| Objective:To investigate the effects of endogenous N/OFQ receptors on ventricular arrhythmia and heart rate variability in early?15min?acute myocardial ischemia rats.And to investigate whether endogenous N/OFQ receptors?using specific N/OFQ receptor antagonist UFP-101?in antagonistic rats can alter the phosphorylation level of RKIP in ischemic tissue of early acute myocardial ischemia rats.To investigate whether endogenous N/OFQ receptors indirectly affect the phosphorylation level of RKIP to regulate?1-AR on the surface of myocardial membrane in the ischemic area of rats with acute myocardial ischemia.Method:Part ?:Antagonistic effects of endogenous N/OFQ receptors on arrhythmia in early?15min?myocardial ischemia rats.30 adult male SD rats with a weight of about 250g were divided into three groups with an average of 10 rats in each group according to the random number table method,respectively in the Sham group?Sham operation group,the rats only had thoracotomy without coronary artery ligation?.CAO group?coronary artery ligation group,in which rats were thoracotomy and coronary artery ligation?;Group UFP-101(thoracotomy and coronary artery ligation,concentration of 10-9mol/L UFP-101 was given via caudal vein before operation with a load of 1ml/kg).The corresponding models were established in each group,and data of ventricular arrhythmia and heart rate variability were observed and recorded from 10min before surgery to 15min after myocardial ischemia.Part ?:Antagonizing the regulation of endogenous N/OFQ receptors on the phosphorylation of RKIP in ischemic tissue of early acute myocardial ischemia ratsIn the first part of the experiment,rat myocardial tissue in the ischemic area was taken 15 minutes after surgery,protein tissue was extracted and BCA protein was quantified,and expressions of phosphorylated RKIP?pRKIP?and total RKIP in rat myocardial tissue in each group were detected by Western-Blot.Part ?:Establishment of RKIP up-regulated rat model in myocardial tissue and occurrence of ventricular arrhythmias30 male SD rats,weighing 100-150mg,were divided into three groups of 10 rats in each group according to the random number table method on average,which were respectively the RKIP up-regulated group?Did group for short?.RKIP up-regulated+RKIP inhibition group?Did+Loc group?;control check group?CK group?.Among them,the rats in the Did group and the Did+LOC group were injected with RKIP non-specific up regulating didymin?corn oil soluble?every day with a load of 5mg/kg×d,while the rats in the CK group were injected with equal volume corn oil every day for 4 weeks.4 weeks later,the rats in each group underwent coronary artery ligation,in which the rats in the Did+Loc group were introperitoneally injected with the RKIP specific antagonist Locostatin?corn oil soluble?2 hours before surgery.All rats in each group were injected with UFP-101(10-9mol/L,1ml/Kg)by tail vein10min before surgery,and the data of arrhythmia in each group were recorded 10min before operation and 15min after operation.The ventricular muscle tissues and myocardial tissues in the same part of ventricular muscle and the ischemic area of each group were taken 15 minutes after the operation,and the expression of total RKIP and pRKIP in the myocardial tissues in the same part of each group was detected,so as to verify the success of model making in each group.Part ?:To investigate whether the up-regulation of RKIP in rat myocardial tissue affects the expression of?1-AR in the myocardial membrane surfaceThe myocardial tissue in the ischemic area of each group of rats in the third part was taken to extract plasma membrane surface proteins,and the expression of?1-AR on the plasma membrane surface of each group was detected by Western-Blot.Results:1.Antagonized the effects of endogenous N/OFQ receptors on arrhythmia in early?15min?myocardial ischemia ratsThe Sham group,the UFP-101 group and the CAO group all presented different degrees of ventricular arrhythmia.Compared with Sham group,the incidence and duration of premature ventricular beats?VEB?,ventricular tachycardia?VT?and ventricular fibrillation?VF?as well as the arrhythmia score were significantly increased in CAO group?P<0.05 or P<0.017?.Compared with CAO group,the frequency of VEB occurrence,the frequency and duration of VT+VF and arrhythmia score in UFP-101 group decreased significantly?P<0.05 or P<0.017?.2.Antagonized the regulation of endogenous N/OFQ receptors on the phosphorylation of RKIP in the ischemic area of early acute myocardial ischemia ratsCompared with Sham group,the relative expression of pRKIP in CAO group was significantly increased?P<0.05?,while the relative expression of pRKIP in UFP-101 group was decreased?P<0.05?.There was no significant difference in the expression of total RKIP?no statistical significance?in the ischemic area in the three groups.3.Establishment of RKIP up-regulated rat model in myocardial tissue and occurrence of ventricular arrhythmiasCompared with the CK group,the number of VEB occurrence in the Did group and the Did+Loc group significantly increased?P<0.05?,and the number of VEB occurrence in the Did+Loc group significantly decreased?P<0.05?.Compared with CK group,the occurrence frequency and duration of VT+VF in the Did group were significantly increased?P<0.017?,and there was no significant statistical difference in the Did+Loc group.Compared with the Did group,there was also no significant statistical difference in the occurrence frequency and duration of VT+VF in the Did+Loc group.Compared with CK group,arrhythmia score in Did group increased?P<0.017?,and there was no significant statistical difference in arrhythmia score in Did+Loc group.Compared with Did group,arrhythmia score in Did+Loc group decreased?P<0.017?.Compared with CK group,the relative expression of total RKIP in the Did+Loc group was significantly increased in the Did group?P<0.05?,and there was no statistical difference in the total RKIP expression between the Did group and the Did+Loc group.Compared with CK group,the relative expression of p-RKIP in Did group and Did+Loc group was significantly increased?p<0.05?,and the relative expression of pRKIP in Did+Loc group was decreased compared with Did group?p<0.05?.4.To investigate whether the up-regulation of RKIP in rat myocardial tissue affects the expression of?1-AR in the myocardial membrane surfaceCompared with CK group,the expression of?1-AR on the myocardial cell membrane surface in the ischemic area was significantly up-regulated in the Did group and the Did+Loc group?P<0.05?.However,compared with the Did group,the expression of?1-AR on the myocardial membrane surface of rats in the Did+loc group was down-regulated?P<0.05?.Conclusion:Antagonized endogenous N/OFQ receptors to reduce ventricular arrhythmias induced by acute myocardial ischemia in rats,affected the phosphorylation level of RKIP in ischemic myocardial tissues and may indirectly affect the expression level of?1-AR on the cell membrane surface of ischemic myocardium by regulating the phosphorylation level of RKIP. |
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