Galang N, Sasaki H, Maulik N
Department of Surgery, University of Connecticut School of Medicine, Farmington, CT 06030-1110, USA.
Toxicology. 2000 Aug 7;148(2-3):111-8. doi: 10.1016/s0300-483x(00)00201-8.
Apoptosis is a form of programmed cell death that can be induced in susceptible cells by a wide variety of normal physiological stimuli as well as by deleterious environmental conditions and cytotoxic agents. 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 how free radicals are directly involved in apoptosis, rats were divided into three groups. The first group of rat hearts were perfused for 15 min with KHB buffer, the second group of rat hearts were perfused with superoxide dismutase plus catalase, and the hearts were subjected to 30 min of ischemia and 120 min of reperfusion. The third group of rat hearts, served as control which were subjected to 165 min of perfusion with KHB buffer (where n=6 rats in each group). At the end of each experiment, hearts were saved (at -70 degrees C) and analysed for apoptosis, DNA laddening and MDA production. During the reperfusion continuous cardiac pressure measurements were recorded in real time with a data acquisition and analysis system (CORDAT II, Triton Technologies). Direct measurements of heart rate, developed pressure and the first derivative of the developed pressure were recorded before the intervention and during the reperfusion. Coronary flow was measured by timed collection of coronary effluent. The results of our study revealed apoptotic cells after 120 min of reperfusion as demonstrated by the intense fluorescence of the immunostained digoxigeninlabeled genomic DNA when observed under fluroscence microscopy. Evaluation of DNA fragmentation showed increased ladders of DNA bands in the same reperfused hearts representing integer multiples of the internucleosomal DNA, about 180 bp. The presence of apoptotic cells and DNA fragmentation in the myocardium were abolished by preperfusing the hearts in the presence of SOD and catalase, which also reduced the oxidative stress as evidenced by the MDA production. In concert, myocardial function was significantly better when compared with the ischemic control. Taken together, these results clearly demonstrate that oxidative stress developed in the ischemic reperfused myocardium induces apoptosis and free radical scavengers can play a crucial role in apoptotic cell death associated with ischemia/reperfusion.
细胞凋亡是一种程序性细胞死亡形式，可由多种正常生理刺激以及有害环境条件和细胞毒性剂在易感细胞中诱导产生。细胞凋亡的常见诱导因素包括氧自由基/氧化应激和Ca(2+)，它们也与心肌缺血再灌注损伤的发病机制有关。为了研究自由基如何直接参与细胞凋亡，将大鼠分为三组。第一组大鼠心脏用KHB缓冲液灌注15分钟，第二组大鼠心脏用超氧化物歧化酶加过氧化氢酶灌注，然后心脏经历30分钟缺血和120分钟再灌注。第三组大鼠心脏作为对照，用KHB缓冲液灌注165分钟（每组n = 6只大鼠）。在每个实验结束时，保存心脏（-70℃）并分析细胞凋亡、DNA梯状条带和丙二醛（MDA）生成情况。在再灌注期间，使用数据采集和分析系统（CORDAT II，Triton Technologies）实时记录连续的心脏压力测量值。在干预前和再灌注期间记录心率、舒张期压力和舒张期压力的一阶导数的直接测量值。通过定时收集冠状动脉流出液来测量冠状动脉血流量。我们的研究结果显示，再灌注120分钟后出现凋亡细胞，在荧光显微镜下观察时，免疫染色的地高辛标记基因组DNA发出强烈荧光即可证明。DNA片段化评估显示，在相同的再灌注心脏中，DNA条带梯状增加，代表核小体间DNA的整数倍，约180 bp。在超氧化物歧化酶和过氧化氢酶存在下预先灌注心脏可消除心肌中凋亡细胞的存在和DNA片段化，这也降低了氧化应激，丙二醛生成情况证明了这一点。与此同时，与缺血对照组相比，心肌功能明显更好。综上所述，这些结果清楚地表明，缺血再灌注心肌中产生的氧化应激诱导细胞凋亡，自由基清除剂可在与缺血/再灌注相关的凋亡细胞死亡中发挥关键作用。
