乙醛脱氢酶2（ALDH2）对大鼠心肌缺氧损伤的保护作用
|
摘要
缺血缺氧引起心肌线粒体的功能和代谢改变，在心肌缺血缺氧的发病机理中处于非常重要的地位，心肌线粒体损伤诱导的凋亡蛋白表达的过程是多因素、多环节参与的病理过程，对它致病机理的研究已成为当今的热点。蛋白质组学是“后基因组时代”蓬勃发展的技术体系新型学科群，其特点是采用高通量、高分辨率的蛋白鉴定技术，全景式研究在特定病理条件下的蛋白表达谱，由此在蛋白质水平上对疾病发生、细胞代谢等过程有更全面的认识，可获得一些传统手段无法得到的新蛋白标志物和新关键分子，极大地丰富诊断标志物的选择及对复杂致病机理的全面阐明。本研究针对大鼠心肌缺血缺氧模型的心肌线粒体，进行全蛋白质组表达谱差异分析，筛出一些有潜在应用价值的差异蛋白，并在心肌组织和心肌细胞对其中的差异分子，用特异的抑制剂和基因转染等细胞和分子生物学技术进行功能的分析和验证。
第一部分大鼠心肌缺血缺氧模型的线粒体乙醛脱氢酶2(ALDH2)蛋白质分析
目的：利用差异蛋白质谱识别缺血缺氧大鼠心肌线粒体代谢异常的蛋白。
材料和方法：实验动物分心梗组和对照组(每组9只)。对大鼠行左冠脉前降支结扎，术后4周，以LVEF达46.4±10.9％左右作为成功建立大鼠心梗模型，将之处死后，分别取左心室：先制作电镜标本，观察形态改变；然后进行心肌线粒体抽提，利用双向凝胶电泳技术显示差异蛋白的表达谱，再进行质谱鉴定和蛋白数据库分析。发现蛋白的表达差异后，分别在心梗3d、7d、14d和28d后，用RT-PCR和western blot方法证实心肌该蛋白的mRNA和蛋白质表达随时间变化的差别。另一方面，在用酶消化法获得心肌细胞后，制作细胞的缺氧模型，测定离体心肌细胞线粒体该蛋白酶在不同缺氧时间内的活性变化。结果：大鼠心梗的心肌线粒体电镜下出现线粒体肿胀，基质变淡，嵴膜疏短破坏等病理变化。在双向凝胶电泳技术显示差异蛋白的表达谱，发现其中有3个点表达有显著差异，经胶内酶解后，行质谱鉴定和蛋白数据库分析，其中一条蛋白被证实为乙醛脱氢酶2(Aldehyde dehydrogenase 2 ALDH2)，它在大鼠缺血缺氧心肌中的表达显著下降，并随心梗时间延长，心肌ALDH2的mRNA和蛋白质的表达呈逐渐下降趋势。在细胞水平的心肌线粒体(纯度达90％以上)，用乙醛代谢法测得ALDH2的酶活性也随缺氧时间的延长而下降。结论：在缺血缺氧大鼠的心肌线粒体中乙醛脱氢酶2表达显著下调，心肌缺血缺氧与ALDH2的表达和活性下降相关。
第二部分乙醛脱氢酶2(ALDH2)抑制剂daidzin对大鼠心肌细胞缺氧损伤作用的机制
目的：检测乙醛脱氢酶2(ALDH2)被daidzin抑制时对大鼠心肌细胞在缺氧状态下发生凋亡和信号转导的影响，验证乙醛脱氢酶2(ALDH2)在此过程中所起的作用。材料和方法：在确定daidzin的最佳抑制浓度后，运用心肌细胞缺氧模型，比较大鼠心肌细胞在单独缺氧和经ALDH2特异性抑制剂daidzin预处理24h后缺氧的凋亡和MAPK信号通路的改变。酶活性检测采用乙醛代谢法、ROS的检测用C400测定，凋亡的测定通过用Hoechest 33324、免疫荧光标记的流式细胞仪和TUNEL试剂盒检测，MAPK信号通路酶(ERK1／2、JNK、P38)磷酸化的改变用western blotting的方法检测。结果：60uM daidzin浓度是ALDH2酶活性被抑制而细胞无凋亡发生的最佳工作浓度。在daidzin(60uM)预处理24h后再经缺氧诱导，比单独缺氧引起的心肌细胞凋亡更为明显：表现为在Hoechest 33324染色中，细胞核溶解和核碎裂(P＜0.05)，在FACS和TUNEL的检测中，凋亡细胞明显增加(P＜0.05)，经凋亡后的死亡细胞有增加趋势但无统计学差异，同时与MAPK信号通路有关的磷酸化ERK1／2、JNK和P38的酶活性均表达增强。结论：daidzin使ALDH2酶活性降低可增加心肌细胞对缺氧导致凋亡的易感性，其机理是与细胞ROS和凋亡的增加及MAPK信号通路的激活有关。ALDH2对缺氧引起的细胞凋亡损伤具有保护作用。
第三部分乙醛脱氢酶2(ALDH2)基因对大鼠心肌细胞缺氧损伤的保护作用的机制
目的：通过检测转染野生和缺失型乙醛脱氢酶2(ALDH2*1／ALDH2*2)基因，对大鼠心肌细胞在缺氧状态下发生凋亡和信号转导的影响，进一步证明乙醛脱氢酶2(ALDH2)在此过程中所起的作用。材料和方法：运用细胞缺氧模型(同上)，比较大鼠心肌细胞在转染野生和缺失型乙醛脱氢酶2(ALDH2*1／ALDH2*2)基因再缺氧后ROS、4—HNE、凋亡、MAPK酶活性、bcl2和P53的改变，酶活性检测采用乙醛代谢法、ROS的检测用C400测定，凋亡的测定通过用Hoechest33324、免疫荧光标记的流式细胞仪和TUNEL试剂盒检测，4—HNE的检测用免疫组织化学法，MAPK信号通路酶(ERK1／2、JNK)磷酸化的改变用westernblotting的方法检测，而bcl2和P53分别用RT-PCR和western blotting的方法检测。结果：转染ALDH2野生型(ALHD2*1)和缺失型(ALDH2*2)基因后再缺氧，表现为转染缺失型(ALDH2*2)的心肌细胞对缺氧性损伤的耐受性明显差于野生型(ALHD2*1)的(P＜0.05)。与运用ALDH2特异性抑制剂daidzin不同的是：转染ALHD2*1和ALDH2*2后再缺氧，都对ROS的产生和MAPK途径(ERK1／2和JNK的磷酸化)没有影响(P＞0.05)；而在转染缺失型ALDH2*2基因再缺氧组，除了有细胞凋亡的增加外(P＜0.05)：表现在Hoechest 33324染色(核溶解和核碎裂)，FACS和TUNEL检测，还有4—HNE的明显增加(P＜0.05)。从bcl2和P53的RT-PCR和western blot结果分析，转染野生型(ALHD2*1)的缺氧心肌细胞bcl2表达增高，P53表达下降，与缺失型(ALDH2*2)的结果相反。结论：4—HNE可能是细胞氧化损伤的重要作用因素。ALDH2*1可降解氧化物质4—HNE，它的抗凋亡作用不以ROS-MAPK途径为主，有可能与bcl2—P53的ROS非依赖性刺激的凋亡途径有关，ALDH2酶活性降低可增加心肌细胞对缺氧导致凋亡的易感性，ALDH2对缺氧引起的细胞凋亡损伤具有保护作用。
结论
1．缺血缺氧性损伤可能与大鼠心肌线粒体中乙醛脱氢酶2(ALDH2)的表达和活性显著下降有关。
2．daidzin使ALDH2酶活性降低后，心肌细胞对缺氧的耐受性下降，并对缺氧导致凋亡的易感性增加，此时细胞ROS和凋亡的增加与MAPK信号通路的激活有关。
3．转染ALHD2*1和ALDH2*2对缺氧损伤的心肌细胞ROS的产生和MAPK(ERK1／2和JNK)途径没有影响。ALDH2*1可降解氧化物质4—HNE，它的抗凋亡作用不以ROS-MAPK途径为主，可能与bcl2—P53的ROS非依赖性刺激的抗凋亡途径有关。
4．ALDH2酶活性降低可增加心肌细胞对缺氧导致凋亡的易感性，ALDH2对缺氧引起的细胞凋亡损伤具有保护作用。
潜在价值和创新点
1．我们首次通过蛋白质组学技术发现乙醛脱氢酶2(ALDH2)在缺血缺氧心肌中表达下降，并初步证实其具有心肌细胞保护作用。
2．基于ALDH2的心肌保护作用，我们推测ALDH2基因缺失型这类人群在心肌梗死或高血压后更易发心力衰竭，预后更差，同时也为临床的药物治疗提供靶点。
Cardiac mitochondria plays a key role in the ischemic pathogenesis of heart failure. The apoptotic regulatory proteins mediated by injuried mitochondria have a multifactorial and multistage pathogenesis. Despite extensive studies, the fundamental mechanisms responsible for the development and progression of heart failure have not yet been fully elucidated. Recent technological advances in proteomics allow us for the first time to examine the expression profiles at protein level on a genome-wide scale. In our study, we used 2-DE and LC- MS to identify the proteins with different expressing level in rat infarction hearts. We got a group of significant differential proteins including ALDH2 protein, which may be related to the pathogenesis of heart failure. We also drew a positive conclusion through further biological analysis and validation with the specific inhibitor and transgene, and performed a series experiments related to cell apoptosis and signal route with cellular and molecular techniques.
PART ONE Aldehyde dehydrogenase 2 (ALDH2) proteomic analysis of ischemic myocardial
mitochondrial protein expression in rats
Abstract Aim To determine the metabolic changes in rat ischemic heart, we applied the method of proteomics to discover the possible protein variation in rat myocardial mitochondria. Methods Myocardial infarction model was established by left anterior descending coronary artery ligation. 4 weeks after operation, we used LVEF 46.4±10.9% as myocardial infarction model. The rats were killed and their left ventricules were cut off. Those mitochondrial morphologies were observed by electron microscope, and the mitochondrial proteins were extracted. Weight-age matched rats acted as control. The mitochondrial protein expression in two groups were analyzed by two-dimensional gel electrophoresis and LC-mass spectrometry, and were identified using database comparision. After found the significant changes, RT-PCR and western blot methods would be used to prove this expression difference at the time of 3d 7d 14d and 28d's myocardial infarction. On the other hand, we made the hypoxia model from myocytes with enzymatic digestion and their mitochondrial protein activity from mycytes was measured at different hypoxia time. Results The mitochondria showed significant pathological changes in rat ischemic
heart tissue under the transmission electron microscope, including shape swelling, matrix thinning and cristea breakdown. And 3 protein spots altered significantly were found. Aldehyde dehydrogenase 2 (ALDH2) was identified with database comparision. Expression of ALDH2 decreased remarkably in ischemic heart tissue compared with control. Furthermore, we detected the ALDH2 mRNA and its protein expression of rats at 3d, 7d, 14d, 28d after LAD ligation, and the results showed it decreased gradually after operation. The decrease of ALHD2 enzyme activity also showed hypoxia-time dependant in vitro by the method of acetaldehyde metabolism (the purity of cardiac mitochondria was more than 90% under the electron microscope) . Conclusion The expression of mitochondrial ALDH2 in rat ischemic myocytes decreased and also its enzyme activity showed hypoxia-time dependant in vitro.
