NRG-1对缺氧复氧心肌细胞凋亡及氧化损伤的影响

Effect of neuregulin-1 on cardiomyocyte apoptosis and oxidative damage under hypoxia reoxygenation

目的:探讨神经调节蛋白1(NRG-1)对缺氧复氧环境下的心肌细胞凋亡及氧化损伤影响及机制。方法:以心肌细胞(HCM)为研究对象,构建缺氧复氧心肌细胞模型,在缺氧前加入0.8 mg/L的NRG-1。噻唑蓝(MTT)法检测细胞活力,流式细胞术检测细胞凋亡,二硝基苯肼显色法检测上清中乳酸脱氢酶(LDH)含量,二氯二氢荧光素-乙酰乙酸酯(DCFH-DA)法检测活性氧簇(ROS)水平,硫代巴比妥酸法检测丙二醛(MDA)水平,黄嘌呤氧化酶法检测超氧化物歧化酶(SOD)水平,Western blot检测蛋白激酶B(Akt)、磷酸化的Akt(p-Akt^(Thr308))、cleaved caspase-3的蛋白水平。结果:心肌细胞缺氧复氧后细胞吸光度(A)从0.66±0.03降低至0.36±0.04,细胞凋亡率从(4.62±0.97)%升高至(29.07±3.43)%,ROS水平从69.29±7.96升高至280.84±20.52,LDH、MDA水平也升高,SOD、p-Akt/Akt水平均下降。而NRG-1处理后的细胞吸光度(A)从0.36±0.04升高至0.47±0.05,细胞凋亡率从(29.07±3.43)%下降至(19.76±3.41)%,ROS水平从280.84±20.52下降至128.23±12.32,LDH、MDA水平也下降,SOD、p-Akt^(Thr308)/Akt水平均升高。结论:NRG-1能够部分抑制缺氧复氧诱导的心肌细胞凋亡和氧化损伤,影响心肌细胞中p-Akt/Akt的水平。

Objective:To investigate the effect of NRG-1 on cardiomyocyte apoptosis and oxidative damage under hypoxia reoxygenation.Methods:The myocardial cell HCM was taken as the object of study.The hypoxia reoxygenation of myocardial cell model was established,and NRG-1 at dose of 0.8 mg/L was added before hypoxia.The cell viability was measured by MTT assay and apoptosis was analyzed by flow cytometry.The level of lactate dehydrogenase(LDH)in the supernatant was detected by 2,4-dinitrophenylhydrazine colorimetry assay,DCFH-DA method was used to detected ROS level,the level of MDA was measured by thiobarbituric acid method,the level of SOD was detected by xanthine oxidase method,and the protein levels of Akt and p-Akt Thr308 were determined by Western blot.Results:The A values of the myocardial cells after hypoxia reoxygenation were changed from 0.66±0.03 to 0.36±0.04,the rate of apoptosis increased from(4.62±0.97)%to(29.07±3.43)%,the level of ROS increased from 69.29±7.96 to 280.84±20.52,the levels of LDH and MDA also increased,and the levels of SOD and p-Akt Thr308/Akt decreased.After NRG-1 treatment,the A values of the cells were from 0.36±0.04 to 0.47±0.05,the rate of apoptosis decreased from(29.07±3.43)%to(19.76±3.41)%,the ROS levels decreased from 280.84±20.52 to 128.23±12.32,the levels of LDH and MDA also decreased,and the levels of SOD and p-Akt/Akt increased.Conclusion:NRG-1partly inhibits cardiomyocyte apoptosis and oxidative damage induced by hypoxia reoxygenation by affecting the protein levels of p-Akt/Akt in the cardiomyocytes.