Effects Of Propofol On Cardiac TRPV1 In Remote Preconditioning Induced Cardioprotection

Background Remote conditioning(RIC)is a highly effective method for myocardial protection.A large number of basic studies have found that RIC can reduce the area of myocardial infarction and improve prognosis,and can also protect many important organs.Remote preconditioning mainly includes: remote ischemic preconditioning(RIPC)and remote preconditioning of trauma(RPCT).As a new protective measure of myocardium,its mechanism mainly involves nerve and humoral.A number of laboratory studies have clearly demonstrated its myocardial protective effect.However,two recent multi-center large sample clinical studies found that the myocardial protective effect of RIPC was cancelled in patients undergoing elective heart surgery,leading to the interruption of research from basic research to clinical research.Subsequent re-analysis and study of these two studies found that more than 90% of patients use propofol intraoperatively,which may be a key factor leading to the cancellation of the protective effect of RIPC.Therefore,it is necessary to study the role of propofol in the myocardial protection of remote ischemic preconditioning.Transient receptor potential vanilloid type 1(TRPV1)is a non-selective cationic channel that plays a vital role in sensory transmission and regulation of injurious stimuli and plays a vital role in ischemia reperfusion injury of many important organs.In myocardial tissue and spinal cord dorsal root ganglia(DRG)in a large number of distribution,when it can be activated after synthesis and secretion of sensory neuropeptides,such as calcitonin gene related peptide(CGRP)and substance P(SP)participation maintain normal physiological function of the heart,and in the process of myocardial ischemia reperfusion play an important protective role.Propofol,as the most commonly used intravenous anesthetic drug in clinical practice,can affect the activity of TRPV1 and may contribute to the peripheral sensitivity of traumatic tissues to harmful stimuli.Our research group has found that TRPV1 is a cardiac protective end-effector,regulating the interaction between TRPV1 protein and calmodulin can reduce reperfusion injury,and significantly reduce the myocardial infarction area of myocardial ischemia reperfusion injury in RPCT.Therefore,we hypothesized that propofol,by affecting the activity of TRPV1,resulted in decreased expression of TRPV1 and decreased CGRP in myocardium,thus eliminating the protective effect of RIC myocardium.The purpose of this study was to explore the following questions: 1.The role of cardiac TRPV1 channel protein in the treatment of myocardial ischemia reperfusion injury by RPCT;2.To investigate the effect and mechanism of propofol on cardiac protection by TRPV1 mediated RPCT;3.To investigate the effect and possible mechanism of propofol at different time points on myocardial protection of RIPCMethods: this study was divided into the following three aspects Part 1: To study the effect of cardiac TRPV1 on myocardial protection of RPCT Methods: Healthy male SD rats were randomly divided into SHAM surgery group(SHAM group),ischemia reperfusion group(CON group),remote preconditioning of trauma group(RPCT group),TRPV1 block-capsazepine group(CPZ group),capsazepine combined with RPCT group(CPZ+RPCT group),a total of 5 groups(n=9).In SHAM group,ischemia reperfusion injury was not performed.In CON group,the myocardial ischemia reperfusion injury model was reduced to 30 min before coronary artery ligation and 120 min after reperfusion.In the RPCT group,a 4-cm long wound incision was pretreated for 15 min in the lower abdomen,followed by ischemia reperfusion injury.In the CPZ group,capsazepine was injected with 3 mg/kg through flat vein for 10 min.In the CPZ+RPCT group,capsazepine was injected with 3 mg/kg through flat vein for 10 min,followed by abdominal incision pretreatment for 15 min,and then ischemia reperfusion injury.The rates of vital signs(systolic blood pressure mean arterial heart rate)and arrhythmias(ventricular tachycardia and ventricular fibrillation)were recorded during the experiment.The rats were sacrificed at the reperfusion time of 120 min,and myocardial tissues were taken.The myocardial infarction area was measured using the TTC method and the weight of myocardial infarction area(IS),left ventricular(LV)weight and area at risk(AAR)weight were measured,and the IS/AAR ratio and ARR/LV ratio were calculated.Immunofluorescence was used to observe the changes of TRPV1 expression in rat myocardium.Meanwhile,the expression of TRPV1 in myocardium was determined by Western blotting.The changes of serum troponin(c Tn I)in peripheral blood and Caspase-3 activity in myocardium containing cysteine were determined by ELISA.TUNEL assay was used to detect the changes of apoptotic cells in myocardium Part 2: To observe the effect of propofol on myocardial protection of RPCT and the possible mechanism.Methods: healthy male SD rats were randomly divided into: ischemia reperfusion group(CON group),remote preconditioning of trauma group(RPCT group)pretreatment group,propofol group(PRO),RPCT propofol group(PRO + RPCT group),a total of four groups(n = 9).CON group and RCPT method with the first part of the solution.PRO group,propofol 12 mg/kg/h for 10 min after intravenous infusion,ischemia-reperfusion injury.PRO+RPCT group,propofol 12 mg/kg/h intravenous infusion for 10 min,abdominal incision preconditioning for 15 min,and ischemia reperfusion injury.The incidence of hemodynamics and arrhythmias in each group were recorded.At the time of reperfusion for 120 min,myocardial tissue was taken from the rats.The infarction area was detected by TTC method and the weight of myocardial infarction area,left ventricular weight and ischemic risk area were measured.The location and distribution of TRPV1 and CGRP in rat myocardium were observed by immunofluorescence double standard method.Western blotting was used to determine the content of TRPV1 in myocardium,and ELISA was used to determine the content of CGRP in myocardium,changes