PART TWO The mechanism study of Daidzin, inhibitor of Aldehyde dehydrogenase 2, in
injuring the rat cardiomyocytes induced by hypoxia
Abstract Aim To observe and verificate the effect of inhibited ALDH2 on cardiac myocyte induced by hypoxia, including apoptosis, MAPK signal route, and the role of ALDH2. Methods Cultured cardiomyocytes of neonatal rats were used. Hypoxia was imposed to the cardiomyocytes with or without daidzin pretreatment beyond the optional daidzin concentration. ALDH2 activity was measured by the method of acetaldehyde metabolism, ROS was measured by C400, apoptosis by Hoechest 33324, Fluorescence Activated Cell Sorting (FACS) and The DeadEnd~(TM) Fluorometric TUNEL System, and MAPK signal pathway (ERK1/2 JNK P38) by western blotting. Results ALHD2 enzyme activity of myocytes could be inhibited by daidzin (24h, 20-100 μ M) without induction of apoptosis, the most effective but no apoptotic concentrantion was 60 μ M. When exposed to hypoxia, however, the ROS and apoptisis was significantly increased in the cells pretreated with daidzin compared to those without the pretreatment (P<0.05), also an increased MAPK phospholyrated activation, including ERK1/2, JNK and P38 (P<0.05). Conclusion Myocytes showed decreased tolerance to hypoxia after ALDH2 specifically inhibited by daidzin, The reduction of ALDH2 activity might increase the susceptivity of myocytes to apoptosis
following hypoxia and the increase of ROS may related to MAPK activation, suggesting a protective role of ALDH2 in hypoxia-induced myocardial injury.
PART THREE The mechanism study of Aldehyde dehydrogenase 2 transgene in preventing rat
cardiomyocytes from apoptosis induced by hypoxia
Abstract Aim To observe the effect of wild and mutant type of ALDH2 gene on cardiomyocyte induced by hypoxia, including apoptosis and MAPK cell signal route. Methods Cultured cardiomyocytes of neonatal rats were used. Hypoxia was imposed to the cardiomyocytes with transgene ALDH2 (wild type-ALDH2*1 and mutant type-ALDH2*2) to observe the change of ROS, 4-HNE, apoptosis, MAPK(ERK1/2 and JNK), bcl2 and P53. ALDH2 activity was measured by the method of acetaldehyde metabolism, ROS was measured by C400, 4-HNE by immonohistology, apoptosis by Hoechest 33324, Fluorescence Activated Cell Sorting (FACS) and The DeadEnd~(TM)Colorimetric TUNEL System, and MAPK signal pathway by western blotting, bcl2 and P53 by RT-PCR and western blot. Results myocytes had a different tolerance to hypoxia after transgene of ALDH2 wild and mutant type. The wild and mutant ALHD2 transgene (ALDH2*1/ALDH2*2) had no effect on ROS and MAPK phosphoryrated activation including ERK1/2 and JNK (P>0. 05) . But in transgenic mutant type-ALDH2*2 myocytes induced by hypoxia (compared with its inhibitor daidzin), besides the apoptotic cells, including Hoechest 33324 staining (karyopyknosis and karyorrhexis), FACS and TUNEL, 4-HNE increased significantly (P<0.05). Furthermore the transgenic wild type-ALDH2*1' induced by hypoxia, their expression of higher bcl2 and lower P53 were quite different from mutant type-ALDH2*2's(P<0.05). Conclusion 4-HNE might be an important factor in myocytes oxidative injury. ALDH2 Transgene had no obvious effect on ROS and MAPK cell signal route. And the anti-apoptotic function from ALDH2*1 might be related to bcl2-P53 without ROS-MAPK dependant route. Myocytes showed decreased tolerance to hypoxia after transgene of ALDH2*2 and might increase the susceptivity of myocytes to apoptosis, suggesting a protective role of ALDH2 in hypoxia-induced myocardial injury.
Conclusion
1. The expression of ALDH2 in rat ischemic hearts decreased and also its enzyme activity showed hypoxia-time dependant in vitro.
2. Myocytes showed decreased tolerance to hypoxia after ALDH2 specifically inhibited by daidzin, The reduction of ALDH2 activity might increase the susceptivity of myocytes to apoptosis following hypoxia and the increase of ROS may related to MAPK activation.
3. The transgenic ALDH2*1/ALDH2*2 myocytes induced by hypoxia had no obvious effect on ROS and MAPK(ERK1/2 and JNK) cell signal route. ALDH2*1 could degrade 4-HNE and its anti-apoptotic function might be related to bcl2- P53 dependant route without ROS-MAPK's.
4. Under the lower ALDH2 enzyme activity circumstances, myocytes showed decreased tolerance to hypoxia and might increase the susceptivity of myocytes to apoptosis, suggesting a protective role of ALDH2*1 in hypoxia-induced myocardial injury.
The potential application and novelty of this project
1. Recent technological advances in proteomics allow us for the first time to find the decreased ALDH2 expression profiles in ischemic and hypoxia rat myocytes. And our study confirmed the ALDH2 protective function in heart.
2. Those with myocardial infarction and hypertension increase the susceptivity to heart failure due to the reduction of ALDH2 activity and also have a worse prognosis. We put forward that ALDH2*1 can prevents myocytes from hypoxia injury, which might discover new possible therapeutic targets in clinic.
第一部分大鼠心肌缺血缺氧模型的线粒体乙醛脱氢酶2(ALDH2)蛋白质分析
目的：利用差异蛋白质谱识别缺血缺氧大鼠心肌线粒体代谢异常的蛋白。
材料和方法：实验动物分心梗组和对照组(每组9只)。对大鼠行左冠脉前降支结扎，术后4周，以LVEF达46.4±10.9％左右作为成功建立大鼠心梗模型，将之处死后，分别取左心室：先制作电镜标本，观察形态改变；然后进行心肌线粒体抽提，利用双向凝胶电泳技术显示差异蛋白的表达谱，再进行质谱鉴定和蛋白数据库分析。发现蛋白的表达差异后，分别在心梗3d、7d、14d和28d后，用RT-PCR和western blot方法证实心肌该蛋白的mRNA和蛋白质表达随时间变化的差别。另一方面，在用酶消化法获得心肌细胞后，制作细胞的缺氧模型，测定离体心肌细胞线粒体该蛋白酶在不同缺氧时间内的活性变化。结果：大鼠心梗的心肌线粒体电镜下出现线粒体肿胀，基质变淡，嵴膜疏短破坏等病理变化。在双向凝胶电泳技术显示差异蛋白的表达谱，发现其中有3个点表达有显著差异，经胶内酶解后，行质谱鉴定和蛋白数据库分析，其中一条蛋白被证实为乙醛脱氢酶2(Aldehyde dehydrogenase 2 ALDH2)，它在大鼠缺血缺氧心肌中的表达显著下降，并随心梗时间延长，心肌ALDH2的mRNA和蛋白质的表达呈逐渐下降趋势。在细胞水平的心肌线粒体(纯度达90％以上)，用乙醛代谢法测得ALDH2的酶活性也随缺氧时间的延长而下降。结论：在缺血缺氧大鼠的心肌线粒体中乙醛脱氢酶2表达显著下调，心肌缺血缺氧与ALDH2的表达和活性下降相关。
第二部分乙醛脱氢酶2(ALDH2)抑制剂daidzin对大鼠心肌细胞缺氧损伤作用的机制
目的：检测乙醛脱氢酶2(ALDH2)被daidzin抑制时对大鼠心肌细胞在缺氧状态下发生凋亡和信号转导的影响，验证乙醛脱氢酶2(ALDH2)在此过程中所起的作用。材料和方法：在确定daidzin的最佳抑制浓度后，运用心肌细胞缺氧模型，比较大鼠心肌细胞在单独缺氧和经ALDH2特异性抑制剂daidzin预处理24h后缺氧的凋亡和MAPK信号通路的改变。酶活性检测采用乙醛代谢法、ROS的检测用C400测定，凋亡的测定通过用Hoechest 33324、免疫荧光标记的流式细胞仪和TUNEL试剂盒检测，MAPK信号通路酶(ERK1／2、JNK、P38)磷酸化的改变用western blotting的方法检测。结果：60uM daidzin浓度是ALDH2酶活性被抑制而细胞无凋亡发生的最佳工作浓度。在daidzin(60uM)预处理24h后再经缺氧诱导，比单独缺氧引起的心肌细胞凋亡更为明显：表现为在Hoechest 33324染色中，细胞核溶解和核碎裂(P＜0.05)，在FACS和TUNEL的检测中，凋亡细胞明显增加(P＜0.05)，经凋亡后的死亡细胞有增加趋势但无统计学差异，同时与MAPK信号通路有关的磷酸化ERK1／2、JNK和P38的酶活性均表达增强。结论：daidzin使ALDH2酶活性降低可增加心肌细胞对缺氧导致凋亡的易感性，其机理是与细胞ROS和凋亡的增加及MAPK信号通路的激活有关。ALDH2对缺氧引起的细胞凋亡损伤具有保护作用。
第三部分乙醛脱氢酶2(ALDH2)基因对大鼠心肌细胞缺氧损伤的保护作用的机制
目的：通过检测转染野生和缺失型乙醛脱氢酶2(ALDH2*1／ALDH2*2)基因，对大鼠心肌细胞在缺氧状态下发生凋亡和信号转导的影响，进一步证明乙醛脱氢酶2(ALDH2)在此过程中所起的作用。材料和方法：运用细胞缺氧模型(同上)，比较大鼠心肌细胞在转染野生和缺失型乙醛脱氢酶2(ALDH2*1／ALDH2*2)基因再缺氧后ROS、4—HNE、凋亡、MAPK酶活性、bcl2和P53的改变，酶活性检测采用乙醛代谢法、ROS的检测用C400测定，凋亡的测定通过用Hoechest33324、免疫荧光标记的流式细胞仪和TUNEL试剂盒检测，4—HNE的检测用免疫组织化学法，MAPK信号通路酶(ERK1／2、JNK)磷酸化的改变用westernblotting的方法检测，而bcl2和P53分别用RT-PCR和western blotting的方法检测。结果：转染ALDH2野生型(ALHD2*1)和缺失型(ALDH2*2)基因后再缺氧，表现为转染缺失型(ALDH2*2)的心肌细胞对缺氧性损伤的耐受性明显差于野生型(ALHD2*1)的(P＜0.05)。与运用ALDH2特异性抑制剂daidzin不同的是：转染ALHD2*1和ALDH2*2后再缺氧，都对ROS的产生和MAPK途径(ERK1／2和JNK的磷酸化)没有影响(P＞0.05)；而在转染缺失型ALDH2*2基因再缺氧组，除了有细胞凋亡的增加外(P＜0.05)：表现在Hoechest 33324染色(核溶解和核碎裂)，FACS和TUNEL检测，还有4—HNE的明显增加(P＜0.05)。从bcl2和P53的RT-PCR和western blot结果分析，转染野生型(ALHD2*1)的缺氧心肌细胞bcl2表达增高，P53表达下降，与缺失型(ALDH2*2)的结果相反。结论：4—HNE可能是细胞氧化损伤的重要作用因素。ALDH2*1可降解氧化物质4—HNE，它的抗凋亡作用不以ROS-MAPK途径为主，有可能与bcl2—P53的ROS非依赖性刺激的凋亡途径有关，ALDH2酶活性降低可增加心肌细胞对缺氧导致凋亡的易感性，ALDH2对缺氧引起的细胞凋亡损伤具有保护作用。
结论
1．缺血缺氧性损伤可能与大鼠心肌线粒体中乙醛脱氢酶2(ALDH2)的表达和活性显著下降有关。
2．daidzin使ALDH2酶活性降低后，心肌细胞对缺氧的耐受性下降，并对缺氧导致凋亡的易感性增加，此时细胞ROS和凋亡的增加与MAPK信号通路的激活有关。
3．转染ALHD2*1和ALDH2*2对缺氧损伤的心肌细胞ROS的产生和MAPK(ERK1／2和JNK)途径没有影响。ALDH2*1可降解氧化物质4—HNE，它的抗凋亡作用不以ROS-MAPK途径为主，可能与bcl2—P53的ROS非依赖性刺激的抗凋亡途径有关。
4．ALDH2酶活性降低可增加心肌细胞对缺氧导致凋亡的易感性，ALDH2对缺氧引起的细胞凋亡损伤具有保护作用。
潜在价值和创新点
1．我们首次通过蛋白质组学技术发现乙醛脱氢酶2(ALDH2)在缺血缺氧心肌中表达下降，并初步证实其具有心肌细胞保护作用。
2．基于ALDH2的心肌保护作用，我们推测ALDH2基因缺失型这类人群在心肌梗死或高血压后更易发心力衰竭，预后更差，同时也为临床的药物治疗提供靶点。
Cardiac mitochondria plays a key role in the ischemic pathogenesis of heart failure. The apoptotic regulatory proteins mediated by injuried mitochondria have a multifactorial and multistage pathogenesis. Despite extensive studies, the fundamental mechanisms responsible for the development and progression of heart failure have not yet been fully elucidated. Recent technological advances in proteomics allow us for the first time to examine the expression profiles at protein level on a genome-wide scale. In our study, we used 2-DE and LC- MS to identify the proteins with different expressing level in rat infarction hearts. We got a group of significant differential proteins including ALDH2 protein, which may be related to the pathogenesis of heart failure. We also drew a positive conclusion through further biological analysis and validation with the specific inhibitor and transgene, and performed a series experiments related to cell apoptosis and signal route with cellular and molecular techniques.
