粉防己碱对大鼠心肌缺血/再灌注和缺氧/复氧损伤心肌细胞凋亡的影响及其机制研究
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摘要
第一部分粉防己碱预处理对大鼠缺血再灌注心肌的保护作用及其与表没食子儿茶素没食子酸酯比较研究
目的:
通过大鼠在体心肌缺血再灌注模型,观察粉防己碱预处理对缺血再灌注损伤心肌的保护作用。表没食子儿茶素没食子酸酯具有抗心肌缺血再灌注损伤和心肌细胞凋亡的作用,与表没食子儿茶素没食子酸酯进行比较,观察粉防己碱预处理对缺血再灌注损伤心肌的保护作用。
方法:
建立在体大鼠心脏I/R损伤模型,将96只大鼠随机分为正常对照组(Sham组, n=16)、I/R损伤组(I/R组, n=20)、粉防己碱1组(TET1组, n=20)、粉防己碱2组(TET2组,n=20)、表没食子儿茶素没食子酸酯组(EGCG组,n=20);各组均测定大鼠心肌I/R损伤后血清乳酸脱氢酶(LDH)、心肌组织丙二醛(MDA)、超氧化物歧化酶(SOD)含量,采用HE染色及电镜观察各组心肌的病理变化。各组均测定心肌梗死范围(IS/AAR%,TTC法),比较各组间差异。
结果:
与Sham组比较,I/R组中MDA值、LDH值、IS/AAR %明显升高,SOD显著降低。与I/R组比较,TET1组、TET2组、EGCG组可显著降低IS/AAR%值[(23.28±4.38)%, (19.96±4.38)%, (24.86±4.67)% vs. (43.76±6.30)% ,均P﹤0.01]; LDH [(1329.54±250.21)U/L, (1305.76±266.44)U/L,(1418.62±276.17)U/L,vs. (2016.97±371.95) U/L,均P﹤0.01)]、MDA [(3.16±0.21) nmol/mg prot,(2.98±0.38) nmol/mg prot,(3.39±0.52)nmol /mg prot vs. (5.33±0.51) nmol/mg prot,均P﹤0.01)]均明显降低,而SOD值明显增高[(138.45±20.74)U/mg prot, (146.38±24.41)U/mg prot, (135.46±20.98) U/mg prot vs. ( 98.69±15.41) U/mg prot,均P﹤0.01)]。TET1组、TET2组、EGCG组可以显著减轻心肌缺血再灌注损伤的病理改变。与EGCG组比较,TET1、TET2、EGCG三组间MDA、LDH和SOD值无显著性差异(P>0.05)。
结论:
大鼠心肌缺血再灌注损伤可导致LDH值、MDA值升高、SOD降低,引起心肌病理改变及IS/AAR %升高,与EGCG比较,粉防己碱预处理可以抗心肌缺血再灌注损伤,保护心肌,其保护效果与EGCG相似。
第二部分粉防己碱预处理对缺血再灌注心肌细胞凋亡及凋亡相关蛋白Bax、Bcl-2、Caspase-3和CytC的影响
目的:
观察粉防己碱对缺血再灌注损伤心肌细胞凋亡的影响及其对Bax、Bcl-2蛋白表达和线粒体中细胞色素C释放的影响,并观察粉防己碱对心肌细胞caspase-3变化的影响,初步探讨粉防己碱预处理对缺血再灌注损伤心肌细胞凋亡及凋亡相关蛋白的作用。
方法:
建立在体大鼠心脏I/R损伤模型,将143只大鼠随机分为正常对照组(Sham组,n=23)、I/R损伤组(I/R组,n=30)、粉防己碱1组(TET1组,n=30)、粉防己碱2组(TET2组,n=30)、表没食子儿茶素没食子酸酯组(EGCG组,n=30);实验结束后,用原位末端标记法(TUNEL)和DNA梯带法(DNA Ladder)检测细胞凋亡,用免疫组化法检测Bax、Bcl-2蛋白表达和Western-blotting法检测胞浆细胞色素C表达,用RT-RCR法检测心肌细胞caspase-3 mRNA表达,并观察caspase-3活性变化,比较各组间差异。
结果:
与I/R组相比,TET1组、TET2组、EGCG组明显抑制心肌细胞凋亡,AI明显降低[(8.62±2.45)%,(7.95±2.28)%,(10.62±3.09)% vs. (19.36±5.28)%,均P﹤0.01];与I/R组相比,TET1组、TET2组、EGCG组Bcl-2表达明显增加,Bax明显减少,且Bcl-2/ Bax值显著高于I/R组(P<0.01);I/R组心肌细胞胞浆细胞色素C显著增多(P<0.01),经TET1、TET2和EGCG预处理后,可明显抑制细胞色素C从线粒体向胞浆的释放(P<0.01);TET1、TET2和EGCG预处理后,可明显减少心肌细胞caspase-3的表达和活性(P<0.01)。
结论:
粉防己碱可通过抑制缺血再灌注心肌细胞凋亡来参与缺血再灌注心肌损伤的保护,粉防己碱抗心肌细胞凋亡可能与上调Bcl-2蛋白、下调Bax蛋白表达,升高Bcl-2/ Bax比值,抑制Cyt C从线粒体的释放,减少caspase-3在心肌组织中表达和活性有关。
第三部分粉防己碱预处理对缺氧/复氧诱导心肌细胞凋亡影响及其机制研究
第一节新生大鼠体外心肌细胞培养和缺氧/复氧模型建立
目的:
通过体外培养新生大鼠心肌细胞,构建新生大鼠心肌缺氧/复氧损伤模型,在细胞水平模拟大鼠心肌缺血再灌注(SI/R)损伤,为研究粉防己碱对新生大鼠缺氧/复氧损伤影响提供平台。
方法:
分离新生1-3天的乳鼠心室肌,在培养基中培养3-4天后,行缺氧2h/复氧24h,建立缺氧/复氧(anoxia/reoxygenation)心肌细胞损伤模型。在缺氧前、缺氧2h末期和复氧24h末期利用2,4二硝基苯肼显色法检测乳酸脱氢酶(LDH)活性,硫代巴比妥酸显色法检测丙二醛(MDA)及黄嘌呤氧化酶法(羟胺法)检测超氧化物歧化酶(SOD)含量并进行比较,以证实缺氧/复氧损伤模型构建成功。
结果:
新生1-3天的乳鼠心室肌,在培养基中培养3-4天后,在倒置显微镜下可观察到有搏动的心肌细胞。培养3-4天的心肌细胞在实施缺氧期2h后,LDH和MDA均较缺氧前有所升高,SOD降低(P<0.01);而给予复氧期24h后,LDH和MDA明显高于缺氧期,而SOD进一步降低(P<0.01),对照组的上述指标在相应时间点未见明显改变(P>0.05)。
结论:
通过给新生大鼠体外培养心肌细胞行缺氧2h/复氧24h后,可明显造成心肌细胞损伤,以此模拟大鼠心肌缺血再灌注损伤。
第二节粉防己碱对缺氧/复氧诱导新生大鼠心肌细胞凋亡、PKC表达的影响及机制研究
目的:
通过建立的乳鼠心肌细胞缺氧/复氧模型,观察粉防己碱对缺氧/复氧诱导的心肌细胞凋亡及PKC的影响,探讨粉防己碱抑制心肌细胞凋亡的信号转导机制。
方法:
通过给原代培养乳鼠心肌细胞行缺氧2h/复氧24h ,建立缺氧/复氧(anoxia/reoxygenation)心肌细胞损伤模型。将培养的心肌细胞分为正常对照(CON)组、缺氧/复氧(A/R)组、缺氧预处理(AP)组、粉防己碱预处理(TET)组、缺氧预处理+PKC抑制剂chelerythrin(eAP+CHE)组、粉防己碱+PKC抑制剂chelerythrine(TET+CHE)组,共6组。于复氧24h后,检测LDH活性和心肌细胞MDA、SOD含量,采用原位末端标记法(TUNEL)和DNA梯带法(DNA Ladder)标测心肌细胞凋亡,检测心肌细胞PKC mRNA的表达量及PKC活性,用免疫印迹法(Western blotting)检测胞浆CytC表达,并检测心肌细胞caspase-3 mRNA表达量及活性,比较各组间差异。
结果:
与A/R组相比,AP组、TET组中LDH活性、MDA含量、凋亡指数(AI)显著降低,SOD显著升高(P<0.01);PKC的mRNA的表达量和活性显著增加,心肌细胞胞浆中CytC减少,心肌细胞caspase-3 mRNA表达量减少、caspase-3活性降低有显著性差异(P<0.01);经chelerythrine处理后,再给予AP或TET预处理,则PKC的mRNA的表达量和PKC活性显著下降,可见胞浆中CytC增加,心肌细胞caspase-3 mRNA表达量增加,caspase-3活性增强,凋亡指数(AI)增加(P<0.01)。
结论:
粉防己碱预处理可减少缺氧/复氧诱导的心肌细胞凋亡,PKC抑制剂chelerythrine处理后,可显著降低粉防己碱的保护作用,因此PKC的信号传导途径可能是粉防己碱抑制缺血再灌注损伤心肌细胞凋亡的关键环节。
PartⅠThe Study of Protective Effect Against Ischemia/Reperfusion Injury Preconditioning with Tetrandrine in Rats and Comparison with Epigallocatechin-3-gallate
Objective:
To observe the protective effects of tetrandrine against myocardial ischemia and reperfusion injury in rats. Previous studies had shown that Epigallocatechin-3-gallate (EGCG) could protect myocardium from ischemia/reperfusion injury and inhibit cardiomyocyte apoptosis. Compared with EGCG, the present study was to investigate whether tetrandrine had protective effects on myocardium in ischemia and reperfusion model.
Methods:
Pentobarbital sodium-anesthetized Sprague-Dawley rats underwent 30 min of left anterior descending (LAD) coronary occlusion and 24 hours reperfusion to make ischemia/reperfusion (I/R) injury model in vivo. Ninety-six rats were divided into five groups randomly: Sham group, Ischemia/reperfusion injury (I/R) group, Tetrandrine pretreatment1 (TET1) group, Tetrandrine pretreatment 2 (TET2) group and Epigallo- catechin-3-gallate (EGCG) group. Sham group underwent only the sham operation; other four groups underwent I/R operation. All groups measured infarcted size (IS/ AAR %). Lactatedehydrogenase (LDH) in serum, the superoxidedismutase (SOD) and malondial- dehyde (MDA) in myocardium tissue were measured in each group. In addition,pathologic changes of myocardial tissue were observed under hematoxylin-eosine staining(HE) and electron microscopy. The results were compared among the groups.
Results:
Compared with Sham group, I/R group markedly increased the activity of LDH in serum, the content of MDA in myocardium and IS/AAR %, but decreased the activity of SOD in myocardium. When compared with these in I/R group, IS/AAR% [(23.28±4.38)% (19.96±4.38)%, (24.86±4.67)% vs. (43.76±6.30)%,respectively P﹤0.01], LDH [(1329.54±250.21)U/L, (1305.76±266.44)U/L,(1418.62±276.17)U/L,vs.(2016.97±371.95)U/L,(P﹤0.01)], MDA [(3.16±0.21) nmol/mg prot,(2.98±0.38) nmol/mg prot,(3.39±0.52) nmol/ mg prot vs. (5.33±0.51) nmol/mg prot,respectively P﹤0.01)] were decreased in TET1 group, TET2 group and EGCG group, while SOD value [(138.45±20.74) U/mg prot, (146.38±24.41) U/mg prot, (135.46±20.98) U/mg prot vs ( 98.69±15.41) U/mg prot] were higher in these groups than that in I/R group( P﹤0.01). The hematoxylin-eosine staining and electron microscopic examination showed that pathologic changes of cardiac tissue in the TET1, TET2 and EGCG group were significantly milder than that of the I/R group. The values of MDA,LDH and SOD in TET1, TET2 and EGCG groups had no significant difference (P>0.05)
Conclusion:
Ischemia and reperfusion could result in growths of MDA、LDH and IS/AAR% in serum and myocardium of rats and decreases of SOD. I/R injury could cause the pathologic changes of myocardium and increase the value of IS/AAR%, Pretreatment with tetrandrine could protect the myocardial form ischemia/reperfusion injury. The protective effects of tetrandrine were similar to that of EGCG.
PartⅡThe Effects of Preconditioning with Tetrandrine on cardiomyocytes Apoptosis and Apoptosis Related protein Bax, Bcl-2, Caspase-3 and Cyt C in Rats of Myocardial Ischemia/Reperfusion Model
Objective:
Our previous studies have shown that tetrandrine could protect myocardium from ischemia/reperfusion (I/R) injury; the aim of present study was to investigate effects of tetrandrine on myocardial apoptosis and the protein expression of Bax, Bcl-2 and caspase-3, releasing of cytochrome C in rat ischemia reperfusion injury model, and to discuss the role of pretreatment with tetrandrine on myocardial apoptosis and apoptosis related protein.
Methods:
Pentobarbital sodium-anesthetized SD rats underwent 30 min of left anterior descending (LAD) coronary occlusion followed by 24 hours of reperfusion. Following the partⅠof this study, 143 rats were divided into five groups randomly: Sham group(Sham, n=23), Ischemia/reperfusion injury group (I/R, n=30), Tetrandrine pretreatment 1 group (TET1, n=30), Tetrandrine pretreatment 2 group (TET2, n=30) and Epigallocatechin- 3-gallate group(EGCG, n=30). The apoptotic cardiomyocytes were detected by TUNEL staining and DNA Fragmentation agarose gel electrophoresis. The expression of Bcl-2 and Bax protein were measured by immuno-histochemistry, cytochrome C in the cytoplasm were detected by western blotting. The expression of caspase-3 mRNA was detected by RT-RCR; the activity of caspase-3 was measured in each group. The results were compared among the groups.