in the expression of CGRP in serum,changes in the content of c Tn I in peripheral blood and the activity of Caspase-3 in myocardium,and serum lactate dehydrogenase(LDH)level was detected.Part 3: Effects of propofol on myocardial protection of RIPC Methods: healthy male SD rats were randomly divided into: ischemia reperfusion group(CON group),distal ischemic preconditioning group(RIPC group),propofol preconditioning combined with RIPC group(PIPC+P-pre group),and propofol post-treatment combined with RIPC group(RIPC+P-post group),a total of 4 groups(n=9).CON group,same as the first part.In the RIPC group,rats were subjected to 3cycles of ischemia for 5 min at the left lower extremity,remote preconditioning for 5min of reperfusion,and ischemia reperfusion injury model(same as the first part).In the RIPC+P-pre group,propofol 12 mg/kg/h was injected intravenously for 10 min,and then PIPC was performed.In the RIPC+P-post group,the model of ischemia reperfusion injury was established after the distal preconditioning of the left lower extremity,and propofol was injected intravenously at 12 mg/kg/h before reperfusion.The incidence of hemodynamics and arrhythmias in each group were recorded.The myocardial tissue was taken out after reperfusion,using the TTC method determination of myocardial infarction area and calculate the weight,left ventricular weight and ischemia of danger weight,observed by immunofluorescence TRPV1 expression in rat myocardial,at the same time Western-blotting determination of TRPV1 expression in myocardium,ELISA method for determining the content of peripheral blood c Tn I and Caspase-3 activityResults Part 1 Compared with SHAM group,the hemodynamic indicators and arrhythmias of CON group were significantly reduced(P<0.05)and the incidence of arrhythmias was significantly increased(P<0.05)at the time points of ischemia for 30 min and reperfusion for 120 min.Compared with CON group,HR MAP and RPP in the RPCT group were significantly increased(P<0.05)and the incidence of arrhythmia was significantly reduced(P<0.05)at the time of ischemia for 30 min and reperfusion for120 min,while no significant changes were observed in the RPCT+CPZ group and CPZ group(P>0.05).Compared with SHAM group,the weight of myocardial infarction area(TTC staining)and cell apoptosis rate(TUNEL staining)of CON group were significantly increased(P<0.05),while the content of troponin in peripheral blood and the activity of myocardial Caspase-3 were significantly increased(P<0.05),and the expression of TRPV1 in myocardial tissue was significantly increased(P<0.05).Myocardial infarction compared with CON group,RPCT group decreased obviously,and myocardial apoptosis significantly decreased(P < 0.05),peripheral blood levels of troponin and myocardial caspase 3 activity without significantly increased(P <0.05),CPZ and CPZ + RPCT group compared with CON group,each group in the area of myocardial infarction,myocardial apoptosis of peripheral blood troponin levels caspase 3 activity and the incidence of arrhythmia was changed(P > 0.05).3.Compared with CON group,the expression of TRPV1 in myocardium of RPCT group was increased by immunofluorescence and Western blotting(P<0.05).Part 2 Compared with CON group,HR MAP and RPP of PRO group and RPCT+PRO group showed no significant changes at the time of ischemia for 30 min and reperfusion for 120 min(P>0.05),and no significant changes in arrhythmia incidence(P>0.05).Compared with the RPCT group,the PRO group and the RPCT+PRO group significantly reduced HR MAP and RPP at the time of ischemia for30 min and reperfusion for 120 min(P<0.05),and the incidence of arrhythmia significantly increased.Compared with the RPCT group,the weight of myocardial infarction area was significantly increased in the PRO group and the PRO+RPCT group,and the content of troponin in peripheral blood and the activity of myocardial Caspase-3were significantly increased(P<0.05).The expression of TRPV1 and CGRP was observed by double-standard immunofluorescence.4.Changes in CGRP content the content of CGRP in myocardial tissue and serum was significantly increased in PRO group and RPCT+PRO group than in RPCT group(P<0.05)Part 3.Compared with CON group,HR MAP and RPP in RIPC group and RIPC+p-post group were significantly increased and the incidence of arrhythmia was significantly reduced at the time points of ischemia for 30 min and reperfusion for 120min(P<0.05),while HR MAP and RPP at each time point of RIPC+P-pre were not significantly changed(P>0.05).Compared with CON group,the weight of myocardial infarction area was significantly reduced in the RIPC group and the RIPC+P-post group,and the content of troponin in peripheral blood and the activity of myocardial Caspase-3were significantly reduced(P<0.05),while the weight of myocardial infarction area was infinitely changed in the RIPC+P-pre group(P>0.05),while the content of troponin in peripheral blood and the activity of Caspase-3 were not significantly changed(P>0.05).Compared with CON group,the expression of TRPV1 in RIPC group and RIPC+P-post group was significantly increased by immunofluorescence and Western blotting(P<0.05),while the expression of TRPV1 in RIPC+ p-pre group was not significantly changed(P>0.05).Conclusion RPCT has a protective effect on myocardial ischemia reperfusion injury,and its mechanism may be related to the involvement of cardiac TRPV1.Propofol,an intravenous anaesthesia,can cancel the myocardial protective effect of remote preconditioning(RPCT and RIPC),which may be related to changing the activity of cardiac TRPV1 and reducing the content of CGRP in the myocardium and serum.Before RIPC,continuous pumping of intravenous anesthetic propofol can abolish the myocardial protective effect of RIPC.However,immediately after RIPC reperfusion,the initiation of continuous pumping of intravenous anesthetic propofol did not affect the myocardial protective effect of RIPC.In summary,the effect of propofol on the expression of cardiac TRPV1 is the key mechanism for propofol to cancel the protective effect of remote preconditioning on myocardium.