PART ONE Aldehyde dehydrogenase 2 (ALDH2) proteomic analysis of ischemic myocardial
mitochondrial protein expression in rats
Abstract Aim To determine the metabolic changes in rat ischemic heart, we applied the method of proteomics to discover the possible protein variation in rat myocardial mitochondria. Methods Myocardial infarction model was established by left anterior descending coronary artery ligation. 4 weeks after operation, we used LVEF 46.4±10.9% as myocardial infarction model. The rats were killed and their left ventricules were cut off. Those mitochondrial morphologies were observed by electron microscope, and the mitochondrial proteins were extracted. Weight-age matched rats acted as control. The mitochondrial protein expression in two groups were analyzed by two-dimensional gel electrophoresis and LC-mass spectrometry, and were identified using database comparision. After found the significant changes, RT-PCR and western blot methods would be used to prove this expression difference at the time of 3d 7d 14d and 28d's myocardial infarction. On the other hand, we made the hypoxia model from myocytes with enzymatic digestion and their mitochondrial protein activity from mycytes was measured at different hypoxia time. Results The mitochondria showed significant pathological changes in rat ischemic
heart tissue under the transmission electron microscope, including shape swelling, matrix thinning and cristea breakdown. And 3 protein spots altered significantly were found. Aldehyde dehydrogenase 2 (ALDH2) was identified with database comparision. Expression of ALDH2 decreased remarkably in ischemic heart tissue compared with control. Furthermore, we detected the ALDH2 mRNA and its protein expression of rats at 3d, 7d, 14d, 28d after LAD ligation, and the results showed it decreased gradually after operation. The decrease of ALHD2 enzyme activity also showed hypoxia-time dependant in vitro by the method of acetaldehyde metabolism (the purity of cardiac mitochondria was more than 90% under the electron microscope) . Conclusion The expression of mitochondrial ALDH2 in rat ischemic myocytes decreased and also its enzyme activity showed hypoxia-time dependant in vitro.
PART TWO The mechanism study of Daidzin, inhibitor of Aldehyde dehydrogenase 2, in
injuring the rat cardiomyocytes induced by hypoxia
Abstract Aim To observe and verificate the effect of inhibited ALDH2 on cardiac myocyte induced by hypoxia, including apoptosis, MAPK signal route, and the role of ALDH2. Methods Cultured cardiomyocytes of neonatal rats were used. Hypoxia was imposed to the cardiomyocytes with or without daidzin pretreatment beyond the optional daidzin concentration. ALDH2 activity was measured by the method of acetaldehyde metabolism, ROS was measured by C400, apoptosis by Hoechest 33324, Fluorescence Activated Cell Sorting (FACS) and The DeadEnd~(TM) Fluorometric TUNEL System, and MAPK signal pathway (ERK1/2 JNK P38) by western blotting. Results ALHD2 enzyme activity of myocytes could be inhibited by daidzin (24h, 20-100 μ M) without induction of apoptosis, the most effective but no apoptotic concentrantion was 60 μ M. When exposed to hypoxia, however, the ROS and apoptisis was significantly increased in the cells pretreated with daidzin compared to those without the pretreatment (P<0.05), also an increased MAPK phospholyrated activation, including ERK1/2, JNK and P38 (P<0.05). Conclusion Myocytes showed decreased tolerance to hypoxia after ALDH2 specifically inhibited by daidzin, The reduction of ALDH2 activity might increase the susceptivity of myocytes to apoptosis
following hypoxia and the increase of ROS may related to MAPK activation, suggesting a protective role of ALDH2 in hypoxia-induced myocardial injury.
PART THREE The mechanism study of Aldehyde dehydrogenase 2 transgene in preventing rat
cardiomyocytes from apoptosis induced by hypoxia
Abstract Aim To observe the effect of wild and mutant type of ALDH2 gene on cardiomyocyte induced by hypoxia, including apoptosis and MAPK cell signal route. Methods Cultured cardiomyocytes of neonatal rats were used. Hypoxia was imposed to the cardiomyocytes with transgene ALDH2 (wild type-ALDH2*1 and mutant type-ALDH2*2) to observe the change of ROS, 4-HNE, apoptosis, MAPK(ERK1/2 and JNK), bcl2 and P53. ALDH2 activity was measured by the method of acetaldehyde metabolism, ROS was measured by C400, 4-HNE by immonohistology, apoptosis by Hoechest 33324, Fluorescence Activated Cell Sorting (FACS) and The DeadEnd~(TM)Colorimetric TUNEL System, and MAPK signal pathway by western blotting, bcl2 and P53 by RT-PCR and western blot. Results myocytes had a different tolerance to hypoxia after transgene of ALDH2 wild and mutant type. The wild and mutant ALHD2 transgene (ALDH2*1/ALDH2*2) had no effect on ROS and MAPK phosphoryrated activation including ERK1/2 and JNK (P>0. 05) . But in transgenic mutant type-ALDH2*2 myocytes induced by hypoxia (compared with its inhibitor daidzin), besides the apoptotic cells, including Hoechest 33324 staining (karyopyknosis and karyorrhexis), FACS and TUNEL, 4-HNE increased significantly (P<0.05). Furthermore the transgenic wild type-ALDH2*1' induced by hypoxia, their expression of higher bcl2 and lower P53 were quite different from mutant type-ALDH2*2's(P<0.05). Conclusion 4-HNE might be an important factor in myocytes oxidative injury. ALDH2 Transgene had no obvious effect on ROS and MAPK cell signal route. And the anti-apoptotic function from ALDH2*1 might be related to bcl2-P53 without ROS-MAPK dependant route. Myocytes showed decreased tolerance to hypoxia after transgene of ALDH2*2 and might increase the susceptivity of myocytes to apoptosis, suggesting a protective role of ALDH2 in hypoxia-induced myocardial injury.
Conclusion
1. The expression of ALDH2 in rat ischemic hearts decreased and also its enzyme activity showed hypoxia-time dependant in vitro.
2. Myocytes showed decreased tolerance to hypoxia after ALDH2 specifically inhibited by daidzin, The reduction of ALDH2 activity might increase the susceptivity of myocytes to apoptosis following hypoxia and the increase of ROS may related to MAPK activation.
3. The transgenic ALDH2*1/ALDH2*2 myocytes induced by hypoxia had no obvious effect on ROS and MAPK(ERK1/2 and JNK) cell signal route. ALDH2*1 could degrade 4-HNE and its anti-apoptotic function might be related to bcl2- P53 dependant route without ROS-MAPK's.
4. Under the lower ALDH2 enzyme activity circumstances, myocytes showed decreased tolerance to hypoxia and might increase the susceptivity of myocytes to apoptosis, suggesting a protective role of ALDH2*1 in hypoxia-induced myocardial injury.
The potential application and novelty of this project
1. Recent technological advances in proteomics allow us for the first time to find the decreased ALDH2 expression profiles in ischemic and hypoxia rat myocytes. And our study confirmed the ALDH2 protective function in heart.
2. Those with myocardial infarction and hypertension increase the susceptivity to heart failure due to the reduction of ALDH2 activity and also have a worse prognosis. We put forward that ALDH2*1 can prevents myocytes from hypoxia injury, which might discover new possible therapeutic targets in clinic.
引文
1. Bryant D, BeckeT L, Richardson J, et al. Cardic failure in transgenic mice with myocardial expression of tumor necrosis factor-α. Circulation. 1998;97:1375-1381
2. Drummond GR, Cai H, Davis ME, et al. Transcriptional and posttranscriptional regulation of endothelial nitric oxide synthase expression by hydrogen peroxide. Circ Res. 2000;86:347-354.
3. Ellis GR, Anderson RA, Lang D, et al. Neutrophil superoxide anion-generating capacity, endothelial function and oxidative stress in chronic heart failure: effects of short- and long-term vitamin C therapy. J AM Coll Cardiol. 2000;36:1474-1482.
4. Uchida K, Stadtman E. R. Modification of histidine residues in proteins by reaction with 4-hydroxynonenal. Proc. Natl Acad. Sci. USA, 1992;89:4544-4548.
5. Liu W., Kato M., Akhand A.A. et al. 4-hydroxynonenal induces a cellular redox status-related activation of the caspase cascade for apoptotic cell death. J. Cell Sci. 2000;113:635-641.
6. Siems W. G., Hapner S. J. and vanKuijk F. J. 4-hydroxynonenal inhibits Na(+)-K(+)-ATPase. Free Radic. Biol. Med. 1996;20(2):215-223.
7. Wei F, Jian Huaf, Yu chunshen, et al. A control study on the polym orphism of the A D H and ALD H genes in high risk alcoholic ram ilies of H an ethic population. Chin J Psychiatry, 1999, 32: 1664-1663.
8. Fernandez-solaj, Estruchr. The relation of alcoholic myopathy to cardiomypathy. Ann Intern Med, 1994, 120: 529-536.
9. Chen Z, Zhang J, Jonathan S et al. From the Cover: Identification of the enzymatic mechanism of nitroglycerin bioactivation. Proc Natl Acad Sci (PNAS), 2002;99(12):8306-8311.
10. Takagi S, Iwai N, Yamanchi R. et al. Aldehyde dehydrogenase 2 gene is a risk factor for myocardial infarction in Japanese men. Hypertens Res, 2002; 25:677-681.