Results:
Compared with that in I/R group, the cardiomyocytes apoptosis was markedly inhibited in TET1 group, TET2 group and EGCG group. Apoptosis index (AI) were significantly decreased ([8.62±2.45)%,(7.95±2.28)%,(10.62±3.09)% vs. (19.36±5.28)%, respectively P﹤0.01];The expression of Bcl-2 protein was increased significantly in these groups compared with that in I/R group, while The expression of Bax protein was decreased significantly. Compared with the ratios of Bcl-2/Bax in I/R group, the ratios in TET1, TET2 and EGCG groups were significantly increased (P<0.01). The cytochrome C in the cytoplasm was significantly increased in I/R group, but the release of cytochrome C from the mitochondria was inhibited in TET1, TET2 and EGCG groups. The expression and activity of caspase-3 was increased prominently in I/R group, which was decreased significantly in TET1, TET2 and EGCG groups (P<0.01).
Conclusion:
The protective effect of preconditioning with tetrandrine was probably achieved through decreasing myocardium apoptosis in I/R injury, and modulating expression of Bcl-2 and Bax, increasing the ratios of Bcl-2/Bax, inhibiting the release of cytochrome C and decreasing the expression and activity of caspase-3.
PartⅢThe Effects and Mechanisms of Preconditioning with Tetrandrine on Cardiomyocytes Apoptosis induced by Anoxia/ Reoxygenation in Neonatal Rat Cardiomyocyte Model
Experiment I Primary cultures of cardiomyocytes and Establishment of Model of Anoxia/Reoxygenation
Objective:
To observe whether anoxia/reoxygenation (A/R) results in cardiomyocytes injury in cultured cardiomyotes through inducing anoxia for 2 hour and reoxygenaion for 24 hour and provide a platform for drugs study in this model.
Methods:
Primary cultures of cardiac myocytes were prepared from ventricles of 1～3 day new born Sprague Dawley (SD) rats and cultured for 3 to 4 days. The model of A/R injury was finished through receiving anoxia for 2 hours and reoxygenation for 24 hours in cultured cardiomyocytes of neonatal rat. The cardiomyocytes were divided randomly into 2 groups: control group (CON), anoxia/reoxygenation group (A/R). Prior to the anoxia, at the end of anoxia of 2 hours and reoxygenation of 24 hours, activities of lactate dehydrogenase (LDH), contents of malondialdehyde (MDA) and the superoxide dismutase (SOD) were assayed through spectrophotometric procedures respectively, to confirm the successful construction of anoxia/reoxygenation model..
Results:
In A/R group, activities of lactate dehydrogenase (LDH) and contents of malondial- dehyde (MDA) were higher, but the superoxide dismutase (SOD) were lower at the end of anoxia of 2 hours than that before anoxia (P<0.01); However reoxygenation resulted in a further increase of LDH and MDA and a further decrease of SOD; whereas there were not significantly different between two corresponding times (P>0.05) in CON group.
Conclusion:
Anoxia for 2 hours and reoxygenation for 24 hours could induce cardiomyocytes injury in cultured cardiomyocytes of neonatal rat.
Experiment II The Effects and Mechanisms of Preconditioning with Tetrandrine on Cardiomyocytes Apoptosis induced by Anoxia/ Reoxygenation Model in Neonatal Rat and its Effects on PKC Expression.
Objective:
To observe the effects of tetrandrine on cardiomyocytes apoptosis induced by Anoxia/ Reoxygenation and its effects on PKC, and explore the signal transduction pathway of tetrandrine inhibiting cardiomyocytes apoptosis through the construction of anoxia/ reoxygenation injury model.
Methods:
The model of anoxia/reoxygenation (A/R) injury was finished through anoxia for 2 hours and reoxygenation for 24 hours in cultured cardiomyocytes of neonatal rat, which were divided randomly to six group: Control group(CON); Anoxia/ Reoxyg- enation group(A/R); Anoxia Preconditioning group(AP), Tetrandrine pretreatment group(TET), Chelerythrine and Anoxia Preconditioning group(AP+CHE) and Chelerythrine and Tetrandrine pretreatment group (TET+CHE). LDH, SOD and MDA were assayed; the apoptotic cardiomyocytes were assessed through TUNEL and DNA Ladder in each group. The value of PKC mRNA and the activity of PKC were detected in all groups, and Western blot was used to analyze expression of cytochrome C of the cytoplasm in each group. The expression of caspase-3 mRNA was detected by RT-RCR; the activity of caspase-3 was measured in each group.
Results:
Compared with A/R group, activities of LDH、contents of MDA、apoptosis index (AI) were significantly decreased, and the activity of SOD was significantly increased in AP and TET groups. The value of PKC mRNA and the activity of PKC were significantly increased, the cytochrome C in the cytoplasm was significantly increased in A/R group, and the release of cytochrome C from the mitochondria was inhibited in AP and TET group. The expression and activity of caspase-3 was decreased significantly in AP and TET groups (P<0.01). After the groups were treated by chelerythrine, the protective effects of AP and TET were abolished or attenuated by chelerythrine. The expression and activity of PKC was prominently decreased, and the cytochrome C in the cytoplasm was significantly increased in AP+CHE and TET+CHE groups. The expression and activity of caspase-3 and the apoptotic index was prominently increased in AP+CHE and TET+CHE groups (P<0.01).
Conclusion:
These data strongly suggest that pretreatment of tetrandrine may inhibit cardiomyocytes apoptosis induced by anoxia/reoxygenation, but the PKC inhibitor can significantly attenuate the effects of tetrandrine, and so the PKC signaling pathway may be a key point in the anti-apoptosis of tetrandrine .
目的:
通过大鼠在体心肌缺血再灌注模型,观察粉防己碱预处理对缺血再灌注损伤心肌的保护作用。表没食子儿茶素没食子酸酯具有抗心肌缺血再灌注损伤和心肌细胞凋亡的作用,与表没食子儿茶素没食子酸酯进行比较,观察粉防己碱预处理对缺血再灌注损伤心肌的保护作用。
方法:
建立在体大鼠心脏I/R损伤模型,将96只大鼠随机分为正常对照组(Sham组, n=16)、I/R损伤组(I/R组, n=20)、粉防己碱1组(TET1组, n=20)、粉防己碱2组(TET2组,n=20)、表没食子儿茶素没食子酸酯组(EGCG组,n=20);各组均测定大鼠心肌I/R损伤后血清乳酸脱氢酶(LDH)、心肌组织丙二醛(MDA)、超氧化物歧化酶(SOD)含量,采用HE染色及电镜观察各组心肌的病理变化。各组均测定心肌梗死范围(IS/AAR%,TTC法),比较各组间差异。
结果:
与Sham组比较,I/R组中MDA值、LDH值、IS/AAR %明显升高,SOD显著降低。与I/R组比较,TET1组、TET2组、EGCG组可显著降低IS/AAR%值[(23.28±4.38)%, (19.96±4.38)%, (24.86±4.67)% vs. (43.76±6.30)% ,均P﹤0.01]; LDH [(1329.54±250.21)U/L, (1305.76±266.44)U/L,(1418.62±276.17)U/L,vs. (2016.97±371.95) U/L,均P﹤0.01)]、MDA [(3.16±0.21) nmol/mg prot,(2.98±0.38) nmol/mg prot,(3.39±0.52)nmol /mg prot vs. (5.33±0.51) nmol/mg prot,均P﹤0.01)]均明显降低,而SOD值明显增高[(138.45±20.74)U/mg prot, (146.38±24.41)U/mg prot, (135.46±20.98) U/mg prot vs. ( 98.69±15.41) U/mg prot,均P﹤0.01)]。TET1组、TET2组、EGCG组可以显著减轻心肌缺血再灌注损伤的病理改变。与EGCG组比较,TET1、TET2、EGCG三组间MDA、LDH和SOD值无显著性差异(P>0.05)。
结论:
大鼠心肌缺血再灌注损伤可导致LDH值、MDA值升高、SOD降低,引起心肌病理改变及IS/AAR %升高,与EGCG比较,粉防己碱预处理可以抗心肌缺血再灌注损伤,保护心肌,其保护效果与EGCG相似。
第二部分粉防己碱预处理对缺血再灌注心肌细胞凋亡及凋亡相关蛋白Bax、Bcl-2、Caspase-3和CytC的影响
目的:
观察粉防己碱对缺血再灌注损伤心肌细胞凋亡的影响及其对Bax、Bcl-2蛋白表达和线粒体中细胞色素C释放的影响,并观察粉防己碱对心肌细胞caspase-3变化的影响,初步探讨粉防己碱预处理对缺血再灌注损伤心肌细胞凋亡及凋亡相关蛋白的作用。
方法:
建立在体大鼠心脏I/R损伤模型,将143只大鼠随机分为正常对照组(Sham组,n=23)、I/R损伤组(I/R组,n=30)、粉防己碱1组(TET1组,n=30)、粉防己碱2组(TET2组,n=30)、表没食子儿茶素没食子酸酯组(EGCG组,n=30);实验结束后,用原位末端标记法(TUNEL)和DNA梯带法(DNA Ladder)检测细胞凋亡,用免疫组化法检测Bax、Bcl-2蛋白表达和Western-blotting法检测胞浆细胞色素C表达,用RT-RCR法检测心肌细胞caspase-3 mRNA表达,并观察caspase-3活性变化,比较各组间差异。
结果:
与I/R组相比,TET1组、TET2组、EGCG组明显抑制心肌细胞凋亡,AI明显降低[(8.62±2.45)%,(7.95±2.28)%,(10.62±3.09)% vs. (19.36±5.28)%,均P﹤0.01];与I/R组相比,TET1组、TET2组、EGCG组Bcl-2表达明显增加,Bax明显减少,且Bcl-2/ Bax值显著高于I/R组(P<0.01);I/R组心肌细胞胞浆细胞色素C显著增多(P<0.01),经TET1、TET2和EGCG预处理后,可明显抑制细胞色素C从线粒体向胞浆的释放(P<0.01);TET1、TET2和EGCG预处理后,可明显减少心肌细胞caspase-3的表达和活性(P<0.01)。
结论:
粉防己碱可通过抑制缺血再灌注心肌细胞凋亡来参与缺血再灌注心肌损伤的保护,粉防己碱抗心肌细胞凋亡可能与上调Bcl-2蛋白、下调Bax蛋白表达,升高Bcl-2/ Bax比值,抑制Cyt C从线粒体的释放,减少caspase-3在心肌组织中表达和活性有关。
第三部分粉防己碱预处理对缺氧/复氧诱导心肌细胞凋亡影响及其机制研究
第一节新生大鼠体外心肌细胞培养和缺氧/复氧模型建立
目的:
通过体外培养新生大鼠心肌细胞,构建新生大鼠心肌缺氧/复氧损伤模型,在细胞水平模拟大鼠心肌缺血再灌注(SI/R)损伤,为研究粉防己碱对新生大鼠缺氧/复氧损伤影响提供平台。
方法:
分离新生1-3天的乳鼠心室肌,在培养基中培养3-4天后,行缺氧2h/复氧24h,建立缺氧/复氧(anoxia/reoxygenation)心肌细胞损伤模型。在缺氧前、缺氧2h末期和复氧24h末期利用2,4二硝基苯肼显色法检测乳酸脱氢酶(LDH)活性,硫代巴比妥酸显色法检测丙二醛(MDA)及黄嘌呤氧化酶法(羟胺法)检测超氧化物歧化酶(SOD)含量并进行比较,以证实缺氧/复氧损伤模型构建成功。
结果:
新生1-3天的乳鼠心室肌,在培养基中培养3-4天后,在倒置显微镜下可观察到有搏动的心肌细胞。培养3-4天的心肌细胞在实施缺氧期2h后,LDH和MDA均较缺氧前有所升高,SOD降低(P<0.01);而给予复氧期24h后,LDH和MDA明显高于缺氧期,而SOD进一步降低(P<0.01),对照组的上述指标在相应时间点未见明显改变(P>0.05)。
结论:
通过给新生大鼠体外培养心肌细胞行缺氧2h/复氧24h后,可明显造成心肌细胞损伤,以此模拟大鼠心肌缺血再灌注损伤。
第二节粉防己碱对缺氧/复氧诱导新生大鼠心肌细胞凋亡、PKC表达的影响及机制研究
目的:
通过建立的乳鼠心肌细胞缺氧/复氧模型,观察粉防己碱对缺氧/复氧诱导的心肌细胞凋亡及PKC的影响,探讨粉防己碱抑制心肌细胞凋亡的信号转导机制。
方法:
通过给原代培养乳鼠心肌细胞行缺氧2h/复氧24h ,建立缺氧/复氧(anoxia/reoxygenation)心肌细胞损伤模型。将培养的心肌细胞分为正常对照(CON)组、缺氧/复氧(A/R)组、缺氧预处理(AP)组、粉防己碱预处理(TET)组、缺氧预处理+PKC抑制剂chelerythrin(eAP+CHE)组、粉防己碱+PKC抑制剂chelerythrine(TET+CHE)组,共6组。于复氧24h后,检测LDH活性和心肌细胞MDA、SOD含量,采用原位末端标记法(TUNEL)和DNA梯带法(DNA Ladder)标测心肌细胞凋亡,检测心肌细胞PKC mRNA的表达量及PKC活性,用免疫印迹法(Western blotting)检测胞浆CytC表达,并检测心肌细胞caspase-3 mRNA表达量及活性,比较各组间差异。
结果:
与A/R组相比,AP组、TET组中LDH活性、MDA含量、凋亡指数(AI)显著降低,SOD显著升高(P<0.01);PKC的mRNA的表达量和活性显著增加,心肌细胞胞浆中CytC减少,心肌细胞caspase-3 mRNA表达量减少、caspase-3活性降低有显著性差异(P<0.01);经chelerythrine处理后,再给予AP或TET预处理,则PKC的mRNA的表达量和PKC活性显著下降,可见胞浆中CytC增加,心肌细胞caspase-3 mRNA表达量增加,caspase-3活性增强,凋亡指数(AI)增加(P<0.01)。
结论:
粉防己碱预处理可减少缺氧/复氧诱导的心肌细胞凋亡,PKC抑制剂chelerythrine处理后,可显著降低粉防己碱的保护作用,因此PKC的信号传导途径可能是粉防己碱抑制缺血再灌注损伤心肌细胞凋亡的关键环节。
PartⅠThe Study of Protective Effect Against Ischemia/Reperfusion Injury Preconditioning with Tetrandrine in Rats and Comparison with Epigallocatechin-3-gallate
Objective:
To observe the protective effects of tetrandrine against myocardial ischemia and reperfusion injury in rats. Previous studies had shown that Epigallocatechin-3-gallate (EGCG) could protect myocardium from ischemia/reperfusion injury and inhibit cardiomyocyte apoptosis. Compared with EGCG, the present study was to investigate whether tetrandrine had protective effects on myocardium in ischemia and reperfusion model.
