<div>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</div>

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Abstract 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 1 000 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 oxidase Ⅳ (COX Ⅳ) in the NECA (10, 100 nmol/L) group were increased, and there was no significant difference between TOM20 and COX Ⅳ after 1 000 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 1 000 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 complex Ⅰ, Ⅱ and Ⅳ of 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.

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Received: 18 December 2023

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	WANG Yunqi1
	HUANG Jiakang1
	WANG Yao1
	TIAN Wei2
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	XU Jingman1
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Cite this article:

WANG Yunqi1,HUANG Jiakang1,WANG Yao1等. Effects of 5′-N-ethylcarboxamido adenosine on myocardial mitophagy and mitochondrial respiratory function[J]. Journal of Jiangsu University(Medicine Edition), 2024, 34(05): 388-394.

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http://zzs.ujs.edu.cn/xbyxb/EN/ OR http://zzs.ujs.edu.cn/xbyxb/EN/Y2024/V34/I05/388

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