| BackgroundMany animal studies have shown that after acute myocardial infarction,the ischemia reperfusion(I/R) injury is responsible for up to 50%of the final infarct size.Although the ischemia preconditioning(IPC) remains the most powerful cardioprotective measure,but its clinical application has been hampered by the requirement of intervention before onset of acute myocardial ischemia,which is clearly impossible in the setting of an acute myocardial infarction.Ischemia postconditioning(IPOC) can be triggered during the clinically applicable time period of reperfusion,but it has the same major limitation as the IPC,i.e.the requirement of an invasive protocol which can only be utilized in the patients undergoing percutaneous coronary intervention(PCI) or special surgery for acute myocardial ischemia. Pharmacological postconditioning(PPOC) is a possible alternative method that may substitute invasive treatments of the short ischemic insults.Also,it has been demonstrated that whenever given before or after myocardial ischemia initiation in many studies,opioid can induce cardioprotective effect,though its effect is somewhat weaker compared with the IPC.Remote preconditioning(RIPC) is another endogenous cardioprotective method,i.e. brief ischemia stimulus on organ or tissue outside of heart would render the heart more tolerant to the subsequential prolonged ischemia.Limb RIPC is triggered by brief limb ischemia,which can be manipulated with the simple tools like tourniquet.Resemble to the IPC,it can produce the cardioprotection by inducing ischemic conditioning stimulus. Because limb RIPC procedure is simple,safe,non-invasive and cost less,it is feasible and has a great value in clinical practice.After the cardioprotection of IPOC against ischemia-reperfusion injury is confirmed,the brief limb ischemia during tong term myocardial ischemia and before coronary artery reperfusion,i.e.the limb remote ischemia postconditioning(RIPOC),has also been immediately shown to produce cardioprotection in some animal models and clinical trials.In contrast to IPC and limb RIPC,limb RIPOC can be triggered after ischemic insult.Also,it doesn't need invasive intervention as IPOC does. Therefore,limb RIPOC may have a promising future in clinical scenario.However,the available data show that cardioprotection induced by RIPOC is weaker compared with IPC, and can be affected by multiple factors.Furthermore,the detailed mechanisms of cardioprotection induced by RIPOC have not been studied extensively.Combination with different interventions to obtain an augmented cardioprotective effect is always one of the most popular research focuses.Moreover,IPOC,RIPOC and fentanyl PPOC are three cardioprotective measures with significant clinical value and are triggered by different mechanisms.Therefore,we designed this randomized,controlled animal trial.Our purposes were:1) to investigate cardioprotective effects of IPOC,RIPOC and fentanyl PPOC;2) to determine whether a combination of IPOC,RIPOC and fentanyl PPOC could obtain synergistic cardioprotective effect,and 3) to assess the roles of ROS, opioid receptors and PI3K/Akt signal pathway in the cardioprotection of combined fentanyl PPOC and RIPOC,in order to explore the inherent mechanism of the interaction in the cardioprotection between fentanyl PPOC and RIPOC.This trial was divided into the three parts.Part 1 Experimental study of protective effects of fentanyl postconditioning,remote postconditioning and ischemia postconditioning against rat myocardial ischemia-reperfusion injury in vivoIn this experiment,an in vivo rat model of myocardial I/R injury was used to compare the cardioprotective effects of fentanyl PPOC,RIPOC and IPOC,and to determine whether there was any synergistic effect in myocardial infarct size sparing among the three interventions.Seventy-three anesthetized male Sprague Dawley rats(weighed 250 to 350 g) were randomly allocated into the nine groups:sham group(group S,n=5),control group(group C, n=7),fentanyl PPOC group(group F,n=9);RIPOC group(group R,n=9),IPOC group (group P,n=8),combined fentanyl PPOC and RIPOC group(group F-R,n=9),combined fentanyl PPOC and IPOC group(group F-P,n=9),combined RIPOC and IPOC group (group R-P,n=9),and combined fentanyl PPOC,RIPOC and IPOC group(group F-R-P, n=9).All rats were opened chest,and the left anterior descending coronary artery(LAD) was encircled with a suture to make a snare.Except for the group S,in the other groups, LAD was ligated for 30 min(ischemia) followed by a 180-min reperfusion(LAD open) in vivo.In group C,no additional intervention was performed.In groups F,F-R,F-P and F-R-P, fentanyl 30μg/kg was slowly injected intravenously at 15 min after LAD ligation.In groups R,F-R,R-P and F-R-P,the bilateral hind limbs underwent a 10-min ischemia which started at 15 min after LAD ligation,and limb blood supply was restored at 5 min before reperfusion.Limb ischemia was produced by placing a thin elastic tourniquet(1-mm diameter) around the upper third of the hind extremity in tight position,closed by a knot to stop the