RESUMO
BACKGROUND:Ischemia/reperfusion injury can cause the necrosis of cardiomyocyte,and it can also induce cell apoptosis.However,cell apoptosis may be the main death type of cardiomyocyte at the early stage of infarction,and it may be one of causes for expanding myocardial infarction area.OBJECTIVE:The goal of this study was to observe the anti-apoptotic effect of adenosine (ADO) preconditioning on cardiomyocytes during the ischemia/reperfusion,and to investigate the role of apoptosis-related gene protein Bcl-2 and Bax.DESIGN:A randomized controlled animal experiment.SETTING:First College of Clinical Medical Science,China Three Gorges University&Department of Cardiology,Yichang Central People's Hospital.MATERIALS:Thirty-six healthy male rabbits of clean grade,weighing 2.5 to 3.0 kg,were provided by Laboratory Animal Department,Tongji Medical College,Huazhong University of Science and Technology.The protocol was carried out in accordance with animal ethics guidelines for the use and care of animals.All rabbits were divided into 3 groups according to random number table.There were 12 animals in either control,ADO or ADO+DPCPX (an adenosine A1 receptor antagonist).METHODS:This experiment was carried out in the Central Laboratory,China Three Gorges University between October 2005 and October 2006.Ex vivo rabbit myocardial ischemia/reperfusion models were prepared.After being anesthetized,the rabbits were performed anticoagulation with heparin and carried out Langendorff retroperfusion.In the control group,the hearts of animals subjected to 40 minutes of ischemia and 60 minutes of reperfusion.Six of them were used for determining myocardial infarct size after reperfusion,another six for cardiomyocyte apoptosis,gene expression and ultrastructural analysis of myocardium.In the ADO group:The ADO hearts were continuously infused with 10 μmol/L of adenosine 30 minutes before ischemia,and operated according to the requirement of control group.In the DPCPX roup:the isolated hearts of animals were infused for 15 minutes with 10 mmol/L of DPCPX 45 minutes before ischemia.and operated in accordance with ADO group.MAIN OUTCOME MEASURES:①The heart rate and blood pressure of animals in 3 groups were measured during ischemia/reperfusion process.②The infarct size was determined by triphenyltetrazolium chloride(TTC) staining.③The apoptotic index of cardiomyocytes was detected by histological TUNEL staining and DNA ladder on agarose gel electrophoresis.④Apoptosis-related protein Bcl-2 and Bax expressions were detected by in situ immunohistochemical staining.RESULTS:Thirty-six rabbits were enrolled in the final analysis.①Comparison of heart rate and blood pressure:During the process of ischemia/reperfusion,both heart rate and blood pressure were persistently decreased significantly (P＜0.01).There were no significant differences in two indexes between any two groups (P＞0.05).②Comparison of myocardial infarct size:There were no significant differences in myocardial infarct size among the control group,ADO group and DPCPX group (P＞0.05).The myocardial infarct size of rabbits in the ADO group was significantly smaller than that in the control group.It suggested that ADO preconditioning could contract the myocardial infarct size of rabbits.The myocardial infarct size of rabbits in the ADO+DPCPX group was significantly larger than that in the ADO group,but significantly smaller than that in the control group (P＜0.01).It suggested that DPCPX could partially inhibit the protective effect of ADO.③Comparison of apoptosis of cardiomyocytes: Apoptotic cells were not found in the non-ischemic myocardial tissue,but found in the infarct tissue and infarct edge ischemic tissue.Apoptotic cells in the control group and ADO+DPCPX group were significantly more than those in the ADO group.Apoptotic index in the control group and ADO+DPCPX group was significantly higher than that in the control group,respectively (P＜0.01).④Comparison of apoptosis-related protein expression:The absorbance of Bax in the ADO group was significantly lower than that in the control group(P＜0.01).The absorbance of Bax in the ADO+DPCPX group was significantly lower than that in the control group,but significantly higher than that in the ADO group (P＜0.01).The value of Bcl-2/Bax in the control group was significantly lower than that in the ADO group (P＜0.01).The value of Bcl-2/Bax in the ADO+DPCPX group was significantly higher than that in the control group,but significantly higher than that in the ADO group(P＜0.01).CONCLUSION:Exogenous ADO inhiblts reperfusion-induced apoptosis of cardiomyocytes,which is partially mediated by DPCPX:Down-regulation of apoptosis-related Bax protein plays an important role in the anti-apoptotic effect of exogenous ADO.