5′-N-乙基酰胺基腺苷对心肌线粒体自噬和呼吸功能的影响

Effects of 5′-N-ethylcarboxamido adenosine on myocardial mitophagy and mitochondrial respiratory function

目的:探讨选择性腺苷A2受体激动剂5′-N-乙基酰胺基腺苷(5′-N-ethylcarboxamido adenosine,NECA)对心肌线粒体自噬和呼吸功能的影响及可能机制。方法:培养大鼠心肌H9c2细胞,给予不同浓度NECA(10、100、1000 nmol/L)处理2 h,对照组给予等体积PBS处理2 h。CCK-8法检测细胞活性;蛋白质印迹实验检测线粒体自噬及相关因子的表达水平;运用线粒体膜电位探针四甲基罗丹明乙酯(TMRE)检测线粒体膜电位;运用线粒体红色荧光探针Mitotracker Red和溶酶体绿色荧光探针Lysotracker Green同时标记细胞,并用激光共聚焦扫描显微镜记录线粒体和溶酶体的共定位情况;应用Oxygraph-2k高分辨率呼吸仪检测细胞线粒体呼吸功能。结果:与对照组相比,不同浓度NECA组细胞活力和线粒体膜电位均无显著变化。蛋白质印迹实验结果显示,与对照组相比,10 nmol/L和100 nmol/L NECA处理组线粒体的线粒体外膜转位酶20(TOM20)和内膜蛋白细胞色素C氧化酶Ⅳ亚型(COXⅣ)增高,1000 nmol/L处理后,TOM20和COXⅣ较对照组无明显差异;而NECA对线粒体内膜转位酶23(TIM23)表达水平无显著影响。与对照组相比,加入10 nmol/L NECA处理后线粒体自噬相关因子FUN14结构域包含蛋白1(FUNDC1)表达明显增加,100、1000 nmol/L NECA处理对FUNDC1表达无显著影响;而NECA对同源性磷酸酶张力蛋白诱导激酶1(PINK1)和帕金森蛋白(Parkin)表达水平无明显影响。共聚焦显微镜可见,与对照组相比,NECA处理组的溶酶体与线粒体共定位降低。Oxygraph-2k高分辨率呼吸仪检测结果显示,NECA处理组心肌H9c2细胞线粒体呼吸链复合体Ⅰ、Ⅱ、Ⅳ的活性与对照组相比无明显变化。结论:NECA抑制心肌H9c2细胞线粒体自噬但对线粒体呼吸功能无影响。

Objective:To investigate the effect and its possible mechanism of 5′-N-ethylcarboxamido adenosine(NECA),a selective adenosine A2 receptor agonist,on myocardial mitophagy and mitochondrial respiratory function.Methods:Rat myocardial H9c2 cells were cultured and treated with different concentrations of NECA,while the control group was treated with equal volume PBS for 2 h.CCK-8 was used to test cell viability.The expression levels of mitophagy related proteins were detected by Western blotting.Mitochondrial membrane potential was measured by Tetramethyl rhodamine ethyl ester(TMRE)fluorescent probe.Mitochondrial red fluorescence probe Mitotracker Red and lysosome green fluorescence probe Lysotracker Green were used to label the cells simultaneously,and the co-localization of mitochondria and lysosome was recorded by laser confocal scanning microscope.The mitochondrial respiratory function was measured with Oxygraph-2k high resolution respiratory apparatus.Results:Comparing to the control group,NECA(10,100 and 1000 nmol/L)could not change both the cell viability and the mitochondrial membrane potential.Western blotting results showed that compared with the control group,translocase of the outer mitochondrial membrane 20(TOM20)and inner membrane protein cytochrome C oxidaseⅣ(COXⅣ)in the NECA(10,100 nmol/L)group were increased,and there was no significant difference between TOM20 and COXⅣafter 1000 nmol/L treatment.There was no significant change in the expression level of translocase of inner mitochondrial membrane 23(TIM23).Compared with the control group,the expression of mitophagy related factor FUN14 domain-containing protein 1(FUNDC1)was significantly increased after the addition of NECA(10 nmol/L),while 100 and 1000 nmol/L NECA treatments had no significant effect on FUNDC1 expression.The expression levels of homologous phosphatase tensin induced kinase 1(PINK1)and Parkinson′s protein(Parkin)were not changed markedly.Confocal results showed that compared with the control group,the co-localization of lysosome to mitochondrial was reduced in the NECA treatment group.The results of Oxygraph-2k high resolution respiratory apparatus showed that compared with the control group,the activity of mitochondrial respiratory chain complexⅠ,ⅡandⅣof myocardial H9c2 cells in the NECA treatment group had no significant change.Conclusion:NECA inhibited mitophagy but had no effect on mitochondrial respiratory function in cardiac H9c2 cells.