11. Moncada S, Higgs A. L-Arginine-nitric oxide pathway, N Engl J Med. 1993:329:2002-2012.
12. Ohsawa I, Nishimaki K, Yasuda, et al. Deficiency in a mitochondrial aldehyde dehydrogenase increases vulnerability to oxidative stress in PC12 cells. J Neurochem, 2003:84:1110-1117.
13. Li S, Gomelsky M, Duan J, et al. Overexpression of aldehyde dehydrogenase-2 transgene prevents acetaldehyde-induced cell injury in human umbilical vein endothelial cells: role of ERK and P38 MAP kinase. J Biol chem, 2004:279:11244-11252.
1 Schoffner J, Wallace DC. Heart disease and mitochondrial DNA mutations. Heart Dis Stroke, 1992; 1(4): 235-241.
2. Wasinger V C, Cordwell S J, Cerpa-Poljak A, et al. Progress with gene-product mapping of the Mollicutes: Mycoplasma genitalium. Electrophoresis, 1995, 16(7): 1090—1094.
3. Blackstock W P, Weir M P. Proteomics: Quantitative andphysical mapping of cellular proteins. Trends in biotechnology, 1999, 17:121—127.
4．俞利荣，王楠，吴高德，等．人肝癌细胞系BEL27404和正常肝细胞系L202表达的蛋白质组双向凝胶电泳分析．科学通报，2000，45(2)：170—178．
5. Hanash S M. Biomedical applications of two-dimensional electrophoresis using immobilized pH gradients: Current status[J], Electrophoresis, 2000, 21: 1202-1209.
6. Murray J, Gilkerson R, Capaldi RA. Quantitative proteomics: the copy number of pyruvate dehydrogenase is more than 10(2)-fold lower than that of complex Ⅲ in human mitochondria. Febs Lett, 2002,529 (2-3): 173-178.
7. Kernec F, Unlu M, Labeikovsky W, et el. Changes in the mitochondrial proteome from mouse hearts deficient in creatine kinase. Physiol Genomics, 2001,6 (2): 117-128.
8. Rabilloud T, Kieffer S, Procaccio V, et al. Two-dimensional electrophoresis of human placental mitochondria and protein identification by mass spectrometry: Toward a human mitochondrial proteome. Electrophoresis, 1998, 19 (6): 1006-1014.
9. Hoit BD, Shao Y, Gabel M, et el. Left atrial mechanical and biochemical adaptation to pacing induced hear failure. Cardiovascular Research, 1995,29 (4): 469-474
10. Tyler DD, Jeanine G. The preparation of heart mitochondria from laborator animals. Methods in Enzymol. 1967,10:75-77.
11. Guerra S, Leri A, Wang X, Finato N, Di Loreto C, Beltrami CA, Kajstura J, Anversa P. Myocyte death in the failing human heart is gender dependent. Circ Res. 1999 Oct 29;85(9):856-866.
12. Daniel NN, Korsmeyer SJ. Cell death: critical control points. Cell. 2004 Jan 23; 116(2):205-219
13. Saraste A, Pulkki K, Kallajoki M, Heikkila P, Laine P, Mattila S, Nieminen MS, Parvinen M, Voipio-Pulkki LM. Cardiomyocyte apoptosis and progression of heart failure to transplantation. Eur J Clin Invest. 1999 May;29(5):380-386.
14. Aebersold R, Goodlett DR. Mass spectrometry in proteomics. Chem Rev. 2001, 101(2): 269-295.
15. Fergusorr RA, Goldberg DM. Genetic markers of alcolol abuse. J Clin Chim Acta. 1997, 257(2): 199-250.
16. Watanabe S, Sasahara K, Kinekawa F, et al. Aldehyde dehydrogenase-2 genotypes and HLA haplotypes in Japanese patients with esophageal cancer. Oncol Rep. 2002, 9(5):1063-1068.
17. Matsuo K, Hamajima N, Hirai T, et al. Aldehyde dehydrogenase 2 (ALDH2) genotype affects rectal cancer susceptibility due to alcohol consumption. J Epidemiol. 2002, 12.(2) :70-6.
18. Wei F, Jian ftF, YU CS, et al. A control study on the polymorphism of the ALDH and ALDH genes in high risk alcoholic families of Han ethic population. Chin J Psychiatry. 1999, 32: 164-1663.
19. Fernandez-Sola j, Estruch R, Josep M. Grau, et al. The relation of alcoholic myopathy to cardiomypathy, Ann Intern Med, 1994, 120: 529-536.
20. Chen ZQ, Zhang J, Stamler JS. Identification of the enzymatic mechanism of nitroglycerin bioactivation. Proc Natl Acad Sci. 2002, 99(12) :8306-8311.
21．李温斌综述，陈室田审校。心肌细胞的分离、培养及其应用。心肺血管病杂志，1994，13(4)：239—242．
1. Kajstura J, Cheng W , Reiss K, et al. Apoptotic and necrotic myocyte cell deaths are independent contributing variables of infarct size in rats. Lab Invest, 1996, 74 (1): 86-107.
2. Nanette HB, Daryl JD, Shari K, et al. Hypoxia — activated apoptosis of cardialmyocytes requires reoxygenatien or pH shift and is independent of P53. J Clin invest, 1999,104 (3): 239-252.
3. Kang PM , Haunstetter A , Aoki H ,et al. Morphological and molecular characterization of adult cardiomyocyte apoptosis during hypoxia and reoxygenation. Circ Res, 2000, 87 (2):118-125.
4. Takagi S, Iwai N, Yamanchi R. et al. Aldehyde dehydrogenase 2 gene is a risk factor for myocardial infarction in Japanese men. Hypertens Res, 2002; 25:677-681.
5. Gill C, MESTRIL R, SAMALI A. Losing heart: the role of apoptosis in heart disease—a novel therapeutic target? FASEB J, 2002,16:135-146.
6. Duilio C, Ambrosio G, Kuppusamy P, et al. Neutrophils are primary source of radicals during reperfusion after prolonged myocardial ischemia. Am J Physiol Heart Circ Physiol 2001, 280:H2649-H2657.
7. Smith JA, Weidemann MJ. Further characterization of the neutrophil oxidative burst by flow cytometry. J Immunol Methods, 1993 , 162 : 261 - 268.
8. Handel NS, McClintock DS , Feliciano CE , et al. Reactive oxygen species generated at mitochondrial complex III stabilize hypoxia2inducible factor21alpha during hypoxia : a mechanism of 02 sensing. J Biol Chem , 2000 , 275 (33) :25-30.
9. Donovan M, Cotter T G. Control of mitochondrial integrity by Bcl22 family members and caspase2 independent cell death. Biochim Biophys Acta , 2004 , 1644 (2-3): 133-147.
10. Cotgreave IA, Gerds RG. Recent trends in glutathione biochemistry—glutathione - protein interaction : a molecular link between oxidative stress and cell proliferation . J Biochem and Biophys Res Commu, 1998 , 242 : 1 - 9.
11. Greenberg AK, Basu S, Hu J, et al. Selectivep38 activation in human non-small cell lung cancer. Am J Respir Cell Mol Biol, 2002 , 26 (5) :558-564.
12. Ohsawa I, Nishimaki K, Yasuda, et al.Deficiency in a mitochondrial aldehyde dehydrogenase increases vulnerability to oxidative stress in PC12 cells. J Neurochemistry, 2003,84,1110-1117
13. Dougherty CJ, Kubasiak LA, Prentice H, et al. Activation of c-Jun N-terminal kinase promotes survival of cardiac myocytes after oxidative stress. Biochem J, 2002, 362(Pt3):561-571.
14. Kajstura J, Cheng W, Reiss K, Clark WA , et al. Apoptotic and necroticmyocyte cell death are independent contributing variables of infarct size in rate. Lab Invest, 1996,74 (1) : 86-107
15. Keung WM, Vallee BL. Daidzin: a potent, selective inhibitor of human mitochondrial aldehyde dehydrogenase. Proc.Natl.Acad.Sci.USA, 1993,90:1247-1251.
16. Jin K, Mao X O , Zhu Y, et al. MEK and ERK protect hypoxic cortical neurons via phosphorylation of Bad. J Neurochem, 2002 ,80(1) :119—125.
17. Jiang Z , Zhang Y, Chen X , et al. Actiyation of Erk1/ 2 and Akt in astrocytes under ischemia. Biochem Biophys Res Commun , 2002 , 294 (3) :726-733.
18. Li SY, Gomelsky M, Duan JH, et al. Overexpression of aldehyde dehydrogenase-2 (ALDH2) transgene prevents acetaldehyde-induced cell injury in human umbilical vein endothelial cells. J Biochemistry, 2004, 279 (12 ): 11244-11252.
1. Chen ZQ, Zhang J, Stamler JS. From the Cover: Identification of the enzymatic mechanism of nitroglycerin bioactivation. Proc Natl Acad Sci (PNAS), 2002 , 99 (12):8306-8311.
2. Ikawa M, Impraim CC, Wang G, Yoshida AS, et al. Isolation and Characterization of Aldehyde Dehydrogenase Isozymes from Usual and Atypical Human Livers. J Biol Chem.,1983, Vol 258(10): 6282-6287.
3. Hitt DC , Booth JL , Dandapani V, et al. A flow cytometric protocol for tittering recombinant adenoviral vectors containing the green fluorescent protein . Mol Biotechol, 2000 , 14 (3): 197 - 203.
4. Ohsawa I, Nishimaki K, Yasuda C, et al. Deficiency in a mitochondrial aldehyde dehydrogenase increases vulnerability to oxidative stress in PC12 cells. J Neurochemistry, 2003,84,1110-1117.
5. Takagi S, Iwai N, Yamanchi R. et al. Aldehyde dehydrogenase 2 gene is a risk factor for myocardial infarction in Japanese men. Hypertens Res, 2002; 25:677-681.
6. Mark RJ,Geddes JW, Uchida K,et al. Amyloid B-pepide impairs glucose transportin hippocampal and cortical neurons:involvement of membrane lipid peroxidation. J Neurosci. 1997,17:1046-1054.
7. Kruman I, Mattson MP. Pivotal role of mitochondrial calcium uptake in neurla cell apoptosis and necrosis. J Neurochem. 1999, 72:529-540.
8. Tjalkens RB, Cook LW, Petersen DR, et al. Formation and export of the glutathione conjugate of 4-hydroxy-2, 3-E-nonenal(4-HNE) in hepatoma cells. Arch Biochem Biophys. 1999, 361:113-119.
9. Stewart MJ, Mlaek K, Crabb DW, et al. Distribution of messenger RNAs for aldehyde dehydrogenase 1, aldehyde dehydrogenase 2, and aldehyde dehydrogenase 5 in human tissues. J Invese Med. 1996,44:42-46.
10. Li SY, Gomelsky M, Duan JH, et al. Overexpression of aldehyde dehydrogenase-2 (ALDH2) transgene prevents acetaldehyde-induced cell injury in human umbilical vein endothelial cells. J Biochemistry, 2004, 279 (12): 11244-11252.
11. Susin SA, Castedo NZ M, Hirsch T, et al. Bcl-2 inhibits the mitochondrial release of an apoptogenic protease.J Exp Med. 1996,184: 1331-1341.
12. M acDonald G, Sh i L , Vande velde C, et al. Mitochondria-dependent and -independent Regulation of Granzyme B-induced Apoptosis. J Exp M ed, 1999, 189: 131～144.
13. Minn AJ, V elez P, Schendel SL, et al. Bcl-x(L) forms an ion channel in synthetic lipid membranes. Nature, 1997, 385:353～357.
