Oxidative stress developed during the reperfusion of ischemic myocardium induces apoptosis
Oxidative stress developed during the reperfusion of ischemic myocardium induces apoptosis
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DOI：
10.1016/s0891-5849(97)00388-2
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发表时间：
1998-03-15
影响因子：
7.4
通讯作者：
Das, DK
中科院分区：
文献类型：
--
作者：
Maulik, N;Yoshida, T;Das, DK
细胞凋亡或程序性细胞死亡是一种遗传控制的细胞自杀反应，与DNA片段化或梯状化有关。细胞凋亡的常见诱导剂包括氧自由基/氧化应激和Ca（2+），它们也参与心肌缺血再灌注损伤的发病机制。为了检查缺血再灌注损伤是否由凋亡性细胞死亡介导，使离体灌注的大鼠心脏经受15、30或60分钟的缺血以及15分钟的缺血随后30、60或120分钟的再灌注。在每个实验结束时，处理心脏用于评估细胞凋亡、DNA梯状化。通过使用唾液酸凋亡检测试剂盒中的APOPTAG直接荧光检测地高辛标记的基因组DNA来可视化凋亡的心肌细胞来研究凋亡。通过将从心脏获得的DNA进行1.8%琼脂糖凝胶电泳并在UV照射下拍照来评价DNA梯状化。我们的研究结果显示，凋亡细胞仅在60和120分钟的再灌注心脏的免疫染色的地高辛标记的基因组DNA的强烈荧光，荧光显微镜下观察时证明。没有一个缺血心脏显示出任何细胞凋亡的证据。这些结果证实了DNA片段化的发现，其显示在相同的再灌注心脏中DNA条带的梯状增加，其代表核小体间DNA长度的整数倍（约180 bp）。通过在依布硒啉存在下对心脏进行预灌注，消除了心肌中凋亡细胞和DNA片段的存在，这也消除了心脏中产生的氧化应激。总之，这些结果清楚地表明，在缺血再灌注心肌中产生的氧化应激诱导细胞凋亡。出版社：Elsevier Science Inc.
Apoptosis or programmed cell death is a genetically controlled response for cells to commit suicide and, is associated with DNA fragmentation or laddering. The common inducers of apoptosis include oxygen free radicals/oxidative stress and Ca(2+) which are also implicated in the pathogenesis of myocardial ischemic reperfusion injury. To examine whether ischemic reperfusion injury is mediated by apoptotic cell death, isolated perfused rat hearts were subjected to 15, 30 or 60 min of ischemia as well as 15 min of ischemia followed by 30, 60 or 120 min of reperfusion. At the end of each experiment, hearts were processed for the evaluation of apoptosis, DNA laddering. Apoptosis was studied by visualizing the apoptotic cardiomyocytes by direct fluorescence detection of digoxigenin-labeled genomic DNA using APOPTAG in sial apoptosis detection kit. DNA laddering was evaluated by subjecting the DNA obtained from the hearts to 1.8% agarose gel electrophoresis and photographed under UV illumination. The results of our study revealed apoptotic cells only in the 60 and 120 min reperfused hearts as demonstrated by the intense fluorescence of the immunostained digoxigenin-labeled genomic DNA when observed under fluorescence microscopy. None of the ischemic hearts showed any evidence of apoptosis. These results corroborated with the findings of DNA fragmentation which showed increased ladders of DNA bands in the same reperfused hearts representing integer multiples of the internucleosomal DNA length (about 180 bp). The presence of apoptotic cells and DNA fragmentation in the myocardium were abolished by preperfusing the hearts in the presence of ebselen, which also removed the oxidative stress developed in the heart. Taken together, these results clearly demonstrate that oxidative stress developed in the ischemic reperfused myocardium induces apoptosis. Published by Elsevier Science Inc.