Methods:
Pentobarbital sodium-anesthetized Sprague-Dawley rats underwent 30 min of left anterior descending (LAD) coronary occlusion and 24 hours reperfusion to make ischemia/reperfusion (I/R) injury model in vivo. Ninety-six rats were divided into five groups randomly: Sham group, Ischemia/reperfusion injury (I/R) group, Tetrandrine pretreatment1 (TET1) group, Tetrandrine pretreatment 2 (TET2) group and Epigallo- catechin-3-gallate (EGCG) group. Sham group underwent only the sham operation; other four groups underwent I/R operation. All groups measured infarcted size (IS/ AAR %). Lactatedehydrogenase (LDH) in serum, the superoxidedismutase (SOD) and malondial- dehyde (MDA) in myocardium tissue were measured in each group. In addition,pathologic changes of myocardial tissue were observed under hematoxylin-eosine staining(HE) and electron microscopy. The results were compared among the groups.
Results:
Compared with Sham group, I/R group markedly increased the activity of LDH in serum, the content of MDA in myocardium and IS/AAR %, but decreased the activity of SOD in myocardium. When compared with these in I/R group, IS/AAR% [(23.28±4.38)% (19.96±4.38)%, (24.86±4.67)% vs. (43.76±6.30)%,respectively P﹤0.01], LDH [(1329.54±250.21)U/L, (1305.76±266.44)U/L,(1418.62±276.17)U/L,vs.(2016.97±371.95)U/L,(P﹤0.01)], MDA [(3.16±0.21) nmol/mg prot,(2.98±0.38) nmol/mg prot,(3.39±0.52) nmol/ mg prot vs. (5.33±0.51) nmol/mg prot,respectively P﹤0.01)] were decreased in TET1 group, TET2 group and EGCG group, while SOD value [(138.45±20.74) U/mg prot, (146.38±24.41) U/mg prot, (135.46±20.98) U/mg prot vs ( 98.69±15.41) U/mg prot] were higher in these groups than that in I/R group( P﹤0.01). The hematoxylin-eosine staining and electron microscopic examination showed that pathologic changes of cardiac tissue in the TET1, TET2 and EGCG group were significantly milder than that of the I/R group. The values of MDA,LDH and SOD in TET1, TET2 and EGCG groups had no significant difference (P>0.05)
Conclusion:
Ischemia and reperfusion could result in growths of MDA、LDH and IS/AAR% in serum and myocardium of rats and decreases of SOD. I/R injury could cause the pathologic changes of myocardium and increase the value of IS/AAR%, Pretreatment with tetrandrine could protect the myocardial form ischemia/reperfusion injury. The protective effects of tetrandrine were similar to that of EGCG.
PartⅡThe Effects of Preconditioning with Tetrandrine on cardiomyocytes Apoptosis and Apoptosis Related protein Bax, Bcl-2, Caspase-3 and Cyt C in Rats of Myocardial Ischemia/Reperfusion Model
Objective:
Our previous studies have shown that tetrandrine could protect myocardium from ischemia/reperfusion (I/R) injury; the aim of present study was to investigate effects of tetrandrine on myocardial apoptosis and the protein expression of Bax, Bcl-2 and caspase-3, releasing of cytochrome C in rat ischemia reperfusion injury model, and to discuss the role of pretreatment with tetrandrine on myocardial apoptosis and apoptosis related protein.
Methods:
Pentobarbital sodium-anesthetized SD rats underwent 30 min of left anterior descending (LAD) coronary occlusion followed by 24 hours of reperfusion. Following the partⅠof this study, 143 rats were divided into five groups randomly: Sham group(Sham, n=23), Ischemia/reperfusion injury group (I/R, n=30), Tetrandrine pretreatment 1 group (TET1, n=30), Tetrandrine pretreatment 2 group (TET2, n=30) and Epigallocatechin- 3-gallate group(EGCG, n=30). The apoptotic cardiomyocytes were detected by TUNEL staining and DNA Fragmentation agarose gel electrophoresis. The expression of Bcl-2 and Bax protein were measured by immuno-histochemistry, cytochrome C in the cytoplasm were detected by western blotting. The expression of caspase-3 mRNA was detected by RT-RCR; the activity of caspase-3 was measured in each group. The results were compared among the groups.
Results:
Compared with that in I/R group, the cardiomyocytes apoptosis was markedly inhibited in TET1 group, TET2 group and EGCG group. Apoptosis index (AI) were significantly decreased ([8.62±2.45)%,(7.95±2.28)%,(10.62±3.09)% vs. (19.36±5.28)%, respectively P﹤0.01];The expression of Bcl-2 protein was increased significantly in these groups compared with that in I/R group, while The expression of Bax protein was decreased significantly. Compared with the ratios of Bcl-2/Bax in I/R group, the ratios in TET1, TET2 and EGCG groups were significantly increased (P<0.01). The cytochrome C in the cytoplasm was significantly increased in I/R group, but the release of cytochrome C from the mitochondria was inhibited in TET1, TET2 and EGCG groups. The expression and activity of caspase-3 was increased prominently in I/R group, which was decreased significantly in TET1, TET2 and EGCG groups (P<0.01).
Conclusion:
The protective effect of preconditioning with tetrandrine was probably achieved through decreasing myocardium apoptosis in I/R injury, and modulating expression of Bcl-2 and Bax, increasing the ratios of Bcl-2/Bax, inhibiting the release of cytochrome C and decreasing the expression and activity of caspase-3.
PartⅢThe Effects and Mechanisms of Preconditioning with Tetrandrine on Cardiomyocytes Apoptosis induced by Anoxia/ Reoxygenation in Neonatal Rat Cardiomyocyte Model
Experiment I Primary cultures of cardiomyocytes and Establishment of Model of Anoxia/Reoxygenation
Objective:
To observe whether anoxia/reoxygenation (A/R) results in cardiomyocytes injury in cultured cardiomyotes through inducing anoxia for 2 hour and reoxygenaion for 24 hour and provide a platform for drugs study in this model.
Methods:
Primary cultures of cardiac myocytes were prepared from ventricles of 1～3 day new born Sprague Dawley (SD) rats and cultured for 3 to 4 days. The model of A/R injury was finished through receiving anoxia for 2 hours and reoxygenation for 24 hours in cultured cardiomyocytes of neonatal rat. The cardiomyocytes were divided randomly into 2 groups: control group (CON), anoxia/reoxygenation group (A/R). Prior to the anoxia, at the end of anoxia of 2 hours and reoxygenation of 24 hours, activities of lactate dehydrogenase (LDH), contents of malondialdehyde (MDA) and the superoxide dismutase (SOD) were assayed through spectrophotometric procedures respectively, to confirm the successful construction of anoxia/reoxygenation model..
Results:
In A/R group, activities of lactate dehydrogenase (LDH) and contents of malondial- dehyde (MDA) were higher, but the superoxide dismutase (SOD) were lower at the end of anoxia of 2 hours than that before anoxia (P<0.01); However reoxygenation resulted in a further increase of LDH and MDA and a further decrease of SOD; whereas there were not significantly different between two corresponding times (P>0.05) in CON group.
Conclusion:
Anoxia for 2 hours and reoxygenation for 24 hours could induce cardiomyocytes injury in cultured cardiomyocytes of neonatal rat.
Experiment II The Effects and Mechanisms of Preconditioning with Tetrandrine on Cardiomyocytes Apoptosis induced by Anoxia/ Reoxygenation Model in Neonatal Rat and its Effects on PKC Expression.
Objective:
To observe the effects of tetrandrine on cardiomyocytes apoptosis induced by Anoxia/ Reoxygenation and its effects on PKC, and explore the signal transduction pathway of tetrandrine inhibiting cardiomyocytes apoptosis through the construction of anoxia/ reoxygenation injury model.
Methods:
The model of anoxia/reoxygenation (A/R) injury was finished through anoxia for 2 hours and reoxygenation for 24 hours in cultured cardiomyocytes of neonatal rat, which were divided randomly to six group: Control group(CON); Anoxia/ Reoxyg- enation group(A/R); Anoxia Preconditioning group(AP), Tetrandrine pretreatment group(TET), Chelerythrine and Anoxia Preconditioning group(AP+CHE) and Chelerythrine and Tetrandrine pretreatment group (TET+CHE). LDH, SOD and MDA were assayed; the apoptotic cardiomyocytes were assessed through TUNEL and DNA Ladder in each group. The value of PKC mRNA and the activity of PKC were detected in all groups, and Western blot was used to analyze expression of cytochrome C of the cytoplasm in each group. The expression of caspase-3 mRNA was detected by RT-RCR; the activity of caspase-3 was measured in each group.
Results:
Compared with A/R group, activities of LDH、contents of MDA、apoptosis index (AI) were significantly decreased, and the activity of SOD was significantly increased in AP and TET groups. The value of PKC mRNA and the activity of PKC were significantly increased, the cytochrome C in the cytoplasm was significantly increased in A/R group, and the release of cytochrome C from the mitochondria was inhibited in AP and TET group. The expression and activity of caspase-3 was decreased significantly in AP and TET groups (P<0.01). After the groups were treated by chelerythrine, the protective effects of AP and TET were abolished or attenuated by chelerythrine. The expression and activity of PKC was prominently decreased, and the cytochrome C in the cytoplasm was significantly increased in AP+CHE and TET+CHE groups. The expression and activity of caspase-3 and the apoptotic index was prominently increased in AP+CHE and TET+CHE groups (P<0.01).
Conclusion:
These data strongly suggest that pretreatment of tetrandrine may inhibit cardiomyocytes apoptosis induced by anoxia/reoxygenation, but the PKC inhibitor can significantly attenuate the effects of tetrandrine, and so the PKC signaling pathway may be a key point in the anti-apoptosis of tetrandrine .
引文
1. Levitsky S. Protecting the myocardial cell during coronary revascularization. The William W.L. Glenn Lecture. Circulation, 2006, 114(1 Suppl): I339-343.
2. Ota S, Nishikawa H, Takeuchi M, et al. Impact of nicorandil to prevent reperfusion injury in patients with acute myocardial infarction: Sigmart Multicenter Angioplasty Revascularization Trial (SMART). Circ J. 2006; 70(9):1099-104.
3. Wolff AA, Rotmensch HH, Stanley WC, et al. Metabolic approaches to treatment of ischemic heart disease: the clinicans’perspective. Heart Fail Rev, 2002, 7(2):187-203.
4. Ambrosio G, Tritto I. Reperfusion injury: experimental evidence and clinical implica- tions. Am Heart J, 1999, 138(2 Pt 2):S69-75.
5. Eefting F, Rensing B, Wigman J, et al. Role of apoptosis in reperfusion injury. Cardiovas Res. 2004 61(3): 414– 426
6. Zhao ZQ, Morris CD, Budde JM, et al. Inhibition of myocardial apoptosis reduces infarct size and improves regional contractile dysfunction during reperfusion. Cardiovas Res, 2003, 59(1): 132-142.
7. Ji ES, Yue H, Wu YM, et al. Effects of phytoestrogen genistein on myocardial ischemia / reperfusion injury and apoptosis in rabbits. Acta Pharmacol Sin. 2004; 25 (3): 306 -312
8. Hausenloy DJ, Yellon DM. New directions for protecting the heart against ischaemia– reperfusion injury: targeting the Reperfusion Injury Salvage Kinase (RISK)-pathway. Cardiovas Res. 2004, 61(3): 448– 460.
9. Loubani M, Hassouna A, Galinanes M. Delayed preconditioning of the human myocardium: signal transduction and clinical implications. Cardiovasc Res, 2004, 61 (3):600-609.
10.敖行述,何华美,张乐之.粉防己碱对心血管的药理作用研究进展。中国药业,2000;9(7);63-65.
11.黄波,冯义柏,朱福等.粉防己碱对兔实验性动脉粥样硬化形成的影响。中国临床药学杂志, 2005;14(5):294-297.
12. Yao WX, Jiang MX. Effects of tetrandrine on cardiovascular electrophysiologic properties. Acta Pharmacol Sin, 2002; 23 (12): 1069 -1074.
13.段世英,王裕勤,孙圣斌.粉防己碱对缺血心脏去甲肾上腺素释放及代谢的影响。华中医学杂志,2005; 9(3): 201-202.
14. Yu XC, Wu S, Wang GY et al. Cardiac effects of the extract and active components of radix stephaniae tetrandrae. II. Myocardial infarct, arrhythmias, coronary arterial flow and heart rate in the isolated perfused rat heart. Life Sci,2001; 68(25):2863 -72.
15.李俊峡,李佃贵,李振彬等.粉防己碱对缺血再灌注心肌顿抑的影响。第四军医大学学报,2000;21(5):92.
16.祝武强,曾秋棠,曹林生等.粉防己碱对缺氧和复氧损伤心室肌细胞内Ca2+超载的作用。临床心血管病杂志,2000,16(2):83-84.
17.夏国瑾,姚伟星,刘燕等.粉防己碱对离体大鼠心脏缺血-再灌注损伤心肌的保护作用。同济医科大学学报,1994,23(6):446-449.
18.钱玲梅,王笑云,冷静.粉防己碱干预大鼠急性缺血再灌注肾损伤中的细胞凋亡变化。中华肾脏病杂志,1998,14(4):252-253.
1. Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation, 1986; 74(5):1124-1136.
2.王裕勤,段世英,孙圣斌等.粉防己碱对电刺激时去甲肾上腺素释放及代谢的影响.临床心血管病杂志,2006,22(2):100-102.
