Cardioprotective Effects And Anti-apoptotic Mechanisms Of Insulin On Anoxia/Reoxygenation And Ischemia/Reperfusion-induced Injury In Rats

Part ICardioprotective Effects and Anti-apoptotic Mechanisms of Insulinon Reoxygenation-induced Injury following Anoxia in culturedcardiomyocytes of neonatal RatExperiment I Primary cultures of cardiac myocytes and Establishment of Modelof Anoxia/ReoxygenationObjective:To observe whether anoxia/reoxygenation (A/R) results in myocytes injury in culturedcardiomyotes through inducing anoxia for 2 hour and reoxygenaion for 4 hour.Methods:Primary cultures of cardiac myocytes were prepared from ventricles oflst-to-3rd-day-old Sprague Dawley (SD) rats and cultured for 3 to 5 days. The modelof A/R injury was finished through receiving anoxia for 2 hours and reoxygenation for4 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 4 hours, activities oflactate dehydrogenase (LDH), contents of malondialdehyde (MDA) were assayed throughspectrophotometric procedures respectively.Results: In A/R group, activities of lactate dehydrogenase (LDH) and contents ofmalondialdehyde (MDA) were higher at the end of anoxia of 2 hours than that beforeanoxia (P<0.01); However reoxygenation resulted in a further growth of LDH andMDA; whereas there were not significantly different between two correspondingtimes(P>0.05) in CON group.Conclusion: Anoxia for 2 hours and reoxygenation for 4 hours could induce myocytesinjury in cultured cardiomyocytes of neonatal rat.Experiment II Anti-apoptotic Mechanisms of Insulin on Reoxygenation-inducedInjury following Anoxia in cultured cardiomyocytes of neonatal RatsObjective:To explore the protective effects and mechanisms of insulin on post-anoxia reoxygenation injury in cultured cardiomyocytes of neonatal rats. Methods:The model of anoxia/reoxygenation (A/R) injury was finished through anoxia for 2 hours and reoxygenation for 4 hours in cultured cardiomyocytes of neonatal rat, which were randomized to receive vehicle (0.9%NaCl), insulin, LY294002, insulin plus LY294002 at onset of reoxygenation following 2 hours of anoxia. At the end of reoxygenation of 4 hours, activities of lactate dehydrogenase (LDH), contents of malondialdehyde (MDA) were measured through spectrophotometric procedures, apoptosis of cardiomyocytes were assessed through TUNEL and DNA Ladder, Western blotting was used to analyse expression of phosphorylated Akt in all groups . Results:Compared with vehicle-treated group, activities of LDH, contents of MDA, apoptosisindex (AI) were significantly decreased, and expression of phosphorylated Akt wasincreased prominently in insulin-treated group. However changes of LDH, MDA,AI and phosphorylated Akt resulted from insulin were attenuated or abolished byLY294002 (PI3K inhibitor).Conclusion:These data strongly suggest that early administration of insulin at reoxygenationprotects cardiomyocytes from anoxia/reoxygenation-induced apoptosis throughPI3K/Akt signaling pathway.Part IICardioprotective Effects of Insulin on Post-IschemiaReperfusion-induced Injury in Rats and inComparison with GIKObjective: To explore role of insulin in cardioprotective effects of GIK(glucose-insulin-potassium) and exceptional mechanisms of insulin for metabolicmodulation through comparison of insulin with GIK during reperfusion followingischemia.Methods:58 Male Sprague-Dawley rats were randomly divided into 5 groups :I group wassubjected to ischemia for 30 min without reperfusion. SHAM group was subjected toplacing a silk around the left anterior descending coronary artery without a slipknot.I/R group was subjected to ischemia for 30min and reperfusion for 180 min. INSgroup was subjected to intravenous infusion of Insulin (60U/L, 4ml/kg.h) at 5 minbefore 3 hours of reperfusion. GIK group was subjected to intravenous infusion of GIK (glucose 150g, insulin60U/L, KCL80mmol/L) at 5 min before 3 hours of reperfusion.. MDA , LDH , IS/AAR% and LVEDP were detected throughspectrophotometric procedures, TTC dyeing, and intracardiac pressure monitor.Results:1) MDA, LDH , IS/AAR% and LVEDP were significantly increased in I/R groupcompared with I group (P>0.01); 2) Insulin and GIK could significantly inhibited thegrowths of MDA, LDH, IS/AAR% and LVEDP in INS group and GIK groupcompared with I/R group; 3) Insulin had a similar protective effect onischemia/reperfusion injury with GIK during reperfusion.Conclusion:1) Ischemia and reperfusion could result in growths of MDA, LDH and IS/AAR% inmyocardium of rats. 2) Insulin had an antioxidant effect during post-ischemiareperfusion. 3) Metabolic manipulation was unlikely to be the major mechanism bywhich insulin exerted its protective effects during reperfusion.Part III Role of Bad in Anti-apoptotic mechanism of insulin duringpost-ischemia reperfusion myocardium injury in ratsObjective:To observe anti-apoptotic effect of insulin, and to explore role of Bad in anti-apoptotic mechanism of insulin during post-ischemia reperfusion injury in myocardium of rats. Methods:46 Male Sprague-Dawley rats were randomized to be divided into 4 groups: 1) I/R group was subjected to ischemia for 30 min and reperfusion for 180 min; 2) INS group was subjected to intravenous infusion of Insulin(60U/L, 4ml/kg.h) at 5 min before 3 hours of reperfusion; 3) INS+LY group was subjected to intravenous infusion of LY294002 (0.3mg/kg) at 10 min before 3 hours of reperfusion and intravenous infusion of insulin and LY294002 (30ug/kg/hr) at 5 min before 3 hours of reperfusion; 4) SHAM group was subjected to placing a silk around the left anterior descending coronary artery without a slipknot. At the end of reoxygenation of 4 hours, apoptosis of cardiomyocytes were assessed through TUNEL and DNA Ladder; Immunochemistry and Western-blotting were used to analyse expression ofphosphorylated Bad in all groups.Results:1) Compared with I/R group, apoptosis index (AI) were significantly decreased, andexpression of phosphorylated Bad was higher prominently in INS group. 2) Therewere a remarkably increase in AI and lower expression of phosphorylated Bad inINS+LY group than that in INS group.Conclusion:1) Ischemia and reperfusion resulted in lower expression of phosphorylated Bad inmyocardium of rats; 2) Insulin exerted an anti-apoptotic effect in myocardium of ratsduring ischemia and reperfusion; its mechanism may be involved in higher expressionof phosphorylated Bad via PI3-K/Akt pathway.