The Study Of The Role Of Caspase-3 On Apoptosis During Myocardial Ischemia Reperfusion In Rats And The Effect Of Ac-DEVD-CHO

Objective The myocardial ischemia reperfusion injury (IRI)is a main interference in the reperfusion therapy for acute myocardial infarction. The mechanism is still not very clear. Although, traditionally, necrosis is regarded as the pathological hallmark of myocardial infarction, there is accumulating evidences that apoptosis also contributes to myocardial cell death during infarction.Apoptosis is crucially dependent on the activation of caspase-3.We therefore examined the effect of caspase-3 inhibitor during reperfusion upon myocardial infarction. The rats of myocardiac IRI were used as models to study the role of apoptosis in IRI, evaluate the effects of the interventions of Ac-DEVD-CHO on IRI and provide the theoretical and experimental evidence for clinical strategy. Methods 132 rats were divided into three groups: IR group, Ac-DEVD-CHO group and sham-operative control group. Samples were observed at 30min after ischemia followed by 1h,3h,6h,12h and 24h reperfusion. The myocardial infarction area of other 50 rats with the same time course including only IR and IR+Ac-DEVD-CHO group were evaluated by TTC dyeing method. The following items were determined and compared to evaluate the degree of IRI: morphological changes of myocardium, area of infarction. The myocardial cell apoptosis was determined with terminal deoxynucleotidyl 　transferase-mediated dUTP-fluorescein nick end labeling (TUNEL) method;The activity ofcaspase-3 was determined with fluorescent assay; The protein expression of Fas gene was studied with S-P immunohisto chemical staining and western blot analysis.The infiltration number of PMNs in myocardium was also observed. For selecting the intervention strategies of anti-IRI and antiapoptotic : caspase-3 inhibitor was used. Results (1) Apoptotic index(AI)had been increasing during the period of reperfusion,and reached the highest point at 12h after reperfusion, and also remained high level at 24h after reperfusion. There was no evidence of myocyte apoptosis found in myocardium with sham-operated control; Apoptotic index was significantly reduced by caspase-3 inhibitor．(2) The infarct size/ischemia area at risk of group IR had been increasing during the period of reperfusion, and did not decline at 24h after reperfusion; The infarct size/ischemia area at risk of group IR+Ac-DEVD-CHO except for 1h after reperfusion was smaller than those of group IR. (3)Caspase-3 activity was detected at 1h after reperfusion, which increased gradually and peaked at 12h, until 24h after reperfusion it also remained high level. In group IR+Ac-DEVD-CHO, caspase-3 activity was inhibited by Ac-DEVD-CHO.(4) The protein expression of Fas gene peaked at 6h after reperfusion, until 24h after reperfusion it also remained high level. After treated with Ac-DEVD-CHO, the protein expression of Fas gene did not alter.(5)In group IR,the infiltration number of PMNs had been increasing during the period of reperfusion, and did not decrease at 24h after reperfusion. Ac-DEVD-CHO was effective in reducing the infiltration number of PMNs.Conclusion1. The activation of Caspase-3 contributed to myocardial cell apoptosis during ischemia reperfusion. Cardiac myocyte death during ischemia reperfusion was mediated, at least in part, by an apoptotic mechanism.Apoptosis is a crucial event that can initiate reperfusion-induced the2. infitration number of PMNs and subsequent tissue injury. 3. When the Fas receptor binds its ligand, this recognition event lead to caspase-3 activation and apoptosis.4. Ac-DEVD-CHO was effective in reducing myocardial injury after ischemia reperfusion, which could at least be partially attributed to the attenuation of cardiomyocyte apoptosis.