目的 研究腺苷A2A和A2B受体是否参与的抗心肌缺血再灌注损伤作用及其相关机制。方法 利用H9c2心肌细胞建立模拟缺血/再灌注损伤模型,给予非选择性腺苷受体激动剂5′-N-乙基酰胺基腺苷及选择性腺苷A2A受体拮抗剂(SCH58261,SCH)、选择性腺苷A2B受体拮抗剂(MRS1706,MRS),采用CCK-8法测定细胞活性、JC-1染色检测细胞线粒体膜电位(△Ψm)、Amplex Red过氧化氢/过氧化物酶试剂盒测定细胞内H2O2水平,活性氧试剂盒检测细胞内活性氧(reactive oxygen species,ROS)水平,MitoSox Red超氧化物指示剂检测线粒体内活性氧水平。结果 5′-N-乙基酰胺基腺苷明显改善缺血/再灌注损伤引起的细胞活性和线粒体膜电位的降低,显著抑制缺血/再灌注损伤导致的细胞和线粒体内活性氧含量的升高,腺苷A2A及A2B受体拮抗剂均可阻断5′-N-乙基酰胺基腺苷的上述作用(P<0.01或P<0.05)。结论 5′-N-乙基酰胺基腺苷可减轻H9c2心肌细胞的缺血/再灌注损伤,腺苷A2A、A2B受体共同参与5′-N-乙基酰胺基腺苷的心肌保护作用,其机制可能与调节线粒体通透性转换孔(mitochondrial permeability transition pore,mPTP)的开放及线粒体活性氧的产生有关。
Abstract
OBJECTIVE To study the roles of adenosine A2A and A2B receptor in 5′-(N-ethylcarboxamido) adenosine (NECA)-induced cardioprotection in vitro against reperfusion injury, and to explore the underlying mechanism. METHODS Simulated ischemia/reperfusion injury model was developed in cardiac H9c2 cells. NECA, an unselective adenosine receptor agonist, the selective antagonists of adenosine A2A receptor antagonist SCH58261 (SCH) and the selective A2B receptor antagonist MRS1706 (MRS) were used. CCK-8 assay was used to evaluate cell viability. Mitochondrial membrane potential (△Ψm) was measured with fluorescence microscope using JC-1. Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit was used to detect the level of intracellular H2O2. Intracellular reactive oxygen species (ROS) levels were determined with DCFH-DA. Mitochondrial ROS were detected with MitoSox Red. RESULTS NECA applied at reperfusion reduced cell death in cells subjected to simulated ischemia/reperfusion. Compared with the ischemia/reperfusion injury group, NECA inhibited the reduction of cell viability and △Ψm, and the elevation of intracellular and mitochondrial ROS, which were all abolished by adenosine A2A and A2B receptor antagonists(P<0.01 or P<0.05 ). CONCLUSION Adenosine A2A and A2B receptors work in concert to mediate the cardioprotective effect of NECA presumably by modulating the mPTP opening and mitochondrial ROS generation.
关键词
5′-N-乙基酰胺基腺苷 /
缺血/再灌注损伤 /
H9c2心肌细胞 /
腺苷A2A受体 /
腺苷A2B受体
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Key words
5′-(N-ethylcarboxamido) adenosine /
ischemia/reperfusion injury /
H9c2 cardiomocytes /
adenosine A2A receptor /
adenosine A2B receptor
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中图分类号：
R965
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参考文献
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脚注
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基金
国家自然科学基金资助项目(81270182,81470397)
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