| Background:Acute myocardial infarction(AMI)is caused by the occlusion of coronary artery.Revascularization should be as early as possible, which can cause recanalization of infarct relative coronary artery(IRCA), achieve satisfactory reperfusion on myocardial level,and relieve the ischemia-reperfusion injury(IRI).It is the key-point therapy.After emergent percutaneous coronary intervention(PCI)to open IRCA in AMI there are still 10-30%patients who have no-reflow or slow-reflow phenomenon.Studies show that even if the blood flow has been reverted to TIMIⅢlevel,some patients can not achieve full reperfusion and worsen the prognosis severely. For this reason,how to prevent IRI has been the emphasis of clinical studies in recent years.Much more drugs to prevent and cure IRI have been investigated such as Ca2+-channel blocker(CCB),free radical scavenger,anti-oxidant,neutrophil inhibitor,adenosine,complement system inhibitor,endothelin receptor inhibitor,anti-apoptosis factor,sodium-hydrogen exchange inhibitor,RAS inhibitor and NO,etc.Carvedilol,a tertiary generationβ-blocker,has been used widely in clinical study to treat patients with hypertension,CAD and arrhythmia.In recent years,researchers have paid close attention to carvedilol for its myocardial protection in IRI.Studies have verified that the mechanisms of myocardial protection of carvedilol in IRI included:blocking Ca2+-channel, inhibiting infiltration of neutrophil,protecting the mitochondria of cardiocyte, anti-injury of endothelium cells,anti-oxidant,lessening the deactivation of NO and avoiding the overdose of NO,etc.For this reason,it is very important to research the relativity of carvedilol and myocardial full reperfusion after AMI.Objective:In this research,we established the reperfusion model on AMI rabbit by the method of obstructing and releasing the Left Anterior Ventricular Branch(LAVB)of left circumflex coronary artery(LCX).Observing the ECG;markers of myocardial injury,area of ischamic and infarcted myocardia and the changes of histopathology in different period after reperfusion.We can explore the effects of carvedilol on myocardial IRI after reperfusion in AMI rabbit.Through testing the activity of NOS,and the concentration of NO, Interliukin-8(IL-8),Tumor Necrosis Factor-α(TNF-α),Malondialdehyde (MDA)and the expression level of NOS-mRNA,we elucidated that carvedilol could benefit the satisfactory reperfusion level on ischemia myocardia because it inhibited the releasing of mediators of inflammation so as to decrease the activition of induced Nitric Oxide Synthase(iNOS),and decrease the deactivation of NO for having removed OFR directly.This study has helped to guide to the application of carvedilol in AMI patients with reperfusion.The experiment was divided into 4 parts.Methods and Results:1 Establishment of the reperfusion model on AMI rabbits:Methods A total of 24 healthy Japanese albino rabbits of both sex were used in this study. After measuring body weight(2.5～3.5kg),rabbits were randomly divided into 3 groups:ischemia-reperfusion group(IR group,8),ischemia-noperfusion group(AMI group,8),sham group(sham,8).After preconditioning the myocardium twice by obstructing the blood flow for 5 minutes,we obstructed the flow of LAVB in IR group for 60 minutes,and then released it to be reperfusion.In AMI group we obstructed the flow permanently by ligating LAVB.And in sham group we only threaded but did not obstruct the flow. Then we killed them 3 days later.Venous blood was gathered.The levels of cTNI,CK and CK-MB were assayed at pre-reperfusion(baseline)and post-reperfusion period(4-hour,8-hour,12-hour,24-hour,48-hour and 72-hour after being reperfusion).The summation changes of ST segment elevation were observed in leadsП,Ш,avF by ECG.Histopathology of myocardia,Evan's Blue and TTC dyeing were taken into notice.STATA8.0 software pack was used for data analysis.Initially the homogeneity of variance between all the groups was analyzed.All the numerical data were expressed as mean±standard deviation.Students t test,analysis of variance(ANOVA)and Scheffe test were used for statistical test in this study. Chi-square test was used for analysis of categorical data.Linear Correlation analysis was used to measure the coefficient of correlation about the correlation data.P＜0.05 was considered as statistic significance.Results The results come out that the ST segment all elevated in each group after LAVB was obstructed.In IR group the ST segment lowered more than 50%within 120 minutes after releasing the artery.This phenomenon had not appeared in other two groups.Compared to baseline,the cTNI,CK and CK-MB were all raised in IR group,and the peak value antedisplaced to 8h,12h and 10h.These three factors were all raised but no antedisplacement of the enzyme peak in AMI group.In sham group the raising of the three factors was slight,and no antedisplacement of the enzyme peak either.The