3.张新平,庞月华,冯义柏等.粉防己碱对内膜损伤后平滑肌细胞表型转化和MKP-1表达的影响.心脏杂志,2006,18(2): 131-134.
4. Rao MR. Effects of tetrandrine on cardiac and vascular remodeling. Acta Pharmacol Sin. 2002;23(12):1075-1085.
5.李庆平,杨解人,饶曼人.预先给予粉防己碱对大鼠工作心脏缺血再灌注损伤的保护作用[J].中国药理学与毒理学杂志,1994,8(2):114~117.
6. Townsend PA, Scarabelli TM, Pasini E, et al . Epigallocatechin-3-gallate inhibits STAT-1 activation and protects cardiac myocytes from ischemia/ reperfusion-induced apoptosis. FASEB J. 2004; 18(13):1621-1623.
7. Shen YC, Chen CF, Sung YJ. Tetrandrine ameliorates ischaemia-reperfusion injury of rat myocardium through inhibition of neutrophil priming and activation. Br J Pharmacol. 1999;128(7):1593-601.
8.胡宗礼,黄晓萍,袁学文.表没食子儿茶素没食子酸醋对缺血再灌注心肌细胞凋亡的作用。中国病理生理杂志,2005,21(10):1923-1926.
9. Ferrari R, Ceconi C, Curello S, et al. Role of oxygen free radicals in ischemic and reperfused myocardium. Am J Clin Nutr, 1991;53(1 Suppl): 215-222S.
10. Ferrandi C, Ballerio R, Gaillard P, et al. Inhibition of c-Jun -terminal kinase decreases cardiomyocyte apoptosis and infarct size after myocardial ischemia and reperfusion in anaesthetized rats. Br J Pharmacol. 2004;142(6), 953-60.
11. Shi CZ, Feng YB, Gu X, et al. Effect of protective myocardium by allitridum from decreasing apoptosis in rats with ischemia/reperfusion injury. Journal of Nanjing Medical University, 2005, 19(2):86-90.
12. Park JL, Lucchesi BR. Mechanisms of myocardial reperfusion injury. Ann Thorac Surg 1999; 68(5): 1905-1912.
13. Hoffman JW Jr, Gilbert TB, Poston RS, et al. Myocardial reperfusion injury: etiology, mechanisms, and therapies. J Extra Corpor Technol. 2004; 36(4):391-411.
14. Ambrosio G, Tritto I. Reperfusion injury: experimental evidence and clinical implications. Am Heart J 1999; 138(2pt2): S69–75.
15. Vanden Hock TL, Li C, Shao Z, et al. significant levels of oxidants are generated by isolated cardiomyocytes during ischemia prior to reperfusion. J Mol Cell Cardiol. 1997; 29(9): 2571-2583
16. Chakraborti T, Ghosh SK, Michael JR, et al. Targets of oxidative stress in cardiovascular system. Mol Cell Biochem, 1998; 187(1-2): 1-10.
17.徐新春.钙超载与心肌缺血再灌注损伤。心血管病学进展, 2000; 21(4):227-230.
18. Baxter GF. Ischemic preconditioning of myocardium. Ann Med 1997; 29: 345–352.
19. Loubani M, Hassouna A, Galinanes M. Delayed preconditioning of the human myo- cardium: signal transduction and clinical implications. Cardiovasc Res, 2004, 61 (3):600-609.
20.李大强,冯义柏,张家明等.粉防己碱防止家兔快速心房起搏间隙连接蛋白40降解.第四军医大学学报,2004,25(2):150-152.
21.张新平,庞月华,冯义柏等.粉防己碱对内膜损伤后血管平滑肌细胞表型转化和p38表达的影响.中国动脉硬化杂志,2006;l4(4):304-308.
22.钟宁,钱家庆. M受体机制在粉防己碱保护心脏缺血再灌注损伤作用中的影响.中国药理学通报,2000,16(3):282-284.
23. Aneja R, Hake PW, Burroughs TJ, et al. Epigallocatechin, a green tea polyphenol, attenuates myocardial ischemia reperfusion injury in rats. Mol Med. 2004; 10(1-6):55-62.
24. Townsend PA, Scarabelli TM, Pasini E, et al . Epigallocatechin-3-gallate inhibits STAT-1 activation and protects cardiac myocytes from ischemia/ reperfusion-induced apoptosis. FASEB J. 2004; 18(13):1621-1623.
25. Sheng R, Gu ZL, Xie ML, et al. EGCG inhibits cardiomyocyte apoptosis in pressure overload-induced cardiac hypertrophy and protects cardiomyocytes from oxidative stress in rats. Acta Pharmacol Sin. 2007; 28(2):191-201.
1. Zhao ZQ, Morris CD, Budde JM, et al. Inhibition of myocardial apoptosis reduces infarct size and improves regional contractile dysfunction during reperfusion. Cardiovasc Res. 2003; 59 (1):132–142.
2. Ji ES, Yue H, Wu YM, et al. Effects of phytoestrogen genistein on myocardial ischemia/reperfusion injury and apoptosis in rabbits. Acta Pharmacol Sin. 2004; 25 (3): 306 -312.
3. Chang TH, Liu XY, Zhang XH, et.al. Effects of dl-praeruptorin A on interleukin-6 level and Fas, bax, bcl-2 protein expression in ischemia-reperfusion myocardium. Acta Pharmacol Sin. 2002; 23(9):769-774.
4. Regula KM, Ens K, Kirshenbaum LA. Mitochondria-assisted cell suicide: a license to kill. J Mol Cell Cardiol. 2003;35(6):559-567.
5. Shen YC, Chen CF, Sung YJ. Tetrandrine ameliorates ischaemia-reperfusion injury of rat myocardium through inhibition of neutrophil priming and activation. Br J Pharmacol. 1999;128(7):1593-601.
6. Xu M, Wang Y, Hirai K, et a1. Calcium preconditioning inhibits mitochondrial permeability transition and apoptosis[J].Am J Physiol Heart Circ Physiol,200l,280(2):H899-908.
7. Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer, 1972; 26(4): 239-257.
8. Majno G, Joris I. Apoptosis, oncosis,and necrosis: an overview of cell death. Am J Pathol. 1995; 146(1):3-15.
9. Petit PX, Lecoeur H, Zorn E, et al. Alteration in mitochondrial structure and function are early events of dexamethasone-induced thymocyte apoptosis. J Cell Biol. 1995; 130 (1):157-167
10. Gottlieb RA, Burleson KO, Kloner RA, et al. Reperfusion injury induces apoptosis in rabbit cardiomyocytes. J Clin Invest, 1994; 94(4): 1621-628.
11. Takashi E, Ashraf M. Pathologic assessment of myocardial cell necrosis and apoptosis after ischemia and reperfusion with molecular and morphological markers.. J Mol CellCardiol, 2000, 32(2):209-224.
12. Fliss H, Gattinger D. Apoptosis in ischemic and reperfused rat myocardium .Circ Res, 1996, 79(5):949-956.
13. Maulik N, Yoshida T, Engelman RM. Ischemia precondition attenuates apoptotic cell death associated with ischemia/reperfusion. Mol Cell Biochem,1998, 186(1-2): 139-145.
14. Mocanu MM, Baxter GF and Yellon DM. Caspase inhibition and limitation of myocardial infarct size: protection against lethal reperfusion injury. Br J Pharmacol, 2000; 130(2):197-200.
15. Freude B, Masters TN, Kostin S, et al. Cardiomyocyte apoptosis in acute and chronic conditions. Basic Res Cardiol. 1998; 93(2):85-9.
16. Buja LM. Myocardial ischemia and reperfusion injury. Cardiovasc Pathol. 2005; 14 (4):170-175.
16. 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.
17. Haunstetter A, Izumo S. Toward antiapoptosis as a new treatment modality. Circ Res, 2000; 86(4): 371–376.
18. Buja LM, Entman ML. Modes of myocardial cell injury and cell death in ischemic heart disease.Circulation,1998,98(14):1355-7.
19.Hausenloy DJ, Yellon DM. New directions for protecting the heart against ischaemia- reperfusion injury: targeting the Reperfusion Injury Salvage Kinase (RISK)-pathway. Cardiovasc Res. 2004; 61(3):448-60.
20. Freude A, Master TN, Robicsek F, et al. Apoptosis is initiated by myocardial ischemia and executed during reperfusion. J Am Coll Cardiol, 2000; 32(2): 197–208.
21. Zhao ZQ, Nakamura M, Wang NP, et al. Reperfusion induces myocardial apoptotic cell death. Cardiovasc Res, 2000; 45(3): 651–660.
22. Peart JN, Gross GJ. Adenosine and opioid receptor-mediated cardioprotection in the rat: evidence for cross-talk between receptors. Am J Physiol Heart Circ Physiol, 2003, 285(1): H81-89.
23. Xu M, Wang Y, Hirai K, et al. Calcium preconditioning inhibits mitochondrial permeability transition and apoptosis. Am J Physiol Heart Circ Physiol, 2001, 280(2): H899-908.
24. Babu PP, Suzuki G, Ono Y, et al. Attenuation of ischemia and/or reperfusion injury during myocardial infarction using mild hypothermia in rats: an immunohistochemical study of Bcl-2, Bax, Bak and TUNEL. Pathol Int, 2004, 54(12):896-903.
25. Lam M, Dubyak G, Chen L, et al .Evidence that BCL-2 represses apoptosis by regulating endoplasmic reticulum-associated Ca2+ fluxes. Proc NatI Acad Sci USA, 1994, 91(14):6569-6573.
26. Ryan JJ, Prochownik E, Gottliep CA, et al. C-myc and bcl-2 modulate p53 function by altering p53 subcellular trafficking during the cell cycle. Proc Natl Acad Sci USA. 1994. 91(13): 5878-5882.
27. Borutaite V, Budriunaite A, Morkuniene R, et al. Release of mitochondrial cytochrome C and activation of cytosolic caspases induced by myocardial ischemia. J Biochimica Biophysica Acta, 2001,1537(11):101-109.
28. Xie Z. Koyama T, Suzuki J, et al. Coronary reperfusion following ischemia: different expression of Bcl-2 and Bax proteins, and cardiomyocyte apoptosis. Jpn heart J, 2001, 42(6):759-770.
29. McClintock DS, Santore MT, Lee VY,et al. Bcl-2 family members and functional electron transport chain regulate oxygen deprivation-induced cell death. Mol Cell Biol, 2002, 22(1):94-104
30. Golstein P. Controlling cell death. Science, 1997; 275(5303): 1081-1082.
31. Nagata S. Apoptosis by death factor. Cell, 1997; 88(3): 355-365.
32. Brenner C, Cadiou H, Vieria HL, et al. Bcl-2 and Bax regulate the channel activity of the mitochondrial adenine nucleotide translocator. Oncogene, 2000, 19(3): 329-336.
33. Enari M, Sakahira H, Yokoyama H, et al. A caspase-actived DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature, 1998, 391(6662): 43-50.
34. Cohen GM. Caspase: the executioners of apoptosis. Biochem J. 1997;326(Pt1 ): 1-16.
35. Zhu XF, Liu ZC, Xie BF, et al. Involvement of caspase-3 activation in squamocin- induced apoptosis in leukemia cell line HL-60. Life Sci, 2002, 70(11): 1259-1269.
36. Townsend PA, Scarabelli TM, Pasini E, et al . Epigallocatechin-3-gallate inhibits STAT-1 activation and protects cardiac myocytes from ischemia/reperfusion-induced apoptosis. FASEB J. 2004; 18(13):1621-1623.
1. Harary I, Farley B. In vitro studies of single isolated beating heart cells. Science. 1960,131: 1674-1675.
2. Rui T, Kvietys PR. NFkappaB and AP-1 differentially contribute to the induction of Mn-SOD and Enos during the development of oxidant tolerance. FASEB J. 2005;19(13):1908-1910.
3. Gu X, Feng YB, Shi CZ, et al. Antiapoptotic mechanism of insulin in reoxygen- ation-induced injury in cultured cardiomyocytes of neonatal rats. J Huazhong Univ Sci Technolog Med Sci. 2005; 25(6):632-635.
4. Yue TL, Wang C, Gu JL, et al. Inhibition of extracellular signal–regulated kinase enhances Ischemia/Reoxygenation–induced apoptosis in cultured cardiac myocytes and exaggerates reperfusion injury in isolated perfused heart. Circ Res. 2000; 86 (6):692-699.
5. Rakhit RD, Mojet MH, Marber MS, et al. Mitochondria as Targets for Nitric Oxide–Induced Protection During Simulated Ischemia and Reoxygenation in Isolated Neonatal Cardiomyocytes. Circulation. 2001;103(21):2617-2623.
6. Tang Y, Wang Q, Li J. Influence of KATP channel inhibitor on the changes of HSP70 expression in sevoflurane-induced neonatal rat cardiomyocytes. Sichuan Da Xue Xue Bao Yi Xue Ban. 2003; 34(4):653-655.
7. Siwik DA, Tzortzis JD, Pimental DR, et al. Inhibition of copper-zinc superoxide dismutase induces cell growth, hypertrophic phenotype, and apoptosis in neonatal rat cardiac myocytes in vitro. Circ Res. 1999 23; 85(2):147-53.
8. Rohr S, Scholly DM, Kleber AG.. Patterned growth of neonatal rat heart cells in culture. Morphological and electrophysiological characterization. Circ Res. 1991; 68 (1): 114-30.
9. Simpson P, McGrath A, Savion S. Myocyte hypertrophy in neonatal rat heart cultures and its regulation by serum and by catecholamines. Circ Res. 1982, 51 (6):787-801.
10. Simpson P, Savion S. Differentiation of rat myocytes in single cell cultures with and without proliferating nonmyocardial cells. Cross-striations, ultrastructure, andchronotropic response to isoproterenol. Circ Res. 1982; 50(1):101-16.
11. Ojamaa K, Samarel AM, Klein I. Identification of a contractile-responsive element in the cardiac alpha-myosin heavy chain gene. J Biol Chem. 1995, 270 (52): 31276 -31281.
12. de Micheli A, Chavez E. Ischemia-reperfusion myocardial injury. Arch Cardiol Mex. 2003; 73(4):284-90.