14. Chinnaiyan AM, Q' Rourke K, L ane BR, et al. Interaction of CED-4 with CED-3 and CED-9: A Molecular Framework for Cell Death. Science,1997, 275: 1122～1126.
15. Kharbanda S, Pandey P, Schofield L, et al. Role for Bcl-xL as an inhibitor of cytosolic cytochrome C accumulation in DNA damage-induced apoptosis P roc Natl Acad Sci (PNAS), 1997, 94: 6939～6942.
16. Shimizu S, Eguchi Y, Kosaka H, et al, Prevention of hypoxia-induced cell death by Bcl-2 and Bcl-xL. Nature, 1995, 374:811～813.
17. Spector MS, Desnoyers S, Hoeppner DJ, et al. Interaction between the C. elegans cell-death regulators CED-9 and CED-4. Nature, 1997; 385(6617): 653-656.
18. Yin XM, Oltvai ZN, Korsmeyer SJ, et al. BH1 and BH2 domains of Bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax. Nature, 1994,369 (6478): 321-323.
19. Sato T, Hanada M, Bodrug S, et al. Interactions among members of the Bcl-2 protein family analyzed with a yeast two-hybrid system. Proc Natl Acad Sci USA, 1994, 91(20):9238-9242.
20. Ishikawa Y, Kusaka E, Enokido Y, et al. Regulation of Bax translocation through phosphorylation at Ser270 of Bcl-2 by MAPkinase in NO-induced neuronal apoptosis. Mol Cell Neurosci, 2003,24(2): 451-459.
21. Bu S, Blaukat A, Fu X, et al. Mechanisms for methoxyestradiol-induced apoptosis of prostate cancer cells. FEBS Lett, 2002,531(2):141-151.
22. Ahn HJ, Kim YS, Kim JU, et al. Mechanism of taxol-induced apoptosis in human SKOV3 ovarian carcinoma cells. J Cell Biochem, 2004,91(5) :1043-1052.
23. Crowe DL, Boardman ML, and Fong KS, et al. Anti-Fas antibody differentially regulates apoptosis in Fas ligand resistant carcinoma lines via the caspase 3 family of cell death proteases but independently of bcl2 expression. Anticancer Res. 1998; 18(5A): 3163-3170.
24. Liadis N, Murakami K, Eweida M, et al. Caspase-3-Dependent β-Cell Apoptosis in the Initiation of Autoimmune Diabetes Mellitus. Mol Cell Biol, 2005; 25:3620-3629.
25. CLARK R. COFFMAN. Cell Migration and Programmed Cell Death of Drosophila Germ Cells. Ann. N. Y. Acad. Sci. 2003, 995:117-126.
26. Long X, Boluyt MO, H ipolito ML, et al. p53 and the hypoxia induced apoptosis of cultured neonatal rat cardiac myocytes. J Clinlnvest, 1997; 99(11): 2635-2643.
27. Samuni AM, Kasid U, Chuang EY, et al. Effects of Hypoxia on Radiation-Responsive Stress-Activated Protein Kinase, p53, and Caspase 3 Signals in TK6 Human Lymphoblastoid Cells. Cancer Res, Jan 2005; 65:579-586.
28. Kroemer G. The protooncognene Bcl-2 and its role in regulating apoptosis. Nature Medicine, 1997, 3:614-620.
29. Kirshenbaum LA, de Moissac D. The bal-2 gene product prorevents programmed cell death of ventricular myocytes. Circulation, 1997:96 (5): 1580-1585.
1. Chapple ILC. Reactive oxygen species and antioxidants in inflamma2tory diseases. J Clin Periodontol, 1997, 24 :287 - 296.
2. Sies H. Oxidative stress: oxidants and antioxidants. Exp Physiol. 1997, 82: 291-295.
3. Finkel T., Holbrook N.J. Oxidants, oxidative stress and the biology of ageing. Science, 2000, 408:239-247.
4. Cotgreave IA , Gerds RG. Recent trends in glutathione biochemistry-glutathione -protein interaction: a molecular link between oxidative stress and cell proliferation. J Biochem and Biophys Res Commu ,1998,242 :1-9.
5. Lander HM. An essential role for free radicals and derived species in signal .FASEB, 1997, 11: 118—124.
6. Davies SM, Poljak A,Duncan MW,et al. Measurements of protein carbonyls, ortho-and meta-tyrosine and oxidative phosphorylation complex activity in mitochondria from young and old rats. Free Radic Biol Med.2001, 31(2): 181-190.
7. Merker K, Stolzing A, Grune T. Proteolysis, caloric restriction and aging. Mech Ageing Dev. 2001, 122(7), 595—615.
8. Squier TC. Oxidative stress and protein aggregation during biological aging. Exp Gerontol. 2001, 36(9):1539-1550.
9. Wickner S, Maurizi MR, Gottesman S. Posttranslational Quality Control: Folding, Refolding, and Degrading Proteins. Science.l999,286:1888-1893.
10. Tabner BJ, El-Agnaf OM, German MJ, et al. Protein aggregation, metals and oxidative stress in neurodegenerative diseases. Biochem Soc Trans. 2005, 33(Pt 5): 1082-1086.
11. Barja G. Endogenous oxidative stress: relationship to aging, longevity and caloric restriction. Ageing Res Rev. 2002; 1(3): 397-411.
12. Barja G. Free radicals and aging. Trends Neurosci. 2004, 27(10): 595-600.
13. Pamplona R, Portero-Otin M, Sanz A, et al. Modification of the longevity-related degree of fatty acid unsaturation modulates oxidative damage to proteins and mitochondrial DNA in liver and brain. Exp Gerontol. 2004, 39(5): 725-733.
14. Bohr VA. Repair of oxidative DNA damage in nuclear and mitochondrial DNA, and some changes with aging in mammalian cells. Free Radic Biol Med. 2002:32(9): 804-812.
15. Sauer H, Wartenberg M, and Hescheler J. Reactive oxygen species as intracellular messengers during cell growth and differentiation. Cell Physiol Biochem. 2001,11(4): 173—86.
16. Finkel T. Redox-dependent signal transduction. FEBS Lett, 2000, 476(1-2): 52-54.
17. Shimizu S, Eguchi Y, Kosaka H, et al. Prevention of hypoxia-induced cell death by Bcl-2 and Bcl-xL. Nature.1995,374(6525):811-813.
18. Jacobson MD, Raft MC. Programmed cell death in animal development. Cell. 1997, 88(3): 347-54.
19. Jing YK, Dai J, Chalmers-Redman RME, et al. Arsenic trioxide selectively induces acute promyelocytic leukemia cell apoptosis via hydrogen peroxid2dependent pathway. Blood, 1999,94: 2102-2111.
20. Guenal I, Fraisse CS, Gaumer S, et al. Bcl-2 and Hsp27 act at different levels to suppress programmed cell death. Oncogene, 1997,15(3):347—360.
21. Mehlen P, Schulze-Osyhoff K, Arrigo AP, et al. Small Stress Proteins as Novel Regulators of Apoptosis. Heat shock protein 27blocks fas/Apo-l-and staurosporine-induced cell death. J Biol Chem, 1996, 271:16510—16514.
22. Quillet-Mary A., Jaffrezou JP, Mausat V., et al. Implication of Mitochondrial Hydrogen Peroxide Generation in Ceramide-induced Apoptosis. J Biol Chem, 1997, 272: 21388—21395.
23. Seger R, Krebs EG. The MAPK signaling cacade. FASEB J,1995, 9(9):726-735.
24. Jians Y(姜勇), Liu AH(刘爱经), Zhao KS(赵克森). The signal transduction pathways of mitogen-activated protein kinase.. J First Mil Med Univ(第一军医大学学报)1999, 19(1):59-62. (in Chinese).
25. Madan P, Calder MD, and Watson AJ. Mitogen-activated protein kinase (MAPK) blockade of bovine preimplantation embryogenesis requires inhibition of both p38 and extracellular signal-regulated kinase (ERK) pathways. Reproduction. 2005,130: 41-51.
26. Xia ZG, Dickens M, Raingeaud J, et al. Opposing Effects of ERK and JNK-p38 MAP Kinases on Apoptosis. Science. 1995, 270(24):1326-1331.
27. Bonni A, Brunet A, West AE, et al. Cell Survival Promoted by the Ras-MAPK Signaling Pathway by Transcription-Dependent and -Independent Mechanisms. Science. 1999, 286(12):1358-1362.
28. Satoh T, Nakatsuka D, Watanabe Y, et al. Neuroproteetion by MAPK/ ERK kinase inhibition with U0126 against oxidative stress in a mouse neuronal cell line and rat primary cultured cortical neurons. Neurosci Lett, 2000 ,288(2) : 163 —166.
29. van Heuten HA, Eskildsen-Helmond YE, de onge HW, et al. Phosphoinositide-generated messengers in cardiac signal transduction. Mol Cell Biochem,.1996; 157(1-2): 5- 14.
30. Page C, Doubell A F. Mitogen-activated protein kinase (MAPK) in cardiac tissues. Mol Cell Biochem, 1996; 157: 49-57.
31. Yazaki Y, Komuro I, Yamazaki T et al. Role of protein kinase system in the signal transduction of stretch-mediated protooncogene expression and hypertrophy of cardiac myocytes. Mol Cell Biochem. 1993; 119(1-2): 11-16.
32. Sadoshima J , Qiu Z, M rgan JP, et al. Angiotensin II and Other Hypertrophic Stimuli Mediated by G Protein-Coupled Receptors Activate Tyrosine Kinase, Mitogen-Activated Protein Kinase, and 90-kD S6 Kinase in Cardiac Myocytes : The Critical Role of Ca2+-Dependent Signaling. Circ Res,.1995;76: 1-15.
33. Schorb W , Conrad KM , Singer HA,et al. Angiotensin II is a potent stimulator of MAP-kinase activity in neonatal rat cardiac fibroblasts. J Mol Cell Cardio., 1995; 27: 1151-1160.
34. Schulze OK, Barkker AC, Vanhaesebroeck B, et al. Cytotoxic activity of tumor necrosis factor is mediated by early damage of mitochondrial functions. Evidence for the involvement of mitochondrial radical generation. J Biol Chem. 1992, 267: 5317— 5323.
35. Frade JM, Michaelidis TM. Origin of eukaryotic programmed cell death: a consequence of aerobic metabolism? Biolessays, 1997, 19(9): 827—832
36. Zou H, Henzel WJ, Liu X, et al. Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3. Cell. 1997, 90(3): 405— 413.
37. Minn AJ, V elez P, Schendel SL, et al. Bcl-x(L) forms an ion channel in synthetic lipid membranes. N ature, 1997, 385:353— 357
38. Kharbanda S, Pandey P, Schofield L, et al. Role for Bcl-xL as an inhibitor of cytosolic cytochrome C accumulation in DNA damage-induced apoptosis P roc Natl Acad Sci (PNAS), 1997, 94: 6939- 6942.
39. CLARK R. COFFMAN. Cell Migration and Programmed Cell Death of Drosophila Germ Cells. Ann. N.Y. Acad. Sci. 2003, 995: 117-126.
40. M acDonald G, Shi L, Vande velde C, et al. Mitochondria-dependent and -independent Regulation of Granzyme B-induced Apoptosis. J Exp M ed,1999, 189: 131～144
41. Spector MS, Desnoyers S, Hoeppner DJ, et al. Interaction between the C. elegans cell-death regulators CED-9 and CED-4. Nature, 1997; 385(6617): 653-656.