experiment of rabbits in IR and AMI groups were consistent with the AMI diagnostic criteria in AMI Guideline of diagnosis and therapy established by Cardiac Disease branch of Chinese Medical Association in 2004.The histopathology examination and TTC staining agree with the AMI diagnosis too.The experiment of rabbits in IR group was in accordance with the recanalization criteria of IRCA recommended by the《Chinese Journal of Cardiology》editorial committee in 1991.The injection of Evan's Blue was in coincidence with the recanalization of IRCA too.2 Changes of NOS-NO system activity in rabbit models of acute myocardial infarction with reperfusion:Methods Twenty-four rabbits were randomly divided into 3 groups with 8 rabbits in each group:IR group,AMI group and sham group.Models were created by the way of above part 1.The concentration of NO and activity of NOS were assayed at pre-reperfusion (baseline)and post-reperfusion period(4-hour,8-hour,12-hour,24-hour, 48-hour and 72-hour after being reperfusion).Using the way of Evan's Blue and TTC dyeing to test myocardial weight of infarction(WOI)and ischemia (WOR).The relationship between the level of NO and the weight of infarction and ischemia was observed.The statistic method was the same with the above part 1.Results There were no statistical significant differences among 3 groups in general features(p＞0.05).The levels of NO,NOS and iNOS showed no significant changes among 3 groups at baseline.The concentration of NO and the activity of NOS in IR group were higher than those in AMI group at 4-hour,8-hour,24-hour,48-hour time points;The difference of NO level between IR group and AMI group was noted at 4-hour,8-hour,24-hour and 72-hour time points(p＜0.05);The activity of NOS between IR group and AMI group at 8-hour,12-hour,24-hour,48-hour and 72-hour time points had significant statistic difference(p＜0.01);The activity of iNOS in IR group was higher than that in AMI group.The activity of iNOS between IR group and AMI group at 4-hour,8-hour,12-hour,24-hour and 48-hour time points had significant statistic difference(p＜0.01);The concentration of NO and the activity of NOS in IR group were lower than those in sham group at 8-hour,12-hour,24-hour,48-hour and 72-hour time points(p＜0.01);The activity of iNOS in IR group was higher than that in sham group(p＜0.01 except at 72-hour time point).The activity of iNOS began to increase at 4-hour and reached the peak level at 24-hour after reperfusion.The levels of NO and NOS showed a downward trend except at 24-hour time point. Negative correlation was found between NO and ratio of WOR(p=0.0096)in IR group.Negative correlation was also found between NO and ratio of WOI(p=0.0027)too.3 Effect of carvedilol on NOS-NO system in rabbit models of acute myocardial infarction with reperfusion:Methods Twenty-four rabbits were randomly divided into 3 groups with 8 rabbits in each group:control group, treatment group and sham group(gavaged with carvedilol 10mg/d for 7 days before creating models).The establishment of models were the same with the above part 1.The levels ofcTnI,CK,CK-MB,NO and activity of NOS were assayed at pre-reperfusion(baseline)and post-reperfusion period(4-hour, 8-hour,12-hour,24-hour,48-hour and 72-hour after being reperfusion). Expression of eNOS and iNOS mRNA was detected by RT-PCR.The changes of ST raising summation were observed by ECG in leadsП,Ш,avF.The changes of left ventricular end-diastolic pressure(LVEDP),left ventricular end systolic pressure(LVESP)and mean arterial pressure(MABP)were observed.Using the examination of histopathology of myocardia,Evan's Blue and TTC dyeing,myocardial WOI and WOR were tested.The statistic method was the same with the above part 1.Results There were no statistical significant differences among 3 groups in general features(p＞0.05).The levels of NO,NOS and iNOS showed no significant changes among three groups at baseline.The concentration of NO and the activity of NOS in treated group were higher than those in control group at 4-hour,8-hour,72-hour time points, but they were lower at 12-hour and 24-hour time points;The difference of NO concentration between treated group and control group was noted at 4-hour,12-hour,24-hour and 72-hour time points(p＜0.01);The activity of NOS between treated group and control group at 4-hour,8-hour,12 -hour,24-hour,48-hour and 72-hour time points had statistic significant difference(p＜0.01);The activity of iNOS in treated group was lower than that in control group.The activity of iNOS between treated group and control group at 8-hour,12-hour,24-hour and 48-hour time points had statistic significant difference(p＜0.01);The concentration of NO and the activity of NOS in treated group were higher than those in sham group at 4-hour and 8-hour time points,but they were lower at other time points(p＜0.01);The activity of iNOS in treated group were higher than that in sham group