13. Zhao ZQ. Oxidative stress-elicited myocardial apoptosis during reperfusion. Curr Opin Pharmacol. 2004; 4(2):159-65.
1. Bandyopadhyay D,Chattopadhyay A,Ghosh G,et al. Oxidative stress- induced ischemic heart disease: protection by antioxidants. Curr Med Chem,2004,11 (3):369-387.
2. Carden DL, Granger DN. Pathophysiology of ischaemia-reperfusion injury. J Pathol, 2000,190(3): 255-266.
3. Townsend PA, Scarabelli TM, Pasini E, et al . Epigallocatechin-3-gallate inhibits STAT-1 activation and protects cardiac myocytes from ischemia/ reperfusion -induced apoptosis. FASEB J. 2004; 18(13):1621-1623.
4. Ma XQ, Fu RF, Feng GQ,et al. Hypoxia-reoxygenation-induced apoptosis in cultured neonatal rat cardiomyocyets and the protective effect of prostaglandin E. Clin Exp Pharmacol Physiol. 2005; 32(12):1124-30.
5. Maekawa N, Abe J, Shishido T, et al. Inhibiting p90 ribosomal S6 kinase prevents (Na+)-H+ exchanger-mediated cardiac ischemia-reperfusion injury. Circulation. 2006; 113 (21):2516-23.
6. Murry CE,Jennings RB,Reimer KA. Preconditioning with ischemia:A delay of lethal cell injury in ischemic myocardium. Circulation,1986,74 (5):1124-1136.
7. Gaudette GR, Krukenkamp IB, Saltman AE, et al. Preconditioning with PKC and the ATP-sensitive potassium channels: a codependent relationship. Ann Thorac Surg, 2000, 70(2):602-608.
8. Nojiri M, Tanonaka K,Yabe K,et al. Involvement of adenosine receptor, potassium channel and protein kinase C in hypoxic preconditioning of isolated cardiomyocytes of adult rat. Jpn J Pharmacol, 1999, 80(1):15-23.
9. Wang GY, Zhou JJ, Shan J, et al. Protein kinase C-epsilon is a trigger of delayed cardioprotection against myocardial ischemia of kappa-opioid receptor stimulation in rat ventricular myocytes. J Pharmacol Exp Ther, 2001, 299(2):603-610.
10. Saurin AT, Pennington D J, Raat NJ, et al. Targeted disruption of the protein kinase C epsilon gene abolishes the infarct size reduction that follows ischemic precondition- ing of isolated buffer-perfused mouse hearts. Cardiovasc Res, 2002,55(3):672-680.
11. Adrain C, Martin SJ. The mitochondrial apoptosome: a killer unleashed by the cytochrome seas. Trends Biochem Sci. 2001; 26(6):390-7.
12. Suleiman MS, Halestrap AP, Griffiths EJ. Mitochondria: a target for myocardial protection. Pharmacol Ther. 2001; 89(1):29-46.
13. Laclau MN, Boudina S, Thambo JB, et al. Cardioprotection by ischemic precon- ditioning preserves mitochondrial function and functional coupling between adenine nucleotide translocase and creatine kinase. J Mol Cell Cardiol. 2001; 33 (5):947-56.
14. Javadov SA, Clarke S, Das M, et al. Ischaemic preconditioning inhibits opening of mitochondrial permeability transition pores in the reperfused rat heart. J Physiol. 2003; 549(Pt 2):513-24.
15. Borutaite V, Jekabsone A, Morkuniene R, et al. Inhibition of mitochondrial permeability transition prevents mitochondrial dysfunction, cytochrome c release and apoptosis induced by heart ischemia.J Mol Cell Cardiol. 2003; 35(4):357-66.
16. Ott M, Robertson JD, Gogvadze V, et al. Cytochrome c release from mitochondria proceeds by a two-step process. Proc Natl Acad Sci U S A. 2002; 99(3):1259-63.
17. Regula KM, Ens K, Kirshenbaum LA. Mitochondria-assisted cell suicide: a license to kill. J Mol Cell Cardiol. 2003; 35(6):559-67.
18. Maulik N, Engelman RM, Rousou JA, et al. Ischemic preconditioning reduces apoptosis by upregulating anti-death gene Bcl-2. Circulation.1999, 100(19Suppl): II369-375.
19. Nakamura M, Wang NP, Zhao ZQ, et al. Preconditioning decreases Bax expression, PMN accumulation and apoptosis in reperfused rat heart. Cardiovasc Res. 2000; 45 (3):661-70.
20. Baghelai K, Graham LJ, Wechsler AS, et al. Delayed myocardial preconditioning by alpha1-adrenoceptors involves inhibition of apoptosis. J Thorac Cardiovasc Surg. 1999; 117(5):980-986.
21. Rajesh KG, Sasaguri S, Zhitian Z, et al. Second window of ischemic preconditioning regulates mitochondrial permeability transition pore by enhancingBcl-2 expression. Cardiovasc Res. 2003; 59(2):297-307.
22. Liu H, McPherson BC, Yao Z. Preconditioning attenuates apoptosis and necrosis: role of protein kinase C epsilon and -delta isoforms. Am J Physiol Heart Circ Physiol. 2001,281(1):H404-410.
23. Argaud L, Gateau-Roesch O, Chalabreysse L, et al. Preconditioning delays Ca2+- induced mitochondrial permeability transition. Cardiovasc Res. 2004; 61 (1):115- 122 .
24. Kim GT, Chun YS, Park JW, et al. Role of apoptosis-inducing factor in myocardial cell death by ischemia-reperfusion. Biochem Biophys Res Commun, 2003, 309(3): 619-624.
25. Uchiyama T, Engelman RM, Maulik N, et al. Role of Akt signaling in mitochondrial survival pathway triggered by hypoxic preconditioning. Circulation. 2004; 109 (24): 3042-3049.
1. Lepran I, Pollesello P, Vajda S, et al. Preconditioning effects of levosimendan in a rabbit cardiac ischemia-reperfusion model. J Cardiovasc Pharmacol, 2006, 48 (4): 148-52.
2. Dominguez-Rodriguez A, Abreu-Gonzalez P, Garcia-Gonzalez MJ, et al. A unicenter, randomized, double-blind, parallel-group, placebo-controlled study of Melatonin as an Adjunct in patients with acute myocardial Infarction undergoing primary Angioplasty The Melatonin Adjunct in the acute myocardial Infarction treated with Angioplasty (MARIA) trial: Study design and rationale. Contemp Clin Trials, 2006, Oct 17.
3. Liang JC, Chen HR, Chiu CC, et al. Protective effect of labedipinedilol-A, a novel dihydropyridine-type calcium channel blocker, on myocardial apoptosis in ischemia- reperfusion injury. Life Sci, 2006, 79(13):1248-1256.
4. Wolff AA, Rotmensch HH, Stanley WC, et al. Metabolic approaches to treatment of ischemic heart disease: the clinicans’perspective. Heart Fail Rev, 2002, 7(2):187-203.
5. Ambrosio G, Tritto I. Reperfusion injury: experimental evidence and clinical implications. Am Heart J, 1999, 138(2 Pt 2):S69-75.
6. Eefting F, Rensing B, Wigman J, et al. Role of apoptosis in reperfusion injury. Cardiovas Res. 2004; 61(3): 414-426
7. Zhao ZQ, Morris CD, Budde JM, et al. Inhibition of myocardial apoptosis reduces infarct size and improves regional contractile dysfunction during reperfusion. Cardiovas Res, 2003; 59(1): 132-142.
8. Ji ES, Yue H, Wu YM, et al. Effects of phytoestrogen genistein on myocardial ischemia / reperfusion injury and apoptosis in rabbits. Acta Pharmacol Sin. 2004; 25 (3): 306-312
9. Scnwartzman PA, Ciatowski JA. Apoptosis: The biochemistry and molecutar biology of programmed cell death. Emdocr Rev.1999, 14(2):133-151.
10. Mellai M, Schiffer D. Apoptosis in brain tumors: prognostic and therapeutic considerations. Anticancer Res. 2007; 27(1A):437-48.
11. Durai R, Yang SY, Sales KM, et al. Insulin-like growth factor binding protein-4 genetherapy increases apoptosis by altering Bcl-2 and Bax proteins and decreases angiogenesis in colorectal cancer. Int J Oncol, 2007, 30(4):883-888.
12. Xie Z. Koyama T, Suzuki J, et al. Coronary reperfusion following ischemia: different expression of Bcl-2 and Bax proteins, and cardiomyocyte apoptosis. Jpn heart J, 2001, 42(6):759-770.
13. McClintock DS, Santore MT, Lee VY,et al. Bcl-2 family members and functional electron transport chain regulate oxygen deprivation-induced cell death. Mol Cell Biol, 2002; 22(1):94-104
14. Kane DJ, Sarafian TA, Anton R, et al. Bcl-2 inhibition of neural death: decreased generation of reactive oxygen species. Science, 1993, 262 (5137): 1274-1277.
15. Evan G, Littlewood T. A matter of life and cell death. Science, 1998, 281(5381):1317-
16. Xie Z, Koyama T, Abe K, et al. Upregulation of p53 protein in rat heart subjected to a transient occlusion of the coronary artery followed by reperfusion. Jpn J physiol, 2000, 50 (1):159-162.
17. Shen JG, Quo XS, Jiang B, et al. Chinonin, a novel drug against cardiomyocyte apoptosis induced by hypoxia and reoxygenation. Biochim Biophys Acta , 2000 ,1500 ( 2 ) : 217-226.
18. Sukhotnik I, Bernshteyn A, Mogilner JG. The basic biology of apoptosis and its implications for pediatric surgery. Eur J Pediatr Surg. 2005; 15(4): 229-235.
19. Ashkenazi A, Dixit VM. Death receptors: Signaling and modulation. Sciense, 1998; 281(5381):1305-1308
20. Takemoto Y, Yoshiyama M, Takeuchi K. Increased JNK, AP-1 and NF-kB DNA binding activities in isoproterenol-induced cardiac remodeling. J Mol Cell Cardiol, 1999; 31(11):2017-2030.
21. Bogoyevitch MA, Gillespie-Brown J, Ketterman AJ, et al. Stimulation of the stress- activated mitogen-activated protein kinase subfamilies in perfused heart, p38/RKmitogen- activated protein kinases and c-Jun N-terminal kinases are activated by ischemia/ rsperfusion. Circ Res, 1996,79(2):162-173
22. Ma XL, Kumar S, Gao F, et al. Inhibition of p38 mitogen-actvated protein kinases decreases cardiomyocyte apoptosis and improve cardiac function after myocardialischemia and reperfusion. Circulation, 1999, 99(13):1685-1691
23. Strohm C, Barancik M, Bruehl ML, et al. Inhibition of the ERKcascade by PD98059 and UO126 counteracts ischemic preconditioning in pig myocardium. J Cardiovasc Pharmacol, 2000,36(2):218-229
24. Ferrandi C, Ballerio R, Gaillard P, et al. Inhibition of c-Jun N-terminal kinase decreases cardiomyocyte apoptosis and infarct size after myocardial ischemia and reperfusion in anaesthetized rats. Br J Pharmacol, 2004, 142(6):953-960
25. Krammer PH. CD95(APO-1/Fas)-mediated apoptosis: live and let die. Adv Immunol, 1999; 71:163-210.
26. Muzio M, Chinnaiyan AM, Kischkel FC, et al. FLICE, a novel FADD-homologous ICE/CED-3-like protease, is re cru it ed to the CD95(Fas/Apo-1) death-inducing signaling complex. Cell, 1996; 85(6): 817-827.
27. Stephanou A , Scarabelli TM, Brar BK, et al. Induction of apoptosis and Fas receptor/ Fas ligand expression by ischemia/reperfusion in cardiacmyocytes requires Serine727of the STAT-1 transcription factor but not Tyrosine 701.J Biol Chem,2001;276 (30):28340-28347.
28. Ashkenazi A, Dixit VM. Death receptors: signaling and modulation. Science, 1998; 281(5381):1305-1308.
29. Beg AA, Baltimore D. An essential role for NF-KB in preventing TNF-α-induced cell death. Science, 1996; 274(5288):782-784.
30. Pan G, Roureke K, Chinnaiyam AM, et al. The receptor for the cytotoxic ligand TRAILP.Science, 1997, 276(5309):111-113.
31. Kandel ES, Hay N. The regulation and activities of the multifunctional serine/ threonine kinase Akt/PKB. Exp Cell Res, 1999; 253(1): 210-229.
32. Brunet A, Bonni A, Zigmond MJ, et al. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell, 1999; 96(6): 857-868.
33. Ciani E, Virgili M, Contestabile A. Akt pathway mediates a cGMP- dependent survival role of nitric oxide in cerebellarneurones. J Neurochem, 2002; 81(2): 218-228.
34. Sugden PH, Clerk A. Akt like a woman: gender differences in susceptibility to cardiovascular disease. Circ Res, 2001; 88(10): 975-977.
35. Plas DR, Talapatra S, Edinger AL,et al. Akt and Bcl-xL promote growth factor- independent survival through distinct effects on mitochondrial physiology. J Biol Chem, 2001; 76(15):12041-12048.
36. Henshall DC, Araki T, Schindler CK, et al. Activation of Bcl-2-associated death protein and counter-response of Akt within cell populations during seizure-induced neuronal death. J Neurosci, 2002; 22(19): 8458-8465.
37. The antiapoptotic mechanism of Insulin on Reoxygenation-induced Injury in Cultured Cardiomyocytes of Neonatal Rat. Journal of Huazhong University of Science and technology. 2005; 6(25): 632~635.
38. Crow MT, Mani K, Nam YJ,et al. The mitochondrial death pathway and cardiac myocyte apoptosis. Circ Res. 2004; 95(10):957-70.
39. BorutaiteV, BrownGC. Mitochondria in apoptosis of ischemic heart. FEBS Lett, 2003, 541(2):1-5.
40. Regula KM, EnsK, Kirschenbaum LA. Mitochondria-assisted cell suicide: a license to kill. J Mol Cell Cardiol, 2003, 35(3):559-567.