42. Chinnaiyan AM, Q 'Rourke K, L ane BR, et al. Interaction of CED-4 with CED-3 and CED-9: A Molecular Framework for Cell Death. Science,1997, 275: 1122～1126
43. Yin XM, Oltvai ZN, Korsmeyer SJ. BH1 and BH2 domains of Bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax. Nature , 1994,369 (6478) :321-323.
44. Ishikawa Y, Kusaka E, Enokido Y, et al. Regulation of Bax translocation through phosphorylation at Ser270 of Bcl-2 by MAPkinase in NO-induced neuronal apoptosis. Mol Cell Neurosci, 2003,24 (2) : 451-459.
45. Bu S, Blaukat A, Fu X, et al. Mechanisms.for methoxyestradiol-induced apoptosis of prostate cancer cells. FEBS Lett. 2002,531 (2): 141-151.
46. Vivat-annah V, You D, Rizzo C, et al. Synergistic cytotoxicity xhibited by combination treatment of selective retinoid ligands ith taxol (Paclitaxel). Cancer Res.2001, 61 (24):8703-8711.
47. Ahn HJ, Kim YS, Kim JU, et al. Mechanism of taxol-induced apoptosis in human SKOV3 ovarian carcinoma cells. J Cell Biochem.2004,91 (5): 1043-1052.
48. Olivetti G, Quaini F, Sala R, et al . Acute myocardial infarction in humans is associated with activation of programmed myocyte cell death in the surviving portion of the heart. J Mol Cell Cardiol. 1996,28:2005-2016.
49. Kajstura J, Cheng W, Reiss K, et al. Apoptotic and necroticmyocyte cell death are independent contributing variables of infarct size in rate. Lab Invest, 1996; 74: 86-91.
50. Yowe D I, Ames BN. Q uantitation of age-related m itochondiral DNA deletions in rat tissues show s that their pattern of accumulation differs from that of humans. Gene, 1998; 209 (122) : 23-30.
51. Agocha A, L ee HW, Eghbali-Webb M. Hypoxia regulates basal and induced DNA synthesis and collagen type 1 production in human cardiac fibroblasts: effects of transform ing growth factor-B1, thyroid hormone, angiotensin E and basic fibroblast grow th factor. J Mol Cell Card iol, 1997; 29 (8): 2233-2244.
52. Wu-Wong JR, Chiou WJ, Dickmson R, et al. Endothelin attenuates apoptosis in human smooth muscle cells. Biochem J. 1997; 328 (Pt3): 733-737.
53. Diez J, Panizo A , Herandez M , et al. Cardiomyocyte apoptosis and cadiac angiotensin-converting enzyme in spontaneousy hypertension rats. Hypertension, 1997; 30 (5): 1029-1034.
54. Aikawa R, Komuro I, Yamazaki T, et al. Oxidative stress activates extracellular signal regulated kinases through Src and Ras in cultured cardiac myocytes of neonatal rats. J Clin Invest, 1997; 100 (7): 1813-1821.
55. Bauriedel G, Schumcking I, Hutter R, et al. Increased apop tosis and necrosis of coronary plaques in unstable angina. Z Kard iol, 1997;86 (11) : 902-910.
56. Chernovsky AV, Wang Y, Wong FS, et al. The role of Fas in autoimmune diabetes. Cell, 1997; 89: 17-24.
57. Ma XL, Kumar S, Gao F, et al. Inhibition of P38 mitogen-activated protein kinase decrease cardiomyocyte apoptosis an improves cardiac function after myocardial ischemia and reperfusion. Circulation. 1999, 99 (23) :1685-1691.
58. Long X, Boluyt MO, H ipolito ML, et al. p53 and the hypoxia2in2 duced apop tosis of cultured neonatal raat cardiac myocytes. J Clinlnvest, 1997; 99 (11): 2635-2643.
59. Samuni AM, Kasid U, Chuang EY, et al. Effects of Hypoxia on Radiation-Responsive Stress-Activated Protein Kinase, p53, and Caspase 3 Signals in TK6 Human Lymphoblastoid Cells. Cancer Res, Jan 2005,65:579-586.
60. Kirshenbaum LA, de-Moissac D. The bal-2 gene product prevents programmed cell death of ventricular myocytes. Circulation, 1997;96(5): 1580-1585.
61. Kroemer G. The proto-oncognene Bcl-2 and its role in regulating apoptosis. Nature Medicine, 1997,3:614-620
62. Los M, Craen MV, Penning LC, et al. Requirement of an ICE/CED23 protease for Fas/APO-1 mediated apoptosis. Nature, 1995,375:81-83.
63．曹立珍，谭焕然．结扎冠状动脉对大鼠心肌凋亡相关基因表达的影响．北京医科大学学报 JOURNAL OF BEU ING M-EDICAL UNIVERSITY．1999，31(6)：501—505．
2. Drummond GR, Cai H, Davis ME, et al. Transcriptional and posttranscriptional regulation of endothelial nitric oxide synthase expression by hydrogen peroxide. Circ Res. 2000;86:347-354.
3. Ellis GR, Anderson RA, Lang D, et al. Neutrophil superoxide anion-generating capacity, endothelial function and oxidative stress in chronic heart failure: effects of short- and long-term vitamin C therapy. J AM Coll Cardiol. 2000;36:1474-1482.
4. Uchida K, Stadtman E. R. Modification of histidine residues in proteins by reaction with 4-hydroxynonenal. Proc. Natl Acad. Sci. USA, 1992;89:4544-4548.
5. Liu W., Kato M., Akhand A.A. et al. 4-hydroxynonenal induces a cellular redox status-related activation of the caspase cascade for apoptotic cell death. J. Cell Sci. 2000;113:635-641.
6. Siems W. G., Hapner S. J. and vanKuijk F. J. 4-hydroxynonenal inhibits Na(+)-K(+)-ATPase. Free Radic. Biol. Med. 1996;20(2):215-223.
7. Wei F, Jian Huaf, Yu chunshen, et al. A control study on the polym orphism of the A D H and ALD H genes in high risk alcoholic ram ilies of H an ethic population. Chin J Psychiatry, 1999, 32: 1664-1663.
8. Fernandez-solaj, Estruchr. The relation of alcoholic myopathy to cardiomypathy. Ann Intern Med, 1994, 120: 529-536.
9. Chen Z, Zhang J, Jonathan S et al. From the Cover: Identification of the enzymatic mechanism of nitroglycerin bioactivation. Proc Natl Acad Sci (PNAS), 2002;99(12):8306-8311.
10. Takagi S, Iwai N, Yamanchi R. et al. Aldehyde dehydrogenase 2 gene is a risk factor for myocardial infarction in Japanese men. Hypertens Res, 2002; 25:677-681.
11. Moncada S, Higgs A. L-Arginine-nitric oxide pathway, N Engl J Med. 1993:329:2002-2012.
12. Ohsawa I, Nishimaki K, Yasuda, et al. Deficiency in a mitochondrial aldehyde dehydrogenase increases vulnerability to oxidative stress in PC12 cells. J Neurochem, 2003:84:1110-1117.
13. Li S, Gomelsky M, Duan J, et al. Overexpression of aldehyde dehydrogenase-2 transgene prevents acetaldehyde-induced cell injury in human umbilical vein endothelial cells: role of ERK and P38 MAP kinase. J Biol chem, 2004:279:11244-11252.
1 Schoffner J, Wallace DC. Heart disease and mitochondrial DNA mutations. Heart Dis Stroke, 1992; 1(4): 235-241.
2. Wasinger V C, Cordwell S J, Cerpa-Poljak A, et al. Progress with gene-product mapping of the Mollicutes: Mycoplasma genitalium. Electrophoresis, 1995, 16(7): 1090—1094.
3. Blackstock W P, Weir M P. Proteomics: Quantitative andphysical mapping of cellular proteins. Trends in biotechnology, 1999, 17:121—127.
4．俞利荣，王楠，吴高德，等．人肝癌细胞系BEL27404和正常肝细胞系L202表达的蛋白质组双向凝胶电泳分析．科学通报，2000，45(2)：170—178．
5. Hanash S M. Biomedical applications of two-dimensional electrophoresis using immobilized pH gradients: Current status[J], Electrophoresis, 2000, 21: 1202-1209.
6. Murray J, Gilkerson R, Capaldi RA. Quantitative proteomics: the copy number of pyruvate dehydrogenase is more than 10(2)-fold lower than that of complex Ⅲ in human mitochondria. Febs Lett, 2002,529 (2-3): 173-178.
7. Kernec F, Unlu M, Labeikovsky W, et el. Changes in the mitochondrial proteome from mouse hearts deficient in creatine kinase. Physiol Genomics, 2001,6 (2): 117-128.
8. Rabilloud T, Kieffer S, Procaccio V, et al. Two-dimensional electrophoresis of human placental mitochondria and protein identification by mass spectrometry: Toward a human mitochondrial proteome. Electrophoresis, 1998, 19 (6): 1006-1014.
9. Hoit BD, Shao Y, Gabel M, et el. Left atrial mechanical and biochemical adaptation to pacing induced hear failure. Cardiovascular Research, 1995,29 (4): 469-474
10. Tyler DD, Jeanine G. The preparation of heart mitochondria from laborator animals. Methods in Enzymol. 1967,10:75-77.
11. Guerra S, Leri A, Wang X, Finato N, Di Loreto C, Beltrami CA, Kajstura J, Anversa P. Myocyte death in the failing human heart is gender dependent. Circ Res. 1999 Oct 29;85(9):856-866.
12. Daniel NN, Korsmeyer SJ. Cell death: critical control points. Cell. 2004 Jan 23; 116(2):205-219
13. Saraste A, Pulkki K, Kallajoki M, Heikkila P, Laine P, Mattila S, Nieminen MS, Parvinen M, Voipio-Pulkki LM. Cardiomyocyte apoptosis and progression of heart failure to transplantation. Eur J Clin Invest. 1999 May;29(5):380-386.
14. Aebersold R, Goodlett DR. Mass spectrometry in proteomics. Chem Rev. 2001, 101(2): 269-295.
15. Fergusorr RA, Goldberg DM. Genetic markers of alcolol abuse. J Clin Chim Acta. 1997, 257(2): 199-250.
16. Watanabe S, Sasahara K, Kinekawa F, et al. Aldehyde dehydrogenase-2 genotypes and HLA haplotypes in Japanese patients with esophageal cancer. Oncol Rep. 2002, 9(5):1063-1068.
17. Matsuo K, Hamajima N, Hirai T, et al. Aldehyde dehydrogenase 2 (ALDH2) genotype affects rectal cancer susceptibility due to alcohol consumption. J Epidemiol. 2002, 12.(2) :70-6.
18. Wei F, Jian ftF, YU CS, et al. A control study on the polymorphism of the ALDH and ALDH genes in high risk alcoholic families of Han ethic population. Chin J Psychiatry. 1999, 32: 164-1663.
19. Fernandez-Sola j, Estruch R, Josep M. Grau, et al. The relation of alcoholic myopathy to cardiomypathy, Ann Intern Med, 1994, 120: 529-536.
20. Chen ZQ, Zhang J, Stamler JS. Identification of the enzymatic mechanism of nitroglycerin bioactivation. Proc Natl Acad Sci. 2002, 99(12) :8306-8311.
21．李温斌综述，陈室田审校。心肌细胞的分离、培养及其应用。心肺血管病杂志，1994，13(4)：239—242．
1. Kajstura J, Cheng W , Reiss K, et al. Apoptotic and necrotic myocyte cell deaths are independent contributing variables of infarct size in rats. Lab Invest, 1996, 74 (1): 86-107.
