| Objective With the improvement of surgical techniques and the aging of the population,cardiac surgery under cardiopulmonary bypass（CPB）has become the main method to treat heart diseases,such as valvular heart disease,coronary atherosclerotic heart disease,and large blood vessel replacement,etc.However,during cardiac surgery under CPB,myocardial ischemia/reperfusion injury（I/RI）is inevitable,which seriously affects the perioperative survival rate and prognosis of patients.Therefore,it is of great significance to find an effective myocardial protection method to reduce I/RI after CPB for improving the prognosis of patients undergoing cardiac surgery during perioperative period.Morphine,as a classic opioid analgesic drug,is widely used in perioperative analgesia and sedation.Clinical and basic studies have also confirmed that morphine preconditioning can protect I/RI not only in normal hearts,but also in some pathological hearts.However,the specific mechanism of action remains unclear.δ-opioid receptor is one of the main targets of morphine,which is involved in the transduction cascade of multiple signaling proteins in cells.Caspase-3,as a key regulatory protein of apoptosis,is involved in the occurrence and development of myocardial injury after reperfusion.Whether morphine preconditioning can alleviate global I/RI throughδ-opioid receptor mediated intracellular caspase-3 apoptotic regulatory proteins remains unclear.This study aims to establish a CPB model of global ischemia/reperfusion injury in isolated rats,and explore the role ofδ-opioid receptor in reducing global I/RI during morphine preconditioning and regulating caspase-3 protein by detecting myocardial enzymology,pathology and apoptotic protein.Methods1.Preparation of global ischemia/reperfusion injury modelMale healthy adult SD rats were killed by decapitating,and their hearts were immediately removed,non-cardiac tissues were removed,and the hearts were suspended on the Langendorff perfusion frame.K-H solution was reverse-injected at constant temperature and constant pressure with constant current.95%O2and 5%CO2mixture was used and p H value was maintained at 7.35-7.45.Power Lab pressure sensing system was switched on,and the left ventricular end-diastolic pressure was maintained between 0 and 10 mm Hg for 15 minutes.If severe arrhythmias or left ventricular developing pressure（LVDP）<70 mm Hg,it should be abandoned.LVDP,heart rate（HR）and the maximum rate of increase/decrease of left ventricular pressure（+dp/dt）were recorded.The isolated global heart I/RI model was prepared by using the clipped perfusion catheter for global ischemia for 30 minutes,and then the perfusion was resumed for 120 minutes.2.Grouping and ProcessingEighty-five male rats with a body weight of 220-260 g were randomly divided into five groups:control group（Sham group）,global cardiac ischemia reperfusion group（IR group）,morphine preconditioning group（1μmol/L,MPC group）,morphine+δ-opioid receptor antagonist（2 mg/L NTD,MNTD group）andδ-opioid receptor antagonist（2mg/L,NTD group）.The Sham group was continuously given K-H solution to the heart until the end of the experiment.In IR group,global heart I/RI model was prepared after55 minutes K-H infusion.MPC group was treated with 1μmol/L morphine hydrochloride and K-H solution for 5 minutes each for 3 cycles after cardiac stabilization for 25 minutes,and I/RI model was prepared.MNTD group was infused with 2 mg/L NTD 10 minutes before MPC until 5 minutes after ischemia,and I/RI model was prepared.In the NTD group,2 mg/L NTD was perfused for 45 minutes after cardiac stabilization for 15 minutes,and I/RI model was prepared.3.Determination of lactate dehydrogenaseThis experiment was divided into three time points:10,60 and 120 minutes of reperfusion.Four times of coronary artery effusion were collected at the time points of15 minutes of cardiac stabilization and three times after reperfusion,respectively,and lactate dehydrogenase（LDH）was measured.4.Detection of Caspase-3 protein and cell apoptosisThe myocardial tissue was collected at three time points of 10,60 and 120 minutes after cardiac reperfusion.The protein expression of Casepase-3 andβ-actin was detected by Western blot and the apoptosis of myocardial cells was determined by TUNEL staining.5.Detection of Caspase-3 protein and cell apoptosisAt the end of 120 minutes of reperfusion,the heart was taken for triphenyltetrazole chloride（TTC）staining to detect the myocardial ischemia Area（IS）,ischemia risk Area（ARR）and the ratio of IS/ARR.Results1.Evaluation of global ischemia-reperfusion injury modelCompared with the Sham group,TTC staining and myocardial enzymology tests after reperfusion showed that myocardial infarction volume IS and IS/AAR ratio were increased in the IR group,and LDH activity,Caspase-3 protein expression and apoptosis rate of coronary outflow fluid were significantly increased at each time point,suggesting that the isolated global heart I/RI model was established successfully.2.Morphine preconditioning decreased the expression of Caspase-3 protein and apoptosis at different time points after reperfusion,and exerted a protective effect on myocardiumCompared with IR group,morphine pretreatment significantly decreased myocardial infarction volume IS and the percentage of IS/AAR and LDH activity level at each time point after reperfusion.The expression of Caspase-3 protein in myocardial cells was reversed at 10,60 and 120 minutes after ischemia reperfusion in isolated rats,and the apoptosis rate of myocardial cells decreased（P<0.05）.The LDH level was the highest 10 min after reperfusion,the relative expression of Caspase-3 protein was the highest at 60 minutes after reperfusion,and TUNEL staining indicated that the apoptosis rate was the highest at 120 minutes after reperfusion（P<0.05）.3.δ-opioid receptor blocking morphine preconditioning decreased Caspase-3expression and apoptosis,blocking its myocardial protective effectCompared with IR group,MNTD group and NTD group showed no statistically significant differences in LDH active caspase-3 protein relative expression and apoptosis rate in myocardial infarction area.Compared with the MPC group,NTD significantly reversed the reduction of myocardial infarction volume and the relative expression of apoptosis protein Caspase-3 and the increase of apoptosis ratio at each time point of reperfusion（P<0.05）.Conclusion Morphine preconditioning can inhibit the expression of caspase-3protein during early reperfusion by activating theδ-opioid receptor,reduce the rate of myocardial injury and apoptosis after ischemia/reperfusion,and reduce the scope of myocardial infarction to play a protective role in myocardium. |
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