arterial blood supply in the hind limbs.In groups P,F-P,R-P and F-R-P,at 30 min after LAD ligation,IPOC was done with successive three cycles of a 20 s LAD open followed by a 20 s LAD re-occlusion.Throughout the experiment,heart rate(HR),mean arterial pressure(MAP),and a leadⅡelectrocardiogram were continuously monitored. Also,rectal temperature was kept between 36.5℃to 37.5℃.At the end of reperfusion, arterial blood sample was obtained to quantify plasma activity of lactate dehydrogenase (LDH),creatine kinase isoenzyme MB(CK-MB) and serum activity of cardiac troponin I (cTnI) using the kits specifically for rat LDH,CK-MB and cTnI,respectively.Also,the infarct size(IS%) was evaluated using the Evans blue and triphenyltetrazolium chloride (TTC) staining.The results showed no significant differences among all groups in weight,rectal temperature,baselines of hemodynamic variables before LAD ligation(P＞0.05),and hemodynamic variables and incidences of ventricular arrhythmias in the first 15 min after LAD ligation(P＞0.05).During the period from 15 min after LAD ligation to initiation of reperfusion,HR,MAP and rate-pressure product(RPP) were significantly lower in groups F, F-R,F-P and F-R-P comparing with other groups.There was no significant difference in HR, MAP and RPP at 60 min after reperfusion among all groups.Except for group S,there were no significant differences in the incidences of ventricular arrhythmias during LAD ligation among all groups,but significant differences in the incidences of ventricular arrhythmias during initial period of reperfusion among all groups.As compared with group C, ventricular arrhythmia during initial period of reperfusion significantly decreased in other groups but no groups F,R and F-R.Also,arrhythmic score during initial period of reperfusion was lower in groups P,F-R,F-P,R-P and F-R-P than in groups F.and R.There was no significant difference in LDH activity among all groups.As compared with group C,the activity levels of CK-MB and cTnI,and IS%decreased significantly in other groups.Among the groups F,R and P,activity levels of CK-MB and cTnI were highest in group P,and lowest in group R.IS%was significantly lower in group R(48.4±1.4%) compared with the groups F and P(55.6±2.2%and 54.2±4.4%,respectively).The restuts of factor analysis showed existence of a synergetic effect in the infract size sparing between fentanyl PPOC and RIPOC.However,there were no significant interactions in the infract size sparing between fentanyl PPOC and IPOC,and between RIPOC with IPOC.As compared with group F-R,in group F-R-P,a combination with fentanyl PPOC,RIPOC and IPOC could not further reduce infarct size,suggesting ho level-2 interaction among the three interventions.Part 2 Roles of reactive oxygen species and opioid receptors in cardioprotection of combined fentanyl PPOC and RIPOCBased the results from part-one experiment,we designed part-two experiment to assess the roles of reactive oxygen species(ROS) and opioid receptors in cardioprotective effect of combined fentanyl PPOC and RIPOC.This part experiment was divided into the three subunits,in which ROS scavenger [N-(2-mercaptopropionyl) glycine,MPG,),selective delta-receptor antagonist(naltrindole, NTD) and selective kappa-receptor antagonist(nor-binaltorphimine,nor-BNI) were used, respectively.Our aims were to explore the roles of ROS,κreceptor andδreceptor in cardioprotective effect of combined fentanyl PPOC and RIPOC.In each subunit,24 anesthetized male Sprague Dawley rats(weighed 250 to 350 g)were randomly allocated into the four groups:control group(group C),fentanyl PPOC group(group F),RIPOC group(group R),and combined fentanyl PPOC and RIPOC group(group F-R).In the three subunits,all rats were treated with the myocardial I/R in vivo and the fentanyl PPOC or/and limb RIPOC according to the corresponding strategies used in part-one experiment.According to subunit assignments,moreover,MPG,NTD and nor-BNI were also administered intravenously to the rats,respectively,before LAD ligationThe results showed that MPG,NTD and nor-BNI did not significantly change the hemodynamic variables and IS%in group C compared with group C in part-one experiment. When MPG was administered,activity levels of CK-MB and cTnI were significantly lower in groups F and R than in group C,but did not significantly differ between groups F and R. The activity level of CK-MB was significantly lower in group F-R compared with group F. However,there was no significant difference in activity level of cTnI among groups F,R and F-R.As compared with group C,IS%in group R did not significantly change,but IS% in groups F and F-R decreased significantly.The IS%was not different between groups F and F-R.When nor-BNI was administered,activity levels of CK-MB and cTnI were significantly lower in groups F,R and F-R than in group C.The activity levels of CK-MB and cTnI in groups F and R were not different,but they were higher than those in group F-R. As compared with group C,IS%in group F did not significantly change,but IS%in groups R and F-R decreased significantly.The