| Objective: 1.The expression of SIRT3 and the changes of myocardial acetylation in myocardial ischemia / reperfusion were studied in isolated perfused heart model.2.Construction of the diabetic rat model,to study the function of SIRT3 on protein acetylation and oxidative stress in myocardial ischemia/reperfusion injury in diabetic model while to investigate the protective effect of SIRT3 against myocardial ischemia/reperfusion injury in Langendorff-perfused diabetic rat hearts.Methods : In part 1 of the study,10 SD rats,2~3 months old,weight(220-250)g,were randomly divided into control(A)and test(B)groups(n=5).The isolated hearts of SD rats in the two groups were perfused in vitro by Langendorff perfusion system.In group A,After parallel perfusion of 20 min when the cardiac function was stabilized and then stable perfusion lasted for 60 min.In group B,perfusion of isolated hearts were interrupted for global ischemia of 40 min after s parallel perfusion of20 min when the cardiac function was stabilized and then reperfusion was established and lasted for 60 min.After modeling of two groups,the Sirt3 expression in myocardium was detected by fluorescence quantitative PCR.Inpart 2 of the study,24 type 1 diabetes mellitus(DM)male SD rats,2~3 months old,weight(220-250)g,were modeled by intraperitoneal injection with streptozotocin(STZ).The tail vein blood of rats was collected to monitor blood glucose at 72 h,7,14 and 28 days after injection?The successful preparation of the model by taking blood glucose > 16.70mmol/L ? urine > +++ as the standard.After the modeling,the DM rats were randomly divided into three groups(n=8).After feeding for 5 weeks,Group A were treated by intragastric administration(gastric infusion)of saline for 4 weeks,Group B were treated with nicotinamide(SIRT3 inhibitor)and Group C were treated with resveratrol(SIRT3 activator)in the same way.After 4 weeks of pretreatment,the isolated hearts of all DM rats were subjected to ischemia/reperfusion process in Langendorff perfusion modeling device.All isolated hearts in three groups were perfused stably for 20 min,then global ischemia(no flow)of 40 min was establishied,followed by reperfusion for 60 min.During modeling,Heart rate,left ventricular pressure(LVDP),and the maximum velocity of ascending and descending in intraventricular pressure(ądp/dtmax),were recorded individually in three groups.Collect coronary effluent when perfused stably for 20 min and60 minutes after reperfusion,the Troponin T(Tn-T)contents in coronary effluent were also detected by ELISA.After perfusion,myocardial infarct sizes in three groups were examined by TTC staining,and the levels of caspase3 reflect cell apoptosis in myocardial tissue and protein acetylation were tested by immunofluorescence and western bolt,respectively.Myocardial SOD activityand malondialdehyde(MDA)content were measured by immunofluorescence of frozen section.Results: In part 1 of this study: Compared the myocardial expression of Sirt3 m RNA after the end of the perfusion in each group,the A group is 1.7 times of that in B group,the difference was statistically significant(p<0.05)?Compared the levels of acetylation of myocardial tissue by immunofluorescence in each group,the B group was significantly higher than the A group.a In part 2 of this study: Comparision of the HR,LVDP,ądp/dtmax among the three groups,the differences were statistically significant(p<0.05),and results showed that these measurements were highest in group C and those in group A were higher than that in group B,the differences were statistically significant(P < 0.05).Compared myocardial infarct size after ischemia / reperfusion in three groups,group B was significantly larger than group A and C,while group C was lowest,the differences were statistically significant(P < 0.05).The expression of caspase 3 protein in myocardium was detected by frozen section immunofluorescence after myocardial ischemia /reperfusion in each group,group B was significantly larger than group A and C,while group C was lowest,the differences were statistically significant(P <0.05).Myocardial acetylation levels were measured by Western blot after myocardial ischemia / reperfusion in each group,and the results showed that group B was significantly larger than group A and C,while group C was lowest,the differences were statistically significant(P < 0.05).Compared SOD activity in myocardial tissue after myocardial ischemia / reperfusion in eachgroup,group C was higher than that in both group A and B,while lowest in group B,the differences were statistically significant(P < 0.05).Compared the level of MDA after myocardial ischemia / reperfusion in each group,group B was significantly larger than group A and C,while group C was lowest,the differences were statistically significant(P < 0.05).Conclusion: 1.The expression of SIRT3 in the hearts of normal adult rats was significantly lowered after isolated ischemia / reperfusion,while the overall level of cardiac acetylation was significantly increased.2.Enhanced activity of SIRT3 can reduce the myocardial ischemia/reperfusion injury in diabetic rats by attenuating myocardial oxdative stress. |
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