41. 41.Kong JY, Rabkin SW. Palmitate-induced apoptosis in cardiomyocytes mediated through alterations in mitochonria: prevention by cyclospori A. Biocim Biophys Acta, 2000, 1485:45-55.
42. Zamzami N, Hirsch T, Dallaporte B, et al. Mitochondrial implication in accidental and programmed cell death: apoptosis and necrosis. J Bioenerg Biomemb, 1997, 29(2): 185- 193.
43. Weiss JN, Korge P, Honda HM, et al. Role of the mitochondrial permeability transition in myocardial disease. Circ Res, 2003; 93(4):292–301.
44. Reed JC. Cytochrome c: can’t live with it—can’t live without it. Cell, 1997, 91(5): 559-562.
45. Green DR, Reed JC. Mitochondria and apoptosis. Science, 1998, 281(5381): 1309-1312.
46. Clostre F. Mitochondrial: recent pathophysiological discoveries and the new therapeutic perspectrues. Ann Pharm Fr, 2000,59(1): 3-21.
47. Vienral HL, Kroemer G. Pathophysilogy of mitochondrial, cell death control. Cell MolLife Sci, 1999, 56(11-12):971-976.
48. Armstrong JS, SteinauserKK, FrenchJ, et al. BCL-2 inhibits apoptosis induced by mitochondrial uncopling but does not prevent mitochondria transmenbrane depolarization. Exp Cell Res, 001, 262(2):170-179.
49. Javadov SA, Lim KH, Kerr M, et al. Protection of hearts from reperfusion injury by propofol is associated with inhibition of the mitochondrial permeability transition. Cardiovasc Res, 2000, 45(2): 360-369.
50. Cain K, Bratton SB, Langlais C, et al. Apaf-1 oligomerizes intobiologically active 700- kD and inactive 1.4-kDa apoptosome complexes. J Biol Chem, 2000, 275(9): 6067 -6070.
51. Cande C, Cohen I, Daugas E, et al. Apoptosis-inducing factor (AIF): a novel caspase- independent death effector released from mitochondria. Bio chimie 2002; 84(2-3): 215-222.
52. Han Z, Li G, Bremner T A, et al. A cytosolic factor is required for mitochondrial cytochrome c efflux during apoptosis. Cell Differ; 1998, 5(6):469-479.
53. Okamura T, Miura T, Takemura G. Effect of caspase inhibitors on myocardial infarct size and myocyte DNA fragmentation in theischemia-reperfused rat heart. Cardiovasc Res, 2000, 45(3):642-650.
54. Yaoita H, Ogawa K, et al. Apoptosis in relevant clinical situations: contribution of apoptosis in myocardial infarction. Cardiovasc Res, 2000, 45(3): 630-641.
55. Marber MS, Latchman DS, Walker JM, et al. Cardiac stress protein elevation 24 hours after brief ischemia or heat stress isassociated with resistance to myocardial infarction. Circulation. 1993; 88(3):1264-1272.
56. Choi H, Kim SH, Chun YS, et al. In vivo hyperoxic preconditioning prevents myocardial infarction by expressing bcl-2. Exp Biol Med (Maywood). 2006; 231 (4):463-72.
57. Piot CA, Padmanaban D, Ursell PC, et al. Ischemic preconditioning decreases apoptosis in rat hearts in vivo. Circulation. 1997; 96(5):1598-1604.
58. Maulik N, Sasaki H, Addya S, et al. Regulation of cardiomyocyte apoptosis by redox-sensitive transcription factors. FEBS Lett. 2000; 485(1):7-12.
59. Nakamura M, Wang NP, Zhao ZQ, et al. Preconditioning dcreases Bax expression, PMN accumulation and apoptosis in reperfuse rat heart. Cardiovasc Res, 2000, 45 (3):661-670.
60. Baghelai K, Graham LJ, Wechsler AS, et al. Cardiopulmonary support and physiology. Delayed myocardial preconditioning byα1-adrenoceptors involves inhibition of apoptosis. J Thorac Cardiovasc Surg. 1999; 117(5):980-6.
61. Okamura T, Miura T, Iwamoto H, et al. Ischemic preconditioning attenuates apoptosis through protein kinase C in rat hearts. Am J Physiol. 1999, 277(5 Pt 2):H1997-2001.
62. Liu H, McPherson BC, Yao Z. Preconditioning attenuates apoptosis and necrosis: role of protein kinase C epsilon and-delta isoforms. Am J Physiol Heart Circ Physiol. 2001; 281 (1):H404-410.
63. Wang Y, Takashi E, Xu M, et al. Downregulation of protein kinase C inhibits activation of mitochondrial K(ATP)channels by diazoxide. Circulation, 2001; 104(1):85-90.
64. Baghelai K, Graham LJ, Wechsler AS,et al. Delayed myocardial preconditioning by alpha1-adrenoceptors involves inhibition of apoptosis. J Thorac Cardiovasc Surg, 1999; 117(5):980-986.
65. Rajesh KG, Sasaguri S, Zhitian Z, et al. Second window of ischemic preconditioning regulates mitochondrial permeability transition pore by enhancing Bcl-2 expression. Cardiovasc Res. 2003; 59(2):297-307.
66. Taimor G. Mitochondria as common endpoints in early and late preconditioning. Cardiovasc Res. 2003; 59(2):266-267.
2. Ota S, Nishikawa H, Takeuchi M, et al. Impact of nicorandil to prevent reperfusion injury in patients with acute myocardial infarction: Sigmart Multicenter Angioplasty Revascularization Trial (SMART). Circ J. 2006; 70(9):1099-104.
3. Wolff AA, Rotmensch HH, Stanley WC, et al. Metabolic approaches to treatment of ischemic heart disease: the clinicans’perspective. Heart Fail Rev, 2002, 7(2):187-203.
4. Ambrosio G, Tritto I. Reperfusion injury: experimental evidence and clinical implica- tions. Am Heart J, 1999, 138(2 Pt 2):S69-75.
5. Eefting F, Rensing B, Wigman J, et al. Role of apoptosis in reperfusion injury. Cardiovas Res. 2004 61(3): 414– 426
6. Zhao ZQ, Morris CD, Budde JM, et al. Inhibition of myocardial apoptosis reduces infarct size and improves regional contractile dysfunction during reperfusion. Cardiovas Res, 2003, 59(1): 132-142.
7. Ji ES, Yue H, Wu YM, et al. Effects of phytoestrogen genistein on myocardial ischemia / reperfusion injury and apoptosis in rabbits. Acta Pharmacol Sin. 2004; 25 (3): 306 -312
8. Hausenloy DJ, Yellon DM. New directions for protecting the heart against ischaemia– reperfusion injury: targeting the Reperfusion Injury Salvage Kinase (RISK)-pathway. Cardiovas Res. 2004, 61(3): 448– 460.
9. Loubani M, Hassouna A, Galinanes M. Delayed preconditioning of the human myocardium: signal transduction and clinical implications. Cardiovasc Res, 2004, 61 (3):600-609.
10.敖行述,何华美,张乐之.粉防己碱对心血管的药理作用研究进展。中国药业,2000;9(7);63-65.
11.黄波,冯义柏,朱福等.粉防己碱对兔实验性动脉粥样硬化形成的影响。中国临床药学杂志, 2005;14(5):294-297.
12. Yao WX, Jiang MX. Effects of tetrandrine on cardiovascular electrophysiologic properties. Acta Pharmacol Sin, 2002; 23 (12): 1069 -1074.
13.段世英,王裕勤,孙圣斌.粉防己碱对缺血心脏去甲肾上腺素释放及代谢的影响。华中医学杂志,2005; 9(3): 201-202.
14. Yu XC, Wu S, Wang GY et al. Cardiac effects of the extract and active components of radix stephaniae tetrandrae. II. Myocardial infarct, arrhythmias, coronary arterial flow and heart rate in the isolated perfused rat heart. Life Sci,2001; 68(25):2863 -72.
15.李俊峡,李佃贵,李振彬等.粉防己碱对缺血再灌注心肌顿抑的影响。第四军医大学学报,2000;21(5):92.
16.祝武强,曾秋棠,曹林生等.粉防己碱对缺氧和复氧损伤心室肌细胞内Ca2+超载的作用。临床心血管病杂志,2000,16(2):83-84.
17.夏国瑾,姚伟星,刘燕等.粉防己碱对离体大鼠心脏缺血-再灌注损伤心肌的保护作用。同济医科大学学报,1994,23(6):446-449.
18.钱玲梅,王笑云,冷静.粉防己碱干预大鼠急性缺血再灌注肾损伤中的细胞凋亡变化。中华肾脏病杂志,1998,14(4):252-253.
1. Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation, 1986; 74(5):1124-1136.
2.王裕勤,段世英,孙圣斌等.粉防己碱对电刺激时去甲肾上腺素释放及代谢的影响.临床心血管病杂志,2006,22(2):100-102.
3.张新平,庞月华,冯义柏等.粉防己碱对内膜损伤后平滑肌细胞表型转化和MKP-1表达的影响.心脏杂志,2006,18(2): 131-134.
4. Rao MR. Effects of tetrandrine on cardiac and vascular remodeling. Acta Pharmacol Sin. 2002;23(12):1075-1085.
5.李庆平,杨解人,饶曼人.预先给予粉防己碱对大鼠工作心脏缺血再灌注损伤的保护作用[J].中国药理学与毒理学杂志,1994,8(2):114~117.
6. Townsend PA, Scarabelli TM, Pasini E, et al . Epigallocatechin-3-gallate inhibits STAT-1 activation and protects cardiac myocytes from ischemia/ reperfusion-induced apoptosis. FASEB J. 2004; 18(13):1621-1623.
7. Shen YC, Chen CF, Sung YJ. Tetrandrine ameliorates ischaemia-reperfusion injury of rat myocardium through inhibition of neutrophil priming and activation. Br J Pharmacol. 1999;128(7):1593-601.
8.胡宗礼,黄晓萍,袁学文.表没食子儿茶素没食子酸醋对缺血再灌注心肌细胞凋亡的作用。中国病理生理杂志,2005,21(10):1923-1926.
9. Ferrari R, Ceconi C, Curello S, et al. Role of oxygen free radicals in ischemic and reperfused myocardium. Am J Clin Nutr, 1991;53(1 Suppl): 215-222S.
10. Ferrandi C, Ballerio R, Gaillard P, et al. Inhibition of c-Jun -terminal kinase decreases cardiomyocyte apoptosis and infarct size after myocardial ischemia and reperfusion in anaesthetized rats. Br J Pharmacol. 2004;142(6), 953-60.
11. Shi CZ, Feng YB, Gu X, et al. Effect of protective myocardium by allitridum from decreasing apoptosis in rats with ischemia/reperfusion injury. Journal of Nanjing Medical University, 2005, 19(2):86-90.
12. Park JL, Lucchesi BR. Mechanisms of myocardial reperfusion injury. Ann Thorac Surg 1999; 68(5): 1905-1912.
13. Hoffman JW Jr, Gilbert TB, Poston RS, et al. Myocardial reperfusion injury: etiology, mechanisms, and therapies. J Extra Corpor Technol. 2004; 36(4):391-411.
14. Ambrosio G, Tritto I. Reperfusion injury: experimental evidence and clinical implications. Am Heart J 1999; 138(2pt2): S69–75.
15. Vanden Hock TL, Li C, Shao Z, et al. significant levels of oxidants are generated by isolated cardiomyocytes during ischemia prior to reperfusion. J Mol Cell Cardiol. 1997; 29(9): 2571-2583
16. Chakraborti T, Ghosh SK, Michael JR, et al. Targets of oxidative stress in cardiovascular system. Mol Cell Biochem, 1998; 187(1-2): 1-10.
17.徐新春.钙超载与心肌缺血再灌注损伤。心血管病学进展, 2000; 21(4):227-230.
18. Baxter GF. Ischemic preconditioning of myocardium. Ann Med 1997; 29: 345–352.
19. Loubani M, Hassouna A, Galinanes M. Delayed preconditioning of the human myo- cardium: signal transduction and clinical implications. Cardiovasc Res, 2004, 61 (3):600-609.
20.李大强,冯义柏,张家明等.粉防己碱防止家兔快速心房起搏间隙连接蛋白40降解.第四军医大学学报,2004,25(2):150-152.
21.张新平,庞月华,冯义柏等.粉防己碱对内膜损伤后血管平滑肌细胞表型转化和p38表达的影响.中国动脉硬化杂志,2006;l4(4):304-308.
22.钟宁,钱家庆. M受体机制在粉防己碱保护心脏缺血再灌注损伤作用中的影响.中国药理学通报,2000,16(3):282-284.
23. Aneja R, Hake PW, Burroughs TJ, et al. Epigallocatechin, a green tea polyphenol, attenuates myocardial ischemia reperfusion injury in rats. Mol Med. 2004; 10(1-6):55-62.
24. Townsend PA, Scarabelli TM, Pasini E, et al . Epigallocatechin-3-gallate inhibits STAT-1 activation and protects cardiac myocytes from ischemia/ reperfusion-induced apoptosis. FASEB J. 2004; 18(13):1621-1623.
25. Sheng R, Gu ZL, Xie ML, et al. EGCG inhibits cardiomyocyte apoptosis in pressure overload-induced cardiac hypertrophy and protects cardiomyocytes from oxidative stress in rats. Acta Pharmacol Sin. 2007; 28(2):191-201.
1. Zhao ZQ, Morris CD, Budde JM, et al. Inhibition of myocardial apoptosis reduces infarct size and improves regional contractile dysfunction during reperfusion. Cardiovasc Res. 2003; 59 (1):132–142.
2. Ji ES, Yue H, Wu YM, et al. Effects of phytoestrogen genistein on myocardial ischemia/reperfusion injury and apoptosis in rabbits. Acta Pharmacol Sin. 2004; 25 (3): 306 -312.
3. Chang TH, Liu XY, Zhang XH, et.al. Effects of dl-praeruptorin A on interleukin-6 level and Fas, bax, bcl-2 protein expression in ischemia-reperfusion myocardium. Acta Pharmacol Sin. 2002; 23(9):769-774.