IS%was not different between groups R and F-R.When NTD was administered,activity levels of CK-MB and cTnI were significantly lower in groups F,R and F-R compared with group C.The activity levels of CK-MB and cTnI in groups F and R were not different,but they were higher than those in group F-R.As compared with group C,IS%in groups F,R and F-R decreased significantly.The IS%in groups F and R did not differ,but the IS%in group F-R was significantly lower than those in groups F and R.Part 3 Role of the PI3K/Akt signal pathway in cardioprotective effect of combined fentanyl PPOC and RIPOCThe aim of part 3 experiment was to explore the modulating role of PI3K/Akt signal pathway in cardioprotective effect of combined fentanyl PPOC and RIPOC.Thirty-two anesthetized male Sprague Dawley rats(weighed 250 to 350 g) rats were randomly allocated into the four groups:control group(group C),fentanyl PPOC group (group F),RIPOC group(group R),and combined fentanyl PPOC and RIPOC group(group F-R).All rats were treated with the myocardial I/R in vivo and the fentanyl PPOC or/and RIPOC according to the corresponding strategies used in part one.At the 60 min after reperfusion by LAD open,hearts of the rats were harvested,and the myocardial samples from the area at risk in the left ventricle were separated.Total RNA were extracted from 5 of the eight subjects.To reduce the individual variability as far as possible,the RNA samples from the same group were equivalently pooled and then hybridized to an oligo GEArray(?) Rat PI3K/Akt signaling pathway microarray.Corrected spot intensities were normalized to averages of the housekeeping genes and the final values were presented as a ratio between the groups.To validate the result of the microarray,the real-time quantitative PCR (qRT-PCR) was used to quantify the expression of two genes(randomly selected from the genes whose expression were significantly different among groups) in three samples randomly selected from acquired RNA samples in each group.For the remaining 3 of the eight myocardial samples,Western-blotting technique was used to analyze the expression of the phosphorylated Akt.The results showed that expression of many detected genes relating to PI3K/Akt signal pathway was significantly different among the four groups.As compared with group C,in group F,expression of 12 genes changed significantly,in which 9 showed a significant up-regulated expression and 3 present a significant down-regulated expression.As compared with group C,in group R,expression of 7 genes changed significantly,in which 2 showed a significant up-regulated expression and 5 present a significant down-regulated expression.As compared with group C,in group F-R,a total number of 33 genes showed a significant up-regulated expression,and no gene present a down-regulated expression.The results of the qRT-PCR test showed similar ratios in expression levels of genes cyclin D1 and IGF1 to those of microarray.The Western-blotting analysis revealed that the expression of phosphorylated Akt in myocardium increased significantly in groups F,R and F-R compared with group C.The expression of phosphorylated Akt in myocardium was also stronger in group F-R than in groups F and R.ConclusionsBased on the results of all experiments,the following conclusions could be drawn:1.Cardioprotective effects induced by fentanyl PPOC,IPOC and limb RIPOC were not all same.As compared with fentanyl PPOC and IPOC,limb RIPOC was more powerful in the infarct-sparing.As compared with fentanyl PPOC,IPOC was more effective in anti-arrhythmia during initial period of reperfusion.However,limb RIPOC didn't show a significant inhibition on arrhythmia during initial period of reperfusion.2.There was a synergistic interaction in infarct-sparing effect between fentanyl PPOC and RIPOC,but no interaction in infarct-sparing effect between fentanyi PPOC and IPOC, or between IPOC and limb RIPOC.Also,a combination of fentanyl PPOC,IPOC and limb RIPOC could not further enhance cardioprotection.3.ROS was an important mediator of the cardioprotection by RIPOC,and it played an important role in the synergistic cardioprotection by combined fentanyl PPOC and RIPOC. However,ROS was not involved in the cardioprotection of fentanyl PPOC.4.Theκreceptor was involved in the cardioprotection of fentanyl PPOC,and it played an important role in the synergistic cardioprotection by combined the fentanyl PPOC and RIPOC.However,it was not involved in the cardioprotection of RIPOC.Theδreceptor was not related to the cardioprotection of fentanyl PPOC,but it was involved in the cardioprotection of RIPOC and was also crucial to synergistic cardioprotection by combined fentanyl PPOC and RIPOC.5.The synergistic cardioprotection by combined fentanyl PPOC and RIPOC might be contributed to enhanced activation of PI3K/Akt signal pathway.Also,modulating expression genes relating to the PI3K/Akt signal pathway was perhaps the important molecular basis of the synergistic cardioprotection by combined fentanyl PPOC and limb RIPOC.
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