| Remifentanil,a novel ultra-short acting opioid analgesics,has been widely used for the anesthesia in the clinical practice.Previous works have suggested that opioid receptors are involved in cardioprotection effect of ischemia preconditioning and this effect can be completely abolished by opioid receptor antagonists.Further studies have revealed that selective opioid receptor agonists can also induce cardioprotection effect.A recent study reported that as a no-selective opioid agonist,remifentanil had early cardioprotection of preconditioning against ischemia and reperfusion(I/R) injury.However,it still remains unknown whether remifentanil can induce delayed cardioprotection against I/R injury.As an important chemical mediator or cellular signaling molecular,the role of nitric oxide(NO) in the myocardial I/R process is a hotspot subject studied. At normal physiological conditions,NO is mainly synthetized by catalysis of the endothelial nitric oxide synthase(eNOS) in the vascular endothelium.It has been shown that NO has a variety of functions,such as endothelium-dependent vasorelaxation,inhibition of platelet adhesion and aggregation,depression of vascular smooth muscle proliferation,reduction of neutrophil adherence and aggregation,and decrease of intracellular Ca2+ overload.There is mounting evidence to suggest that inducible nitric oxide synthase(iNOS) exerts important roles in the cardioprotection effect of pharmacological preconditioning and is an essential trigger and/or mediator of delayed cardioprotection. But there are few studies to investigate the relation between opioids-induced cardioprotection and nitric oxide synthase(NOS),and there is also no available data about the effects of eNOS and/or iNOS in remifentanil -induced cardioprotection.This experiment was designed to investigate the relation between delayed cardioprotection of remifentanil and NO,and the role of eNOS and/or iNOS in the remifentanil-induced delayed cardioprotection.Our purpose was to further clarity the mechanism of delayed cardioprotection of remifentanil.This study consists of the following three parts:Part One:Study of Delayed Cardioprotection of Remifentanil against Ischemia-Reperfusion InjuryThe healthy male Wistar rats weighing 250—350 g were randomly divided into 7 groups as follows:In group 1,rats received pretreatment with normal saline via trail vein according to the regime of 3×5-min intravenous(Ⅳ) infusion at a rate of 0.1 ml/kg/min 24 h before I/R process.In group 2,rats received pretreatment with normal saline according to the regime of 3×5-minⅣinfusion at a rate of 0.1 ml/kg/min 24 h before I/R process,and naloxone 0.1 mg/kgⅣ10 min before preconditioning with normal saline.In group 3,rats received pretreatment with remifentanilⅣinfusion 12 h before I/R process.In group 4,rats received pretreatment with remifentanilⅣinfusion 24 h before I/R process.In group 5,rats received pretreatment with remifentanilⅣinfusion 48 h before I/R process.In group 6,rats received pretreatment with remifentanilⅣinfusion 72 h before I/R process.In group 7,rats received pretreatment with remifentanilⅣinfusion 24 h before I/R process,and naloxone (0.1 mg/kgⅣ) 10 min before preconditioning with remifentanil.Remifentanil solutions at concentration of 20μg/ml was prepared in normal saline and the regime of remifentanil preconditioning was three 5-min cycles of infusion of remifentanil at a rate of 2μg/kg/min(equivalent to 0.1 ml/kg/min) using an infusion pump,interspersed with 5-min drug-free periods.All animals were subjected to a 30-min occlusion of the left anterior descending coronary artery (LAD) followed by a 120-min reperfusion in vivo.Six rates,which fited the experimental requirements,must at least be included in each group.Heart rate(HR),mean arterial pressure(MAP),and a leadⅡelectrocardiogram were continuously monitored during ischemia-reperfusion process.To determine plasma concentration of CK-MB,arterial blood samples were obtained immediately before ischemia,and at the end of ischemia and reperfusion.After a reperfusion periodof 120 min,heart was removed for myocardial infarct size measurement. Infarct size was expressed as a percentage of the area at risk.The results showed no significant differences among all groups in the baselines of HR,MAP,rate-pressure product(RPP) and plasma concentration of CK-MB before ischemia(P＞0.05).There were also no significant differences among all groups in HR,MAP and RPP at every measurement point during ischemia-reperfusion(P＞0.05).As compared to group 1,plasma concentrations of CK-MB at the end of ischemia and reperfusion and myocardial infarct size were not significantly different in groups 2,3,6 and 7(P＞0.05).The plasma concentrations of CK-MB at the end of ischemia and reperfusion and myocardial infarct size were significantly less in groups 4 and 5 than in group 1(P＜0.05). The plasma concentrations of CK-MB at the end of ischemia and reperfusion and myocardial infarct size were significantly less in groups 4 and 5 than in group 3 and 6(P＜0.05).There was no significant difference between groups 4 and 5 in