,the cell survival rate and the activity of SOD in the pcDNA-lncRNA-4231 group were increased,while the apoptosis rate,the expression level of cleaved caspase-3 and the level of MDA were decreased(all P<0.05).Conclusions:Clopidogrel may alleviate H/R-induced cardiomyocyte damage by up-regulating lncRNA-4231 expression.