【作者】 常超；

【导师】 冯义柏；

【作者基本信息】 华中科技大学 ， 内科学， 2007， 博士

【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。更多还原

【Abstract】 PartⅠThe Study of Protective Effect Against Ischemia/Reperfusion Injury Preconditioning with Tetrandrine in Rats and Comparison with Epigallocatechin-3-gallateObjective: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 ModelObjective: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 ModelExperiment I Primary cultures of cardiomyocytes and Establishment of Model of Anoxia/ReoxygenationObjective: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 .更多还原