plasma concentrations of CK-MB and myocardial infarct size(P＞0.05). Part Two:Influence of Remifentanil on Myocardial Endogenous NO systemThe healthy male Wistar rats were randomly assigned into 2 groups:In group 1,rats received normal saline via trail vein according to the regime of 3×5-minⅣinfusion at a rate of 0.1 ml/kg/min 12 h,24 h,48 h and 72 h,respectively, before the heart was removed.In group 2,rats received remifentanil via trail Vein according to the regime of 3×5-minⅣinfusion at a rate of 2μg/kg/min 12h,24h,48h and 72h,respectively,before the heart was removed.The heart was removed after pretreatment to measure the NO metabolic product in the myocardium supplied by LAD using a electrochemical microsensor method,and quantify the expression of eNOS and iNOS using the Western blot techniques. At every measurement point,6 rats were used.In addition,6 other rat hearts were removed as normal control.The electrochemical microsensor method showed that the myocardial contents of NO metabolic product at 24 h and 48 h after remifentanil pretreatment were significantly higher in group 2 than in group 1(P＜0.05).But there was not significant difference between group 1 and 2 in the myocardial content of NO metabolic product at 12h and 72h after remifentanil pretreatment(P＞0.05). In group 2,the myocardial contents of NO metabolic product were significantly higher at 24h and 48h than at 12h and 72h.Western blot analysis showed that the myocardial expression of eNOS in the two groups did not change significantly compared with the normal value at all measurement points(P＞0.05). As compared to group 1,the myocardial expression of iNOS in group 2 increased significantly at 24 h and 48 h after remifentanil pretreatment(P＜0.05).The myocardial expression of iNOS in group 2 were also significantly higher at 24 h and 48h than at 12h and 72h(P＜0.05). Part Three:Roles of iNOS in Remifentanil-induced Delayed CardioprotectionThe healthy male Wistar rats were randomly assigned into 6 groups:In group 1,rats were pretreated with normal saline via trail vein according to the regime of 3×5-minⅣinfusion at a rate of 0.1 ml/kg/min 24 h before I/R process. In group 2,rats were pretreated with normal saline according to the regime of 3×5-minⅣinfusion at a rate of 0.1 ml/kg/min 24 h before I/R process and SMT(10 mg/kgⅣ) 10 min before occlusion of the LAD.In group 3,rats were pretreated with normal saline according to the regime of 3×5-minⅣinfusion at a rate of 0.1 ml/kg/min 24 h before I/R process,following by SMT 10 mg/kgⅣ.In group 4,rats were pretreated with remifentanil according to the regime of 3×5-minⅣinfusion at a rate of 2μg/kg/min 24 h before I/R process.In group 5,rats were pretreated with remifentanil according to the regime of 3×5-minⅣinfusion at a rate of 2μg/kg/min 24h before I/R process and SMT 10mg/kgⅣ10 min before occlusion of the LAD.In group 6,rats were pretreated with remifentanil according to the regime of 3×5-minⅣinfusion at a rate of 2μg/kg/min,following by SMT 10 mg/kgⅣ.All animals were subjected to a 30-min occlusion of the LAD followed by a 120-min reperfusion in vivo.HR, MAP,and a leadⅡelectrocardiogram were continuously monitored during ischemia-reperfusion process.To determine plasma concentration of CK-MB, arterial blood samples were obtained immediately before ischemia,and at the end of ischemia and reperfusion.After a reperfusion period of 120 min,heart was removed for myocardial infarct size measurement.Infarct size was expressed as a percentage of the area at risk.The results showed no significant differences among all groups in the baselines of HR,MAP,RPP and plasma concentrations of CK-MB before ischemia (P＞0.05).There were also no significant differences among all groups in HR, MAP and RPP at every measurement point during ischemia-reperfusion(P＞0.05). As compared to group 1,plasma concentrations of CK-MB at the end of ischemia-reperfusion in groups 2,3 and 5 were not significantly different, the plasma concentrations of CK-MB at the ends of ischemia and reperfusion and myocardial infarct size were significantly less in groups 4 and 6(P＜0.05). As compared to group 4,plasma concentration of CK-MB at the end of ischemia and reperfusion in group 5 were significantly higher(P＜0.05),plasma concentrations of CK-MB at the end of ischemia and reperfusion in groups 6 were not significantly different(P＞0.05).On the basis of the results from this experiment,the following conclusions can be drawn:1.Pretreatment with remifentanil induces delayed cardioprotection,which occurs in 24 to 48h after pretreatment,and opioid receptors possess an indispensable role in mediating remifentanil-induced delayed cardioprotection.2.Pretreatment with remifentanil can significantly improve production of NO and myocardial expression of iNOS,but does not significantly affect the myocardial expression of eNOS.3.The effect of remifentanil preconditioning against myocardial ischemic injury is relevant to NO,and iNOS is an effector but not a trigger in mediating remifentanil-induced delayed cardioprotection.
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