摘要
目的研究褪黑素抗心肌缺血再灌注损伤的新分子机制。方法 H9c2细胞在接受模拟缺血再灌注损伤前,用不同浓度褪黑素(Mel)(10μmol/L、50μmol/L、100μmol/L)预处理12 h。分别用CCK-8法检测各实验组细胞活力,酶活性测定法检测细胞中丙二醛(MDA)含量、超氧化物歧化酶(SOD)活性,DHE荧光探针检测细胞中活性氧(ROS)的含量,Fluo-4AM荧光探针检测细胞内钙离子浓度,Western blot法检测衰老标记蛋白30(SMP30)及凋亡与氧化应激相关蛋白的表达水平,免疫荧光染色检测细胞SMP30表达量。转染siRNA抑制SMP30的表达,进一步探究SMP30分子是否介导Mel的保护作用。结果 Mel预处理可显著提高再灌注损伤后细胞活力,降低细胞凋亡相关蛋白的表达;降低细胞中MDA含量,增强SOD活性及抗氧化应激相关蛋白表达量,减少ROS的产生;并可显著上调SMP30的表达,降低细胞内钙离子浓度,而使用SMP30 siRNA明显逆转Mel的上述保护作用(均P<0.05)。结论 Mel通过激活SMP30分子,增强细胞抗氧化应激能力,减轻细胞钙超载,进而减少细胞凋亡和坏死,最终改善心肌细胞缺血再灌注损伤。
Objective To study the new molecular mechanism in the protective effects of melatonin（ Mel） against myocardial ischemia reperfusion injury. Methods The cultured H9 c2 cells were pretreated with Mel（ 10,50,100 μmol/L） for 12 h and then subjected to simulated ischemia reperfusion（ SIR）. Cell viability was detected by CCK-8 kit,and the intracellular malondialdehyde（ MDA） content,superoxide dismutase（ SOD） activity were assessed by enzyme activity determination methods. Dihydroethidium（ DHE）,a fluorescence probe,was used to detect the presence of reactive oxygen species（ ROS）. Fluo-4 AM,a Ca2＋fluorescence probe,was used to measure the intracellular calcium concentration. The expression levels of senescence marker protein-30（ SMP30）,apoptosis-related and oxidative stress-related proteins were detected by western blot and immunofluorescent staining. SMP30 siRNA was administered to further study the role of SMP30 in the protective process of Mel. Results Mel pretreatment significantly improved the cell viability and down-regulated the expression of apoptosis-related proteins in SIR-treated H9 c2 cells（ P 〈0.05）. In addition,the intracellular MDA content and ROS production were largely reduced after Mel supplementation,with markedly up-regulated expression of SOD activity and anti-oxidative stress-related proteins（ P 〈0.05）. Furthermore,Mel pretreatment obviously increased the expression and immunofluorescent intensity of SMP30,as well as reducing the intracellular Ca2＋concentration. However,these protective effects of Mel mentioned above were all markedly attenuated by SMP30 siRNA transfection（ P 〈0.05）. Conclusion Mel pretreatment can protect against SIR-induced oxidative stress,calcium overload and apoptosis on H9 c2 cells via enhancing SMP30 expression.
作者
张彬
翟蒙恩
李凯峰
李步潆
刘振华
陈正希
江丽青
梁宏亮
段维勋
金振晓
俞世强
Zhang Bin;Zhai Mengen;Li Kaifeng;Li Buying;Liu Zhenhua;Chen Zhengxi;Jiang Liqing;Liang Hongliang;Duan Weixun;Jin Zhenxiao;Yu Shiqiang(Department of Cardiovascular Surgery,Xijing Hospital,Fourth Military Medical University,Shaan＇xi Xi＇an 710032,China)
出处
《中国体外循环杂志》
2018年第3期177-183,共7页
Chinese Journal of Extracorporeal Circulation
基金
国家自然科学基金(81570230
81570231
81770373)
关键词
褪黑素
缺血再灌注损伤
衰老标记蛋白30
氧化应激
凋亡
Melatonin
Ischemia reperfusion injury
Senescence marker protein-30
Oxidative stress
Apoptosis