2. Nanette HB, Daryl JD, Shari K, et al. Hypoxia — activated apoptosis of cardialmyocytes requires reoxygenatien or pH shift and is independent of P53. J Clin invest, 1999,104 (3): 239-252.
3. Kang PM , Haunstetter A , Aoki H ,et al. Morphological and molecular characterization of adult cardiomyocyte apoptosis during hypoxia and reoxygenation. Circ Res, 2000, 87 (2):118-125.
4. Takagi S, Iwai N, Yamanchi R. et al. Aldehyde dehydrogenase 2 gene is a risk factor for myocardial infarction in Japanese men. Hypertens Res, 2002; 25:677-681.
5. Gill C, MESTRIL R, SAMALI A. Losing heart: the role of apoptosis in heart disease—a novel therapeutic target? FASEB J, 2002,16:135-146.
6. Duilio C, Ambrosio G, Kuppusamy P, et al. Neutrophils are primary source of radicals during reperfusion after prolonged myocardial ischemia. Am J Physiol Heart Circ Physiol 2001, 280:H2649-H2657.
7. Smith JA, Weidemann MJ. Further characterization of the neutrophil oxidative burst by flow cytometry. J Immunol Methods, 1993 , 162 : 261 - 268.
8. Handel NS, McClintock DS , Feliciano CE , et al. Reactive oxygen species generated at mitochondrial complex III stabilize hypoxia2inducible factor21alpha during hypoxia : a mechanism of 02 sensing. J Biol Chem , 2000 , 275 (33) :25-30.
9. Donovan M, Cotter T G. Control of mitochondrial integrity by Bcl22 family members and caspase2 independent cell death. Biochim Biophys Acta , 2004 , 1644 (2-3): 133-147.
10. Cotgreave IA, Gerds RG. Recent trends in glutathione biochemistry—glutathione - protein interaction : a molecular link between oxidative stress and cell proliferation . J Biochem and Biophys Res Commu, 1998 , 242 : 1 - 9.
11. Greenberg AK, Basu S, Hu J, et al. Selectivep38 activation in human non-small cell lung cancer. Am J Respir Cell Mol Biol, 2002 , 26 (5) :558-564.
12. Ohsawa I, Nishimaki K, Yasuda, et al.Deficiency in a mitochondrial aldehyde dehydrogenase increases vulnerability to oxidative stress in PC12 cells. J Neurochemistry, 2003,84,1110-1117
13. Dougherty CJ, Kubasiak LA, Prentice H, et al. Activation of c-Jun N-terminal kinase promotes survival of cardiac myocytes after oxidative stress. Biochem J, 2002, 362(Pt3):561-571.
14. Kajstura J, Cheng W, Reiss K, Clark WA , et al. Apoptotic and necroticmyocyte cell death are independent contributing variables of infarct size in rate. Lab Invest, 1996,74 (1) : 86-107
15. Keung WM, Vallee BL. Daidzin: a potent, selective inhibitor of human mitochondrial aldehyde dehydrogenase. Proc.Natl.Acad.Sci.USA, 1993,90:1247-1251.
16. Jin K, Mao X O , Zhu Y, et al. MEK and ERK protect hypoxic cortical neurons via phosphorylation of Bad. J Neurochem, 2002 ,80(1) :119—125.
17. Jiang Z , Zhang Y, Chen X , et al. Actiyation of Erk1/ 2 and Akt in astrocytes under ischemia. Biochem Biophys Res Commun , 2002 , 294 (3) :726-733.
18. Li SY, Gomelsky M, Duan JH, et al. Overexpression of aldehyde dehydrogenase-2 (ALDH2) transgene prevents acetaldehyde-induced cell injury in human umbilical vein endothelial cells. J Biochemistry, 2004, 279 (12 ): 11244-11252.
1. Chen ZQ, Zhang J, Stamler JS. From the Cover: Identification of the enzymatic mechanism of nitroglycerin bioactivation. Proc Natl Acad Sci (PNAS), 2002 , 99 (12):8306-8311.
2. Ikawa M, Impraim CC, Wang G, Yoshida AS, et al. Isolation and Characterization of Aldehyde Dehydrogenase Isozymes from Usual and Atypical Human Livers. J Biol Chem.,1983, Vol 258(10): 6282-6287.
3. Hitt DC , Booth JL , Dandapani V, et al. A flow cytometric protocol for tittering recombinant adenoviral vectors containing the green fluorescent protein . Mol Biotechol, 2000 , 14 (3): 197 - 203.
4. Ohsawa I, Nishimaki K, Yasuda C, et al. Deficiency in a mitochondrial aldehyde dehydrogenase increases vulnerability to oxidative stress in PC12 cells. J Neurochemistry, 2003,84,1110-1117.
5. Takagi S, Iwai N, Yamanchi R. et al. Aldehyde dehydrogenase 2 gene is a risk factor for myocardial infarction in Japanese men. Hypertens Res, 2002; 25:677-681.
6. Mark RJ,Geddes JW, Uchida K,et al. Amyloid B-pepide impairs glucose transportin hippocampal and cortical neurons:involvement of membrane lipid peroxidation. J Neurosci. 1997,17:1046-1054.
7. Kruman I, Mattson MP. Pivotal role of mitochondrial calcium uptake in neurla cell apoptosis and necrosis. J Neurochem. 1999, 72:529-540.
8. Tjalkens RB, Cook LW, Petersen DR, et al. Formation and export of the glutathione conjugate of 4-hydroxy-2, 3-E-nonenal(4-HNE) in hepatoma cells. Arch Biochem Biophys. 1999, 361:113-119.
9. Stewart MJ, Mlaek K, Crabb DW, et al. Distribution of messenger RNAs for aldehyde dehydrogenase 1, aldehyde dehydrogenase 2, and aldehyde dehydrogenase 5 in human tissues. J Invese Med. 1996,44:42-46.
10. Li SY, Gomelsky M, Duan JH, et al. Overexpression of aldehyde dehydrogenase-2 (ALDH2) transgene prevents acetaldehyde-induced cell injury in human umbilical vein endothelial cells. J Biochemistry, 2004, 279 (12): 11244-11252.
11. Susin SA, Castedo NZ M, Hirsch T, et al. Bcl-2 inhibits the mitochondrial release of an apoptogenic protease.J Exp Med. 1996,184: 1331-1341.
12. M acDonald G, Sh i L , Vande velde C, et al. Mitochondria-dependent and -independent Regulation of Granzyme B-induced Apoptosis. J Exp M ed, 1999, 189: 131～144.
13. Minn AJ, V elez P, Schendel SL, et al. Bcl-x(L) forms an ion channel in synthetic lipid membranes. Nature, 1997, 385:353～357.
14. Chinnaiyan AM, Q' Rourke K, L ane BR, et al. Interaction of CED-4 with CED-3 and CED-9: A Molecular Framework for Cell Death. Science,1997, 275: 1122～1126.
15. Kharbanda S, Pandey P, Schofield L, et al. Role for Bcl-xL as an inhibitor of cytosolic cytochrome C accumulation in DNA damage-induced apoptosis P roc Natl Acad Sci (PNAS), 1997, 94: 6939～6942.
16. Shimizu S, Eguchi Y, Kosaka H, et al, Prevention of hypoxia-induced cell death by Bcl-2 and Bcl-xL. Nature, 1995, 374:811～813.
17. Spector MS, Desnoyers S, Hoeppner DJ, et al. Interaction between the C. elegans cell-death regulators CED-9 and CED-4. Nature, 1997; 385(6617): 653-656.
18. Yin XM, Oltvai ZN, Korsmeyer SJ, et al. BH1 and BH2 domains of Bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax. Nature, 1994,369 (6478): 321-323.
19. Sato T, Hanada M, Bodrug S, et al. Interactions among members of the Bcl-2 protein family analyzed with a yeast two-hybrid system. Proc Natl Acad Sci USA, 1994, 91(20):9238-9242.
20. Ishikawa Y, Kusaka E, Enokido Y, et al. Regulation of Bax translocation through phosphorylation at Ser270 of Bcl-2 by MAPkinase in NO-induced neuronal apoptosis. Mol Cell Neurosci, 2003,24(2): 451-459.
21. Bu S, Blaukat A, Fu X, et al. Mechanisms for methoxyestradiol-induced apoptosis of prostate cancer cells. FEBS Lett, 2002,531(2):141-151.
22. Ahn HJ, Kim YS, Kim JU, et al. Mechanism of taxol-induced apoptosis in human SKOV3 ovarian carcinoma cells. J Cell Biochem, 2004,91(5) :1043-1052.
23. Crowe DL, Boardman ML, and Fong KS, et al. Anti-Fas antibody differentially regulates apoptosis in Fas ligand resistant carcinoma lines via the caspase 3 family of cell death proteases but independently of bcl2 expression. Anticancer Res. 1998; 18(5A): 3163-3170.
24. Liadis N, Murakami K, Eweida M, et al. Caspase-3-Dependent β-Cell Apoptosis in the Initiation of Autoimmune Diabetes Mellitus. Mol Cell Biol, 2005; 25:3620-3629.
25. CLARK R. COFFMAN. Cell Migration and Programmed Cell Death of Drosophila Germ Cells. Ann. N. Y. Acad. Sci. 2003, 995:117-126.
26. Long X, Boluyt MO, H ipolito ML, et al. p53 and the hypoxia induced apoptosis of cultured neonatal rat cardiac myocytes. J Clinlnvest, 1997; 99(11): 2635-2643.
27. Samuni AM, Kasid U, Chuang EY, et al. Effects of Hypoxia on Radiation-Responsive Stress-Activated Protein Kinase, p53, and Caspase 3 Signals in TK6 Human Lymphoblastoid Cells. Cancer Res, Jan 2005; 65:579-586.
28. Kroemer G. The protooncognene Bcl-2 and its role in regulating apoptosis. Nature Medicine, 1997, 3:614-620.
29. Kirshenbaum LA, de Moissac D. The bal-2 gene product prorevents programmed cell death of ventricular myocytes. Circulation, 1997:96 (5): 1580-1585.
1. Chapple ILC. Reactive oxygen species and antioxidants in inflamma2tory diseases. J Clin Periodontol, 1997, 24 :287 - 296.
2. Sies H. Oxidative stress: oxidants and antioxidants. Exp Physiol. 1997, 82: 291-295.
3. Finkel T., Holbrook N.J. Oxidants, oxidative stress and the biology of ageing. Science, 2000, 408:239-247.
4. Cotgreave IA , Gerds RG. Recent trends in glutathione biochemistry-glutathione -protein interaction: a molecular link between oxidative stress and cell proliferation. J Biochem and Biophys Res Commu ,1998,242 :1-9.
5. Lander HM. An essential role for free radicals and derived species in signal .FASEB, 1997, 11: 118—124.
6. Davies SM, Poljak A,Duncan MW,et al. Measurements of protein carbonyls, ortho-and meta-tyrosine and oxidative phosphorylation complex activity in mitochondria from young and old rats. Free Radic Biol Med.2001, 31(2): 181-190.
7. Merker K, Stolzing A, Grune T. Proteolysis, caloric restriction and aging. Mech Ageing Dev. 2001, 122(7), 595—615.
8. Squier TC. Oxidative stress and protein aggregation during biological aging. Exp Gerontol. 2001, 36(9):1539-1550.
9. Wickner S, Maurizi MR, Gottesman S. Posttranslational Quality Control: Folding, Refolding, and Degrading Proteins. Science.l999,286:1888-1893.