except at 24-hour time point.The difference of iNOS between treated group and sham group was noted at 4-hour,12-hour,48-hour and 72-hour time points(p＜0.01);The activity of iNOS began to increase at 4-hour and reached the peak level at 24-hour after reperfusion.The levels of NO and NOS showed a downward trend except 24-hour time point.The difference of eNOS and iNOS mRNA expression between treated group and control group was noted at 72-hour time point.The results come out that the ST segment all raised in each group after LAVB was obstructed.In control group and treatment group,the ST segment lowered more than 50%within 120 minutes after the artery was released,but such changes did not occur in the sham group.The levels of cTnI, CK and CK-MB were significantly increased,and the peaks of the levels were respectively advanced to 8,12 and 10 hours in the control group and treatment group after being reperfusion.Compared with sham group,LVSP,MABP decreased significantly with LVEDP significantly increased in the control group and the treatment group after coronary artery occlusion.It was suggested that myocardial systolic and diastolic function were damaged. Compared with the control group,LVSP and MABP of the treatment group were higher,while LVEDP was reduced.It was suggested that carvedilol has improved myocardial systolic and diastolic function.In the treatment group pathological examination showed that cell swelling and inflammatory cell infiltration were slighter than in the control group.TTC and Evan's Blue staining showed that the ratios of myocardial WOR in the treatment group and the control group were respectively 14.09%and 10.31%.The ratios of myocardial necrosis were respectively 11.10%and 8.01%.The difference between the two groups had statistical significance(p=0.0000).The differences of eNOS and iNOS mRNA expression between treated group and control group was noted at 72-hour time point.4 The relationship between the activity of NOS-NO system and the changes of IL-8,TNF-α,MDA levels in rabbits of acute myocardial infarction with reperfusion:Methods Twenty-four rabbits were randomly divided into 3 groups with 8 rabbits in each group:control group,sham group and treatment group(gavaged with carvedilol 10mg/d for 7 days before creating models). The establishment of models were the same with the above part 1.The concentration of NO,IL-8,TNF-αMDA and activity of NOS,iNOS and eNOS were assayed at pre-reperfusion(baseline)and post-reperfusion period (4-hour,8-hour,12-hour,24-hour,48-hour and 72-hour after being reperfusion).The relationship between the activity of iNOS and the changes of IL-8,TNF-αlevels was observed.The relationship between the concentration of NO and the changes of MDA level was also analyzed.The statistic method was the same with the above part 1.Results There were no statistical significant differences among 3 groups in general features(p＞0.05).The activity of NOS,iNOS and eNOS and the levels of NO were the same as the above part 3.The concentration of IL-8,TNF-αand MDA in treatment group at different time point was lower than that in control group(p＜0.05)except at 72-hour time point;The levels of IL-8,TNF-αand MDA in control and treatment groups were higher than those in sham group at different time points except at 72 hour time point(p＜0.05);The concentration of IL-8,TNF-αand MDA began to increase at 4-hour and reached the peak level at 24-hour after reperfusion.Then they showed a downward trend.Positive correlation was found between the activity of iNOS and the changes of IL-8,TNF-αlevels(p＜0.01).Negative correlation was also found between the concentration of NO and the change of MDA level in control and treatment group(p＜0.01).Conclusions:1 AMI-reperfusion rabbit models can be successfully established by the application of this method.It has proved to be very effective.2 The concentration of NO decreases at earlier period because of the decrease of the eNOS activity,but it increases at later period because of the increase of the iNOS activity.The change of NOS-NO system activity is closely related with IRI.Negative correlation was found between NO and the ratio of WOR and WOI in IR group.3 Carvedilol can reduce inactivation of NO and activation of iNOS in rabbit models of AMI with reperfusion.It can maintain NOS-NO system activity to the ideal level for myocardial reperfusion and reduce myocardial IRI in AMI rabbits with reperfusion.It not only improves myocardial systolic and diastolic function,but also reduces ischemic and necrotic area after reperfusion.4 Positive correlation was found between the iNOS activity and the IL-8, TNF-αlevels.Negative correlation was also found between the concentration of NO and the change of MDA level.Carvedilol regulates the activity of NO-NOS system by inhibiting the release of inflammatory cytokines and removal of OFR.
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