4. Regula KM, Ens K, Kirshenbaum LA. Mitochondria-assisted cell suicide: a license to kill. J Mol Cell Cardiol. 2003;35(6):559-567.
5. Shen YC, Chen CF, Sung YJ. Tetrandrine ameliorates ischaemia-reperfusion injury of rat myocardium through inhibition of neutrophil priming and activation. Br J Pharmacol. 1999;128(7):1593-601.
6. Xu M, Wang Y, Hirai K, et a1. Calcium preconditioning inhibits mitochondrial permeability transition and apoptosis[J].Am J Physiol Heart Circ Physiol,200l,280(2):H899-908.
7. Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer, 1972; 26(4): 239-257.
8. Majno G, Joris I. Apoptosis, oncosis,and necrosis: an overview of cell death. Am J Pathol. 1995; 146(1):3-15.
9. Petit PX, Lecoeur H, Zorn E, et al. Alteration in mitochondrial structure and function are early events of dexamethasone-induced thymocyte apoptosis. J Cell Biol. 1995; 130 (1):157-167
10. Gottlieb RA, Burleson KO, Kloner RA, et al. Reperfusion injury induces apoptosis in rabbit cardiomyocytes. J Clin Invest, 1994; 94(4): 1621-628.
11. Takashi E, Ashraf M. Pathologic assessment of myocardial cell necrosis and apoptosis after ischemia and reperfusion with molecular and morphological markers.. J Mol CellCardiol, 2000, 32(2):209-224.
12. Fliss H, Gattinger D. Apoptosis in ischemic and reperfused rat myocardium .Circ Res, 1996, 79(5):949-956.
13. Maulik N, Yoshida T, Engelman RM. Ischemia precondition attenuates apoptotic cell death associated with ischemia/reperfusion. Mol Cell Biochem,1998, 186(1-2): 139-145.
14. Mocanu MM, Baxter GF and Yellon DM. Caspase inhibition and limitation of myocardial infarct size: protection against lethal reperfusion injury. Br J Pharmacol, 2000; 130(2):197-200.
15. Freude B, Masters TN, Kostin S, et al. Cardiomyocyte apoptosis in acute and chronic conditions. Basic Res Cardiol. 1998; 93(2):85-9.
16. Buja LM. Myocardial ischemia and reperfusion injury. Cardiovasc Pathol. 2005; 14 (4):170-175.
16. 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.
17. Haunstetter A, Izumo S. Toward antiapoptosis as a new treatment modality. Circ Res, 2000; 86(4): 371–376.
18. Buja LM, Entman ML. Modes of myocardial cell injury and cell death in ischemic heart disease.Circulation,1998,98(14):1355-7.
19.Hausenloy DJ, Yellon DM. New directions for protecting the heart against ischaemia- reperfusion injury: targeting the Reperfusion Injury Salvage Kinase (RISK)-pathway. Cardiovasc Res. 2004; 61(3):448-60.
20. Freude A, Master TN, Robicsek F, et al. Apoptosis is initiated by myocardial ischemia and executed during reperfusion. J Am Coll Cardiol, 2000; 32(2): 197–208.
21. Zhao ZQ, Nakamura M, Wang NP, et al. Reperfusion induces myocardial apoptotic cell death. Cardiovasc Res, 2000; 45(3): 651–660.
22. Peart JN, Gross GJ. Adenosine and opioid receptor-mediated cardioprotection in the rat: evidence for cross-talk between receptors. Am J Physiol Heart Circ Physiol, 2003, 285(1): H81-89.
23. Xu M, Wang Y, Hirai K, et al. Calcium preconditioning inhibits mitochondrial permeability transition and apoptosis. Am J Physiol Heart Circ Physiol, 2001, 280(2): H899-908.
24. Babu PP, Suzuki G, Ono Y, et al. Attenuation of ischemia and/or reperfusion injury during myocardial infarction using mild hypothermia in rats: an immunohistochemical study of Bcl-2, Bax, Bak and TUNEL. Pathol Int, 2004, 54(12):896-903.
25. Lam M, Dubyak G, Chen L, et al .Evidence that BCL-2 represses apoptosis by regulating endoplasmic reticulum-associated Ca2+ fluxes. Proc NatI Acad Sci USA, 1994, 91(14):6569-6573.
26. Ryan JJ, Prochownik E, Gottliep CA, et al. C-myc and bcl-2 modulate p53 function by altering p53 subcellular trafficking during the cell cycle. Proc Natl Acad Sci USA. 1994. 91(13): 5878-5882.
27. Borutaite V, Budriunaite A, Morkuniene R, et al. Release of mitochondrial cytochrome C and activation of cytosolic caspases induced by myocardial ischemia. J Biochimica Biophysica Acta, 2001,1537(11):101-109.
28. Xie Z. Koyama T, Suzuki J, et al. Coronary reperfusion following ischemia: different expression of Bcl-2 and Bax proteins, and cardiomyocyte apoptosis. Jpn heart J, 2001, 42(6):759-770.
29. McClintock DS, Santore MT, Lee VY,et al. Bcl-2 family members and functional electron transport chain regulate oxygen deprivation-induced cell death. Mol Cell Biol, 2002, 22(1):94-104
30. Golstein P. Controlling cell death. Science, 1997; 275(5303): 1081-1082.
31. Nagata S. Apoptosis by death factor. Cell, 1997; 88(3): 355-365.
32. Brenner C, Cadiou H, Vieria HL, et al. Bcl-2 and Bax regulate the channel activity of the mitochondrial adenine nucleotide translocator. Oncogene, 2000, 19(3): 329-336.
33. Enari M, Sakahira H, Yokoyama H, et al. A caspase-actived DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature, 1998, 391(6662): 43-50.
34. Cohen GM. Caspase: the executioners of apoptosis. Biochem J. 1997;326(Pt1 ): 1-16.
35. Zhu XF, Liu ZC, Xie BF, et al. Involvement of caspase-3 activation in squamocin- induced apoptosis in leukemia cell line HL-60. Life Sci, 2002, 70(11): 1259-1269.
36. Townsend PA, Scarabelli TM, Pasini E, et al . Epigallocatechin-3-gallate inhibits STAT-1 activation and protects cardiac myocytes from ischemia/reperfusion-induced apoptosis. FASEB J. 2004; 18(13):1621-1623.
1. Harary I, Farley B. In vitro studies of single isolated beating heart cells. Science. 1960,131: 1674-1675.
2. Rui T, Kvietys PR. NFkappaB and AP-1 differentially contribute to the induction of Mn-SOD and Enos during the development of oxidant tolerance. FASEB J. 2005;19(13):1908-1910.
3. Gu X, Feng YB, Shi CZ, et al. Antiapoptotic mechanism of insulin in reoxygen- ation-induced injury in cultured cardiomyocytes of neonatal rats. J Huazhong Univ Sci Technolog Med Sci. 2005; 25(6):632-635.
4. Yue TL, Wang C, Gu JL, et al. Inhibition of extracellular signal–regulated kinase enhances Ischemia/Reoxygenation–induced apoptosis in cultured cardiac myocytes and exaggerates reperfusion injury in isolated perfused heart. Circ Res. 2000; 86 (6):692-699.
5. Rakhit RD, Mojet MH, Marber MS, et al. Mitochondria as Targets for Nitric Oxide–Induced Protection During Simulated Ischemia and Reoxygenation in Isolated Neonatal Cardiomyocytes. Circulation. 2001;103(21):2617-2623.
6. Tang Y, Wang Q, Li J. Influence of KATP channel inhibitor on the changes of HSP70 expression in sevoflurane-induced neonatal rat cardiomyocytes. Sichuan Da Xue Xue Bao Yi Xue Ban. 2003; 34(4):653-655.
7. Siwik DA, Tzortzis JD, Pimental DR, et al. Inhibition of copper-zinc superoxide dismutase induces cell growth, hypertrophic phenotype, and apoptosis in neonatal rat cardiac myocytes in vitro. Circ Res. 1999 23; 85(2):147-53.
8. Rohr S, Scholly DM, Kleber AG.. Patterned growth of neonatal rat heart cells in culture. Morphological and electrophysiological characterization. Circ Res. 1991; 68 (1): 114-30.
9. Simpson P, McGrath A, Savion S. Myocyte hypertrophy in neonatal rat heart cultures and its regulation by serum and by catecholamines. Circ Res. 1982, 51 (6):787-801.
10. Simpson P, Savion S. Differentiation of rat myocytes in single cell cultures with and without proliferating nonmyocardial cells. Cross-striations, ultrastructure, andchronotropic response to isoproterenol. Circ Res. 1982; 50(1):101-16.
11. Ojamaa K, Samarel AM, Klein I. Identification of a contractile-responsive element in the cardiac alpha-myosin heavy chain gene. J Biol Chem. 1995, 270 (52): 31276 -31281.
12. de Micheli A, Chavez E. Ischemia-reperfusion myocardial injury. Arch Cardiol Mex. 2003; 73(4):284-90.
13. Zhao ZQ. Oxidative stress-elicited myocardial apoptosis during reperfusion. Curr Opin Pharmacol. 2004; 4(2):159-65.
1. Bandyopadhyay D,Chattopadhyay A,Ghosh G,et al. Oxidative stress- induced ischemic heart disease: protection by antioxidants. Curr Med Chem,2004,11 (3):369-387.
2. Carden DL, Granger DN. Pathophysiology of ischaemia-reperfusion injury. J Pathol, 2000,190(3): 255-266.
3. Townsend PA, Scarabelli TM, Pasini E, et al . Epigallocatechin-3-gallate inhibits STAT-1 activation and protects cardiac myocytes from ischemia/ reperfusion -induced apoptosis. FASEB J. 2004; 18(13):1621-1623.
4. Ma XQ, Fu RF, Feng GQ,et al. Hypoxia-reoxygenation-induced apoptosis in cultured neonatal rat cardiomyocyets and the protective effect of prostaglandin E. Clin Exp Pharmacol Physiol. 2005; 32(12):1124-30.
5. Maekawa N, Abe J, Shishido T, et al. Inhibiting p90 ribosomal S6 kinase prevents (Na+)-H+ exchanger-mediated cardiac ischemia-reperfusion injury. Circulation. 2006; 113 (21):2516-23.
6. Murry CE,Jennings RB,Reimer KA. Preconditioning with ischemia:A delay of lethal cell injury in ischemic myocardium. Circulation,1986,74 (5):1124-1136.
7. Gaudette GR, Krukenkamp IB, Saltman AE, et al. Preconditioning with PKC and the ATP-sensitive potassium channels: a codependent relationship. Ann Thorac Surg, 2000, 70(2):602-608.
8. Nojiri M, Tanonaka K,Yabe K,et al. Involvement of adenosine receptor, potassium channel and protein kinase C in hypoxic preconditioning of isolated cardiomyocytes of adult rat. Jpn J Pharmacol, 1999, 80(1):15-23.
9. Wang GY, Zhou JJ, Shan J, et al. Protein kinase C-epsilon is a trigger of delayed cardioprotection against myocardial ischemia of kappa-opioid receptor stimulation in rat ventricular myocytes. J Pharmacol Exp Ther, 2001, 299(2):603-610.
10. Saurin AT, Pennington D J, Raat NJ, et al. Targeted disruption of the protein kinase C epsilon gene abolishes the infarct size reduction that follows ischemic precondition- ing of isolated buffer-perfused mouse hearts. Cardiovasc Res, 2002,55(3):672-680.
11. Adrain C, Martin SJ. The mitochondrial apoptosome: a killer unleashed by the cytochrome seas. Trends Biochem Sci. 2001; 26(6):390-7.
12. Suleiman MS, Halestrap AP, Griffiths EJ. Mitochondria: a target for myocardial protection. Pharmacol Ther. 2001; 89(1):29-46.
13. Laclau MN, Boudina S, Thambo JB, et al. Cardioprotection by ischemic precon- ditioning preserves mitochondrial function and functional coupling between adenine nucleotide translocase and creatine kinase. J Mol Cell Cardiol. 2001; 33 (5):947-56.
14. Javadov SA, Clarke S, Das M, et al. Ischaemic preconditioning inhibits opening of mitochondrial permeability transition pores in the reperfused rat heart. J Physiol. 2003; 549(Pt 2):513-24.
15. Borutaite V, Jekabsone A, Morkuniene R, et al. Inhibition of mitochondrial permeability transition prevents mitochondrial dysfunction, cytochrome c release and apoptosis induced by heart ischemia.J Mol Cell Cardiol. 2003; 35(4):357-66.
16. Ott M, Robertson JD, Gogvadze V, et al. Cytochrome c release from mitochondria proceeds by a two-step process. Proc Natl Acad Sci U S A. 2002; 99(3):1259-63.
17. Regula KM, Ens K, Kirshenbaum LA. Mitochondria-assisted cell suicide: a license to kill. J Mol Cell Cardiol. 2003; 35(6):559-67.
18. Maulik N, Engelman RM, Rousou JA, et al. Ischemic preconditioning reduces apoptosis by upregulating anti-death gene Bcl-2. Circulation.1999, 100(19Suppl): II369-375.
19. Nakamura M, Wang NP, Zhao ZQ, et al. Preconditioning decreases Bax expression, PMN accumulation and apoptosis in reperfused rat heart. Cardiovasc Res. 2000; 45 (3):661-70.
20. Baghelai K, Graham LJ, Wechsler AS, et al. Delayed myocardial preconditioning by alpha1-adrenoceptors involves inhibition of apoptosis. J Thorac Cardiovasc Surg. 1999; 117(5):980-986.
21. Rajesh KG, Sasaguri S, Zhitian Z, et al. Second window of ischemic preconditioning regulates mitochondrial permeability transition pore by enhancingBcl-2 expression. Cardiovasc Res. 2003; 59(2):297-307.
22. Liu H, McPherson BC, Yao Z. Preconditioning attenuates apoptosis and necrosis: role of protein kinase C epsilon and -delta isoforms. Am J Physiol Heart Circ Physiol. 2001,281(1):H404-410.
