Redistribution of phosphatidylethanolamine and phosphatidylserine precedes reperfusion-induced apoptosis
Redistribution of phosphatidylethanolamine and phosphatidylserine precedes reperfusion-induced apoptosis
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DOI：
10.1152/ajpheart.1998.274.1.h242
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发表时间：
1998-01-01
影响因子：
4.8
通讯作者：
Das, DK
中科院分区：
文献类型：
--
作者：
Maulik, N;Kagan, VE;Das, DK
尽管传统上认为与缺血再灌注相关的心肌细胞死亡和梗塞是通过坏死引起的，但最近的研究表明缺血再灌注组织中存在细胞凋亡。为了检查心肌缺血再灌注损伤是否是由细胞凋亡介导的，对离体灌注大鼠心脏进行15分钟和30分钟的缺血以及15分钟缺血后30、90或120分钟的再灌注。在每次实验结束时，对心脏进行处理以评估细胞凋亡和DNA梯状图。通过使用 APOPTAG 原位细胞凋亡检测试剂盒直接荧光检测地高辛标记的基因组 DNA 来观察凋亡的心肌细胞，从而研究细胞凋亡。通过将从心肌细胞获得的DNA进行1.8％琼脂糖凝胶电泳并在紫外线照射下拍照来评估DNA梯状图。此外，还对 2,4,6-三硝基苯磺酸盐标记的氨基磷脂进行了高效薄层色谱 (HPTLC)，以评估心肌细胞中的磷脂形态。我们的研究结果显示，仅在再灌注 90 分钟和 120 分钟的心脏中出现凋亡细胞，在荧光显微镜下观察时，免疫染色的地高辛标记的基因组 DNA 发出强烈的荧光即可证明这一点。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 片段化的结果，即在 120 分钟再灌注的心脏中 DNA 带的阶梯增加，代表核小体间 DNA 长度的整数倍（类似于 180 bp）。从心肌细胞获得的磷脂以及心肌细胞中磷脂酰乙醇胺 (PE) 和磷脂酰丝氨酸 (PS) 的跨双层组织的二维 HPTLC 表明，早在缺血 20 分钟后，PE 和 PS 就从膜的内叶易位到外叶。这些结果表明，PS和PE的重新分布先于与缺血心肌再灌注相关的细胞凋亡和DNA断裂，这表明缺血可能触发细胞凋亡信号，尽管在再灌注过程中这一信号变得明显。
Although cardiomyocyte death and infarction associated with ischemia-reperfusion are traditionally believed to be induced via necrosis, recent studies implicated apoptotic cell death in ischemic reperfused tissue. To examine whether myocardial ischemic reperfusion injury is mediated by apoptotic cell death, isolated perfused rat hearts were subjected to 15 and 30 min of ischemia as well as 15 min of ischemia followed by 30, 90, or 120 min of reperfusion. At the end of each experiment, hearts were processed for the evaluation of apoptosis and DNA laddering. Apoptosis was studied by visualizing the apoptotic cardiomyocytes by direct fluorescence detection of digoxigenin-labeled genomic DNA using APOPTAG in situ apoptosis detection kit. DNA laddering was evaluated by subjecting the DNA obtained from cardiomyocytes to 1.8% agarose gel electrophoresis and photographed under ultraviolet illumination. In addition, high-performance thin-layer chromatography (HPTLC) of aminophospholipids labeled with 2,4,6-trinitrobenzenesulfonate was performed to evaluate phospholipid topography in cardiomyocytes. The results of our study revealed apoptotic cells only in the 90- and 120-min reperfused hearts as demonstrated by the intense fluorescence of the immunostained digoxigenin-labeled genomic DNA when observed under fluorescence microscope. None of the ischemic hearts showed any evidence of apoptosis. These results corroborated with the findings of DNA fragmentation that showed increased ladders of DNA bands in the 120-min reperfused hearts, representing integer multiples of the internucleosomal DNA length (similar to 180 bp). Two-dimensional HPTLC of the phospholipids obtained from the cardiomyocytes and transbilayer organization of the phosphatidylethanolamine (PE) and phosphatidylserine (PS) in the myocytes indicated translocation of both PE and PS from the inner leaflet to the outer leaflet of the membrane as early as after 20 min of ischemia. These results demonstrate that the redistribution of PS and PE precedes the apototic cell death and DNA fragmentation associated with the reperfusion of ischemic myocardium, suggesting that ischemia may trigger the signal for apoptosis although it becomes evident during reperfusion.