The Effects And Mechanisms Of Tetrandrine On Cardiomyocytes Apoptosis Induced By Ischemia/Reperfusion And Anoxia/Reoxygenation Injury In Rats

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 .