23. Argaud L, Gateau-Roesch O, Chalabreysse L, et al. Preconditioning delays Ca2+- induced mitochondrial permeability transition. Cardiovasc Res. 2004; 61 (1):115- 122 .
24. Kim GT, Chun YS, Park JW, et al. Role of apoptosis-inducing factor in myocardial cell death by ischemia-reperfusion. Biochem Biophys Res Commun, 2003, 309(3): 619-624.
25. Uchiyama T, Engelman RM, Maulik N, et al. Role of Akt signaling in mitochondrial survival pathway triggered by hypoxic preconditioning. Circulation. 2004; 109 (24): 3042-3049.
1. Lepran I, Pollesello P, Vajda S, et al. Preconditioning effects of levosimendan in a rabbit cardiac ischemia-reperfusion model. J Cardiovasc Pharmacol, 2006, 48 (4): 148-52.
2. Dominguez-Rodriguez A, Abreu-Gonzalez P, Garcia-Gonzalez MJ, et al. A unicenter, randomized, double-blind, parallel-group, placebo-controlled study of Melatonin as an Adjunct in patients with acute myocardial Infarction undergoing primary Angioplasty The Melatonin Adjunct in the acute myocardial Infarction treated with Angioplasty (MARIA) trial: Study design and rationale. Contemp Clin Trials, 2006, Oct 17.
3. Liang JC, Chen HR, Chiu CC, et al. Protective effect of labedipinedilol-A, a novel dihydropyridine-type calcium channel blocker, on myocardial apoptosis in ischemia- reperfusion injury. Life Sci, 2006, 79(13):1248-1256.
4. Wolff AA, Rotmensch HH, Stanley WC, et al. Metabolic approaches to treatment of ischemic heart disease: the clinicans’perspective. Heart Fail Rev, 2002, 7(2):187-203.
5. Ambrosio G, Tritto I. Reperfusion injury: experimental evidence and clinical implications. Am Heart J, 1999, 138(2 Pt 2):S69-75.
6. Eefting F, Rensing B, Wigman J, et al. Role of apoptosis in reperfusion injury. Cardiovas Res. 2004; 61(3): 414-426
7. Zhao ZQ, Morris CD, Budde JM, et al. Inhibition of myocardial apoptosis reduces infarct size and improves regional contractile dysfunction during reperfusion. Cardiovas Res, 2003; 59(1): 132-142.
8. Ji ES, Yue H, Wu YM, et al. Effects of phytoestrogen genistein on myocardial ischemia / reperfusion injury and apoptosis in rabbits. Acta Pharmacol Sin. 2004; 25 (3): 306-312
9. Scnwartzman PA, Ciatowski JA. Apoptosis: The biochemistry and molecutar biology of programmed cell death. Emdocr Rev.1999, 14(2):133-151.
10. Mellai M, Schiffer D. Apoptosis in brain tumors: prognostic and therapeutic considerations. Anticancer Res. 2007; 27(1A):437-48.
11. Durai R, Yang SY, Sales KM, et al. Insulin-like growth factor binding protein-4 genetherapy increases apoptosis by altering Bcl-2 and Bax proteins and decreases angiogenesis in colorectal cancer. Int J Oncol, 2007, 30(4):883-888.
12. Xie Z. Koyama T, Suzuki J, et al. Coronary reperfusion following ischemia: different expression of Bcl-2 and Bax proteins, and cardiomyocyte apoptosis. Jpn heart J, 2001, 42(6):759-770.
13. McClintock DS, Santore MT, Lee VY,et al. Bcl-2 family members and functional electron transport chain regulate oxygen deprivation-induced cell death. Mol Cell Biol, 2002; 22(1):94-104
14. Kane DJ, Sarafian TA, Anton R, et al. Bcl-2 inhibition of neural death: decreased generation of reactive oxygen species. Science, 1993, 262 (5137): 1274-1277.
15. Evan G, Littlewood T. A matter of life and cell death. Science, 1998, 281(5381):1317-
16. Xie Z, Koyama T, Abe K, et al. Upregulation of p53 protein in rat heart subjected to a transient occlusion of the coronary artery followed by reperfusion. Jpn J physiol, 2000, 50 (1):159-162.
17. Shen JG, Quo XS, Jiang B, et al. Chinonin, a novel drug against cardiomyocyte apoptosis induced by hypoxia and reoxygenation. Biochim Biophys Acta , 2000 ,1500 ( 2 ) : 217-226.
18. Sukhotnik I, Bernshteyn A, Mogilner JG. The basic biology of apoptosis and its implications for pediatric surgery. Eur J Pediatr Surg. 2005; 15(4): 229-235.
19. Ashkenazi A, Dixit VM. Death receptors: Signaling and modulation. Sciense, 1998; 281(5381):1305-1308
20. Takemoto Y, Yoshiyama M, Takeuchi K. Increased JNK, AP-1 and NF-kB DNA binding activities in isoproterenol-induced cardiac remodeling. J Mol Cell Cardiol, 1999; 31(11):2017-2030.
21. Bogoyevitch MA, Gillespie-Brown J, Ketterman AJ, et al. Stimulation of the stress- activated mitogen-activated protein kinase subfamilies in perfused heart, p38/RKmitogen- activated protein kinases and c-Jun N-terminal kinases are activated by ischemia/ rsperfusion. Circ Res, 1996,79(2):162-173
22. Ma XL, Kumar S, Gao F, et al. Inhibition of p38 mitogen-actvated protein kinases decreases cardiomyocyte apoptosis and improve cardiac function after myocardialischemia and reperfusion. Circulation, 1999, 99(13):1685-1691
23. Strohm C, Barancik M, Bruehl ML, et al. Inhibition of the ERKcascade by PD98059 and UO126 counteracts ischemic preconditioning in pig myocardium. J Cardiovasc Pharmacol, 2000,36(2):218-229
24. Ferrandi C, Ballerio R, Gaillard P, et al. Inhibition of c-Jun N-terminal kinase decreases cardiomyocyte apoptosis and infarct size after myocardial ischemia and reperfusion in anaesthetized rats. Br J Pharmacol, 2004, 142(6):953-960
25. Krammer PH. CD95(APO-1/Fas)-mediated apoptosis: live and let die. Adv Immunol, 1999; 71:163-210.
26. Muzio M, Chinnaiyan AM, Kischkel FC, et al. FLICE, a novel FADD-homologous ICE/CED-3-like protease, is re cru it ed to the CD95(Fas/Apo-1) death-inducing signaling complex. Cell, 1996; 85(6): 817-827.
27. Stephanou A , Scarabelli TM, Brar BK, et al. Induction of apoptosis and Fas receptor/ Fas ligand expression by ischemia/reperfusion in cardiacmyocytes requires Serine727of the STAT-1 transcription factor but not Tyrosine 701.J Biol Chem,2001;276 (30):28340-28347.
28. Ashkenazi A, Dixit VM. Death receptors: signaling and modulation. Science, 1998; 281(5381):1305-1308.
29. Beg AA, Baltimore D. An essential role for NF-KB in preventing TNF-α-induced cell death. Science, 1996; 274(5288):782-784.
30. Pan G, Roureke K, Chinnaiyam AM, et al. The receptor for the cytotoxic ligand TRAILP.Science, 1997, 276(5309):111-113.
31. Kandel ES, Hay N. The regulation and activities of the multifunctional serine/ threonine kinase Akt/PKB. Exp Cell Res, 1999; 253(1): 210-229.
32. Brunet A, Bonni A, Zigmond MJ, et al. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell, 1999; 96(6): 857-868.
33. Ciani E, Virgili M, Contestabile A. Akt pathway mediates a cGMP- dependent survival role of nitric oxide in cerebellarneurones. J Neurochem, 2002; 81(2): 218-228.
34. Sugden PH, Clerk A. Akt like a woman: gender differences in susceptibility to cardiovascular disease. Circ Res, 2001; 88(10): 975-977.
35. Plas DR, Talapatra S, Edinger AL,et al. Akt and Bcl-xL promote growth factor- independent survival through distinct effects on mitochondrial physiology. J Biol Chem, 2001; 76(15):12041-12048.
36. Henshall DC, Araki T, Schindler CK, et al. Activation of Bcl-2-associated death protein and counter-response of Akt within cell populations during seizure-induced neuronal death. J Neurosci, 2002; 22(19): 8458-8465.
37. The antiapoptotic mechanism of Insulin on Reoxygenation-induced Injury in Cultured Cardiomyocytes of Neonatal Rat. Journal of Huazhong University of Science and technology. 2005; 6(25): 632~635.
38. Crow MT, Mani K, Nam YJ,et al. The mitochondrial death pathway and cardiac myocyte apoptosis. Circ Res. 2004; 95(10):957-70.
39. BorutaiteV, BrownGC. Mitochondria in apoptosis of ischemic heart. FEBS Lett, 2003, 541(2):1-5.
40. Regula KM, EnsK, Kirschenbaum LA. Mitochondria-assisted cell suicide: a license to kill. J Mol Cell Cardiol, 2003, 35(3):559-567.
41. 41.Kong JY, Rabkin SW. Palmitate-induced apoptosis in cardiomyocytes mediated through alterations in mitochonria: prevention by cyclospori A. Biocim Biophys Acta, 2000, 1485:45-55.
42. Zamzami N, Hirsch T, Dallaporte B, et al. Mitochondrial implication in accidental and programmed cell death: apoptosis and necrosis. J Bioenerg Biomemb, 1997, 29(2): 185- 193.
43. Weiss JN, Korge P, Honda HM, et al. Role of the mitochondrial permeability transition in myocardial disease. Circ Res, 2003; 93(4):292–301.
44. Reed JC. Cytochrome c: can’t live with it—can’t live without it. Cell, 1997, 91(5): 559-562.
45. Green DR, Reed JC. Mitochondria and apoptosis. Science, 1998, 281(5381): 1309-1312.
46. Clostre F. Mitochondrial: recent pathophysiological discoveries and the new therapeutic perspectrues. Ann Pharm Fr, 2000,59(1): 3-21.
47. Vienral HL, Kroemer G. Pathophysilogy of mitochondrial, cell death control. Cell MolLife Sci, 1999, 56(11-12):971-976.
48. Armstrong JS, SteinauserKK, FrenchJ, et al. BCL-2 inhibits apoptosis induced by mitochondrial uncopling but does not prevent mitochondria transmenbrane depolarization. Exp Cell Res, 001, 262(2):170-179.
49. Javadov SA, Lim KH, Kerr M, et al. Protection of hearts from reperfusion injury by propofol is associated with inhibition of the mitochondrial permeability transition. Cardiovasc Res, 2000, 45(2): 360-369.
50. Cain K, Bratton SB, Langlais C, et al. Apaf-1 oligomerizes intobiologically active 700- kD and inactive 1.4-kDa apoptosome complexes. J Biol Chem, 2000, 275(9): 6067 -6070.
51. Cande C, Cohen I, Daugas E, et al. Apoptosis-inducing factor (AIF): a novel caspase- independent death effector released from mitochondria. Bio chimie 2002; 84(2-3): 215-222.
52. Han Z, Li G, Bremner T A, et al. A cytosolic factor is required for mitochondrial cytochrome c efflux during apoptosis. Cell Differ; 1998, 5(6):469-479.
53. Okamura T, Miura T, Takemura G. Effect of caspase inhibitors on myocardial infarct size and myocyte DNA fragmentation in theischemia-reperfused rat heart. Cardiovasc Res, 2000, 45(3):642-650.
54. Yaoita H, Ogawa K, et al. Apoptosis in relevant clinical situations: contribution of apoptosis in myocardial infarction. Cardiovasc Res, 2000, 45(3): 630-641.
55. Marber MS, Latchman DS, Walker JM, et al. Cardiac stress protein elevation 24 hours after brief ischemia or heat stress isassociated with resistance to myocardial infarction. Circulation. 1993; 88(3):1264-1272.
56. Choi H, Kim SH, Chun YS, et al. In vivo hyperoxic preconditioning prevents myocardial infarction by expressing bcl-2. Exp Biol Med (Maywood). 2006; 231 (4):463-72.
57. Piot CA, Padmanaban D, Ursell PC, et al. Ischemic preconditioning decreases apoptosis in rat hearts in vivo. Circulation. 1997; 96(5):1598-1604.
58. Maulik N, Sasaki H, Addya S, et al. Regulation of cardiomyocyte apoptosis by redox-sensitive transcription factors. FEBS Lett. 2000; 485(1):7-12.
59. Nakamura M, Wang NP, Zhao ZQ, et al. Preconditioning dcreases Bax expression, PMN accumulation and apoptosis in reperfuse rat heart. Cardiovasc Res, 2000, 45 (3):661-670.
60. Baghelai K, Graham LJ, Wechsler AS, et al. Cardiopulmonary support and physiology. Delayed myocardial preconditioning byα1-adrenoceptors involves inhibition of apoptosis. J Thorac Cardiovasc Surg. 1999; 117(5):980-6.
61. Okamura T, Miura T, Iwamoto H, et al. Ischemic preconditioning attenuates apoptosis through protein kinase C in rat hearts. Am J Physiol. 1999, 277(5 Pt 2):H1997-2001.
62. Liu H, McPherson BC, Yao Z. Preconditioning attenuates apoptosis and necrosis: role of protein kinase C epsilon and-delta isoforms. Am J Physiol Heart Circ Physiol. 2001; 281 (1):H404-410.
63. Wang Y, Takashi E, Xu M, et al. Downregulation of protein kinase C inhibits activation of mitochondrial K(ATP)channels by diazoxide. Circulation, 2001; 104(1):85-90.
64. Baghelai K, Graham LJ, Wechsler AS,et al. Delayed myocardial preconditioning by alpha1-adrenoceptors involves inhibition of apoptosis. J Thorac Cardiovasc Surg, 1999; 117(5):980-986.
65. Rajesh KG, Sasaguri S, Zhitian Z, et al. Second window of ischemic preconditioning regulates mitochondrial permeability transition pore by enhancing Bcl-2 expression. Cardiovasc Res. 2003; 59(2):297-307.
66. Taimor G. Mitochondria as common endpoints in early and late preconditioning. Cardiovasc Res. 2003; 59(2):266-267.