| Objective: This study was taken to assess the activation and contribution ofautophagy to ischemia postconditioning (IPost) in normoglycemic, diabetic and diabetictreated with insulin rat hearts. And, this study was also designed to explore the changes ofthe autophagy activation in the normoglycemic and diabetic IPost patients.Methods: Rats were subjected to IPost established by three cycles of10-secondreperfusion followed by10-second ischemia at the end of30min ischemia. The effects ofIPost on myocardial infarct size were assessed with2,3,5-triphenyltetrazolium chloride(TTC) and evans blue staining after120min reperfusion. The activation of autophagy wasassessed by electron microscopy and biochemical examinations after120min reperfusion.The contribution of autophagic mechanisms to IPost were assessed with co-administrationof the autophagy inhibitor3-methyladenine (3-MA). Western blot analysis was employedto determine alterations in LC3-II, Beclin1, lysosome-associated membrane protein2(LAMP-2),cathepsin D, Bcl-2and Bad protein levels in rat hearts, RT-PCR was used tomeasure LC3and beclin1mRNA levels in rats and patients.Results:1. The cardiac arrhythmia scoresCompared with Control group,In normoglycemic rat (2.3±1.0vs1.6±1.0, P=0.023)，2-week diabetic rat（2.1±1.0vs1.5±1.0, P=0.027）,2-week diabetic treated with insulin rat（2.2±1.0vs1.4±1.0, P=0.021）and4-week diabetic treated with insulin rat（2.7±1.0vs1.8±1.0, P=0.019）,the cardiac arrhythmia scores were significantly decreased in IPostgroup(P<0.05).Compared with IPost group, the cardiac arrhythmia scores were obviously increased in IPost+3-MA group (P<0.05).In4-week diabetic rat,6-week diabetic rat and6-week diabetic treated with insulin rat,There were no difference between Control group and IPost group in cardiac arrhythmiascores(P＞0.05).2. The effects of IPost on myocardial infarct sizeCompared with Control group,In normoglycemic rat (60.05±3.75%vs34.43±1.81%,P<0.01)，2-week diabetic rat（54.45±2.03%vs42.61±2.53%, P<0.01）,2-week diabetictreated with insulin rat(53.34±3.3%vs39.61±1.57%, P<0.01)and4-week diabetic treatedwith insulin rat(58.17±3.8%vs48.57±2.23%, P<0.01),the infarct size in IPost group wereobviously decreased.In4-week diabetic rat,6-week diabetic rat and6-week diabetic treated with insulinrat, There were no difference between Control group and IPost group in myocardial infarctsize (P＞0.05).3. The results of electron microscopyIn normoglycemic rat,2-week diabetic rat,2-week diabetic treated with insulin rat,4-week diabetic rat and4-week diabetic treated with insulin rat, There were few or noautophagic vacuoles(AVs) observed in Sham and Control ventricular tissue. In contrast,IPost ventricular tissue displayed more AVs(P<0.05).In6-week diabetic rat and6-week diabetic treated with insulin rat, the IPostventricular tissue displayed altered nuclear morphology including chromatin condensationand fragmentation, minor changes in cytoplasmic organelles, cell shrinkage, and plasmamembrane blebbing, which characteristic by apoptosis.4. The expression of LC3, Beclin1in IPost ventricular tissueIn normoglycemic rat，2-week diabetic rat,2-week diabetic treated with insulin rat,4-week diabetic rat and4-week diabetic treated with insulin rat,IPost-induced obviouslyincreased in protein levels of LC3-II, Beclin1and mRNA levels of LC3,Beclin1(P<0.05).In6-week diabetic rat and In6-week diabetic treated with insulin rat, there were nodifference between Control,Sham group and IPost group in protein levels of LC3-II, Beclin1and mRNA levels of LC3,Beclin1(P＞0.05).5. The expression of LAMP-2, cathepsin D in IPost ventricular tissueIn normoglycemic rat,2-week diabetic rat,2-week diabetic treated with insulin rat,4-week diabetic rat hearts and4-week diabetic treated with insulin rat, IPost-inducedobviously increased in protein levels of LAMP-2and cathepsin D (P<0.05).In6-week diabetic rat and6-week diabetic treated with insulin rat,there were nodifference between Control,Sham group and IPost group in protein levels of LAMP-2andcathepsin D (P＞0.05).6. The effect of autophagy inhibitor3-MA on infarct sizeCompared with the IPost group,in normoglycemic rat (58.95±3.25%vs34.43±1.81%,P<0.01)，2-week diabetic rat(51.28±2.03%vs42.61±2.53%, P<0.01）,2-week diabetictreated with insulin rat (52.47±2.3%vs39.61±1.57%, P<0.01),4-week diabetic treated withinsulin rat（55±3.2%vs48.57±2.23%, P<0.01）, the infarction volume in the IPost+3-MAgroup was obviously increased.On the other hand, In normoglycemic rat (60.05±3.75%vs53.41±3.01%, P<0.01) and2-week diabetic treated with insulin rat(53.34±3.3%vs40.61±1.57%, P<0.01),compared with the Control group,the infarction volume in the3-MA group was decreased.In4-week diabetic rat, compared with Control group, the infarction volume in theIPost+3-MA group was decreased (p<0.05).7. The expression of LC3, Beclin1in cardiomyocytes after3-MA treatmentIn normoglycemic rat,2-week diabetic rat,2-week diabetic treated with insulin rat,4-week diabetic rat and4-week diabetic treated with insulin rat,compared with IPostgroup,the expression of LC3, Beclin1protein and mRNA levels in IPost+3-MA groupwere significantly blunted by3-MA(p<0.05).8. Expression of Bcl-2and Bad protein levels in cardiomyocytesIn normoglycemic rat,2-week diabetic rat,2-week diabetic treated with insulinrat,4-week diabetic treated with insulin rat,compared with Control and Sham group,theexpression of Bcl-2protein levels in IPost group were obviously increased (p<0.05); theexpression of Bad protein levels in IPost group were obviously decreased (p<0.05).Compared with IPost group,the expression of Bcl-2protein levels in IPost+3-MAgroup were obviously decreased(p<0.05); the expression of Bad protein levels wereobviously increased(p<0.05).In4-week diabetic rat,6-week diabetic rat and6-week diabetic treated with insulinrat,compared with Control and Sham group, the expression of Bcl-2protein levels in IPostgroup were obviously decreased(p<0.05);the expression of Bad protein levels in IPostgroup were obviously increased(p<0.05).9. The levels of LC3, Beclin1mRNA in patientsCompared with Control group, the levels of LC3, Beclin1mRNA were obviouslyincreased in normoglycemic and diabetic patients (p<0.05).Conclusion: The present findings provide the evidence that the protective effects ofIPost are associated with the activation of autophagy in normoglycemic rat,2-weekdiabetic rat,2-week diabetic treated with insulin rat,4-week diabetic treated with insulinrat;while, an autophagic mechanism may contribute to myocardial cell injury in4-weekdiabetic rat;in6-week diabetic treated with insulin rat and6-week diabetic rat hearts, lossof the protective effects of IPost are associated with the activation of apoptosis.The role ofthe IPsot-induced increased in the levels of LC3, Beclin1mRNA in normoglycemic anddiabetic patients need further study. |
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