10. Tabner BJ, El-Agnaf OM, German MJ, et al. Protein aggregation, metals and oxidative stress in neurodegenerative diseases. Biochem Soc Trans. 2005, 33(Pt 5): 1082-1086.
11. Barja G. Endogenous oxidative stress: relationship to aging, longevity and caloric restriction. Ageing Res Rev. 2002; 1(3): 397-411.
12. Barja G. Free radicals and aging. Trends Neurosci. 2004, 27(10): 595-600.
13. Pamplona R, Portero-Otin M, Sanz A, et al. Modification of the longevity-related degree of fatty acid unsaturation modulates oxidative damage to proteins and mitochondrial DNA in liver and brain. Exp Gerontol. 2004, 39(5): 725-733.
14. Bohr VA. Repair of oxidative DNA damage in nuclear and mitochondrial DNA, and some changes with aging in mammalian cells. Free Radic Biol Med. 2002:32(9): 804-812.
15. Sauer H, Wartenberg M, and Hescheler J. Reactive oxygen species as intracellular messengers during cell growth and differentiation. Cell Physiol Biochem. 2001,11(4): 173—86.
16. Finkel T. Redox-dependent signal transduction. FEBS Lett, 2000, 476(1-2): 52-54.
17. Shimizu S, Eguchi Y, Kosaka H, et al. Prevention of hypoxia-induced cell death by Bcl-2 and Bcl-xL. Nature.1995,374(6525):811-813.
18. Jacobson MD, Raft MC. Programmed cell death in animal development. Cell. 1997, 88(3): 347-54.
19. Jing YK, Dai J, Chalmers-Redman RME, et al. Arsenic trioxide selectively induces acute promyelocytic leukemia cell apoptosis via hydrogen peroxid2dependent pathway. Blood, 1999,94: 2102-2111.
20. Guenal I, Fraisse CS, Gaumer S, et al. Bcl-2 and Hsp27 act at different levels to suppress programmed cell death. Oncogene, 1997,15(3):347—360.
21. Mehlen P, Schulze-Osyhoff K, Arrigo AP, et al. Small Stress Proteins as Novel Regulators of Apoptosis. Heat shock protein 27blocks fas/Apo-l-and staurosporine-induced cell death. J Biol Chem, 1996, 271:16510—16514.
22. Quillet-Mary A., Jaffrezou JP, Mausat V., et al. Implication of Mitochondrial Hydrogen Peroxide Generation in Ceramide-induced Apoptosis. J Biol Chem, 1997, 272: 21388—21395.
23. Seger R, Krebs EG. The MAPK signaling cacade. FASEB J,1995, 9(9):726-735.
24. Jians Y(姜勇), Liu AH(刘爱经), Zhao KS(赵克森). The signal transduction pathways of mitogen-activated protein kinase.. J First Mil Med Univ(第一军医大学学报)1999, 19(1):59-62. (in Chinese).
25. Madan P, Calder MD, and Watson AJ. Mitogen-activated protein kinase (MAPK) blockade of bovine preimplantation embryogenesis requires inhibition of both p38 and extracellular signal-regulated kinase (ERK) pathways. Reproduction. 2005,130: 41-51.
26. Xia ZG, Dickens M, Raingeaud J, et al. Opposing Effects of ERK and JNK-p38 MAP Kinases on Apoptosis. Science. 1995, 270(24):1326-1331.
27. Bonni A, Brunet A, West AE, et al. Cell Survival Promoted by the Ras-MAPK Signaling Pathway by Transcription-Dependent and -Independent Mechanisms. Science. 1999, 286(12):1358-1362.
28. Satoh T, Nakatsuka D, Watanabe Y, et al. Neuroproteetion by MAPK/ ERK kinase inhibition with U0126 against oxidative stress in a mouse neuronal cell line and rat primary cultured cortical neurons. Neurosci Lett, 2000 ,288(2) : 163 —166.
29. van Heuten HA, Eskildsen-Helmond YE, de onge HW, et al. Phosphoinositide-generated messengers in cardiac signal transduction. Mol Cell Biochem,.1996; 157(1-2): 5- 14.
30. Page C, Doubell A F. Mitogen-activated protein kinase (MAPK) in cardiac tissues. Mol Cell Biochem, 1996; 157: 49-57.
31. Yazaki Y, Komuro I, Yamazaki T et al. Role of protein kinase system in the signal transduction of stretch-mediated protooncogene expression and hypertrophy of cardiac myocytes. Mol Cell Biochem. 1993; 119(1-2): 11-16.
32. Sadoshima J , Qiu Z, M rgan JP, et al. Angiotensin II and Other Hypertrophic Stimuli Mediated by G Protein-Coupled Receptors Activate Tyrosine Kinase, Mitogen-Activated Protein Kinase, and 90-kD S6 Kinase in Cardiac Myocytes : The Critical Role of Ca2+-Dependent Signaling. Circ Res,.1995;76: 1-15.
33. Schorb W , Conrad KM , Singer HA,et al. Angiotensin II is a potent stimulator of MAP-kinase activity in neonatal rat cardiac fibroblasts. J Mol Cell Cardio., 1995; 27: 1151-1160.
34. Schulze OK, Barkker AC, Vanhaesebroeck B, et al. Cytotoxic activity of tumor necrosis factor is mediated by early damage of mitochondrial functions. Evidence for the involvement of mitochondrial radical generation. J Biol Chem. 1992, 267: 5317— 5323.
35. Frade JM, Michaelidis TM. Origin of eukaryotic programmed cell death: a consequence of aerobic metabolism? Biolessays, 1997, 19(9): 827—832
36. Zou H, Henzel WJ, Liu X, et al. Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3. Cell. 1997, 90(3): 405— 413.
37. Minn AJ, V elez P, Schendel SL, et al. Bcl-x(L) forms an ion channel in synthetic lipid membranes. N ature, 1997, 385:353— 357
38. Kharbanda S, Pandey P, Schofield L, et al. Role for Bcl-xL as an inhibitor of cytosolic cytochrome C accumulation in DNA damage-induced apoptosis P roc Natl Acad Sci (PNAS), 1997, 94: 6939- 6942.
39. CLARK R. COFFMAN. Cell Migration and Programmed Cell Death of Drosophila Germ Cells. Ann. N.Y. Acad. Sci. 2003, 995: 117-126.
40. M acDonald G, Shi L, Vande velde C, et al. Mitochondria-dependent and -independent Regulation of Granzyme B-induced Apoptosis. J Exp M ed,1999, 189: 131～144
41. Spector MS, Desnoyers S, Hoeppner DJ, et al. Interaction between the C. elegans cell-death regulators CED-9 and CED-4. Nature, 1997; 385(6617): 653-656.
42. Chinnaiyan AM, Q 'Rourke K, L ane BR, et al. Interaction of CED-4 with CED-3 and CED-9: A Molecular Framework for Cell Death. Science,1997, 275: 1122～1126
43. Yin XM, Oltvai ZN, Korsmeyer SJ. BH1 and BH2 domains of Bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax. Nature , 1994,369 (6478) :321-323.
44. Ishikawa Y, Kusaka E, Enokido Y, et al. Regulation of Bax translocation through phosphorylation at Ser270 of Bcl-2 by MAPkinase in NO-induced neuronal apoptosis. Mol Cell Neurosci, 2003,24 (2) : 451-459.
45. Bu S, Blaukat A, Fu X, et al. Mechanisms.for methoxyestradiol-induced apoptosis of prostate cancer cells. FEBS Lett. 2002,531 (2): 141-151.
46. Vivat-annah V, You D, Rizzo C, et al. Synergistic cytotoxicity xhibited by combination treatment of selective retinoid ligands ith taxol (Paclitaxel). Cancer Res.2001, 61 (24):8703-8711.
47. Ahn HJ, Kim YS, Kim JU, et al. Mechanism of taxol-induced apoptosis in human SKOV3 ovarian carcinoma cells. J Cell Biochem.2004,91 (5): 1043-1052.
48. Olivetti G, Quaini F, Sala R, et al . Acute myocardial infarction in humans is associated with activation of programmed myocyte cell death in the surviving portion of the heart. J Mol Cell Cardiol. 1996,28:2005-2016.
49. Kajstura J, Cheng W, Reiss K, et al. Apoptotic and necroticmyocyte cell death are independent contributing variables of infarct size in rate. Lab Invest, 1996; 74: 86-91.
50. Yowe D I, Ames BN. Q uantitation of age-related m itochondiral DNA deletions in rat tissues show s that their pattern of accumulation differs from that of humans. Gene, 1998; 209 (122) : 23-30.
51. Agocha A, L ee HW, Eghbali-Webb M. Hypoxia regulates basal and induced DNA synthesis and collagen type 1 production in human cardiac fibroblasts: effects of transform ing growth factor-B1, thyroid hormone, angiotensin E and basic fibroblast grow th factor. J Mol Cell Card iol, 1997; 29 (8): 2233-2244.
52. Wu-Wong JR, Chiou WJ, Dickmson R, et al. Endothelin attenuates apoptosis in human smooth muscle cells. Biochem J. 1997; 328 (Pt3): 733-737.
53. Diez J, Panizo A , Herandez M , et al. Cardiomyocyte apoptosis and cadiac angiotensin-converting enzyme in spontaneousy hypertension rats. Hypertension, 1997; 30 (5): 1029-1034.
54. Aikawa R, Komuro I, Yamazaki T, et al. Oxidative stress activates extracellular signal regulated kinases through Src and Ras in cultured cardiac myocytes of neonatal rats. J Clin Invest, 1997; 100 (7): 1813-1821.
55. Bauriedel G, Schumcking I, Hutter R, et al. Increased apop tosis and necrosis of coronary plaques in unstable angina. Z Kard iol, 1997;86 (11) : 902-910.
56. Chernovsky AV, Wang Y, Wong FS, et al. The role of Fas in autoimmune diabetes. Cell, 1997; 89: 17-24.
57. Ma XL, Kumar S, Gao F, et al. Inhibition of P38 mitogen-activated protein kinase decrease cardiomyocyte apoptosis an improves cardiac function after myocardial ischemia and reperfusion. Circulation. 1999, 99 (23) :1685-1691.
58. Long X, Boluyt MO, H ipolito ML, et al. p53 and the hypoxia2in2 duced apop tosis of cultured neonatal raat cardiac myocytes. J Clinlnvest, 1997; 99 (11): 2635-2643.
59. Samuni AM, Kasid U, Chuang EY, et al. Effects of Hypoxia on Radiation-Responsive Stress-Activated Protein Kinase, p53, and Caspase 3 Signals in TK6 Human Lymphoblastoid Cells. Cancer Res, Jan 2005,65:579-586.
60. Kirshenbaum LA, de-Moissac D. The bal-2 gene product prevents programmed cell death of ventricular myocytes. Circulation, 1997;96(5): 1580-1585.
61. Kroemer G. The proto-oncognene Bcl-2 and its role in regulating apoptosis. Nature Medicine, 1997,3:614-620
62. Los M, Craen MV, Penning LC, et al. Requirement of an ICE/CED23 protease for Fas/APO-1 mediated apoptosis. Nature, 1995,375:81-83.
63．曹立珍，谭焕然．结扎冠状动脉对大鼠心肌凋亡相关基因表达的影响．北京医科大学学报 JOURNAL OF BEU ING M-EDICAL UNIVERSITY．1999，31(6)：501—505．