Role Of ERK Signaling Pathway And MicroRNA-133b-5p In Morphine Preconditioning Induced Cardioprotection Against Ischemia Reperfusion Injury In Rats With Heart Failure

Background and Objective Heart failure(HF)patients are prone to perioperative myocardial ischemia-reperfusion injury when undergoing cardiac or non-cardiac surgery.It is a difficult problem that the clinical anesthesiologist needs to face.Our recent study has found that the activation of extracellular signal-regulated kinase(ERK)signaling pathway plays an important role in morphine preconditioning(MPC)to alleviate myocardial ischemia-reperfusion(I/R)injury in rats with heart failure.We found that the down-regulation of miR-133b-5p was the most obvious in heart failure cardiomyocytes compared with normal cardiomyocytes,and this down-regulation could be antagonized by MPC.Fas is a key target gene of miR-133b-5p and MPC attenuates myocardial I/R injury,which may be due to the inhibition of Fas mediated cardiomyocyte apoptosis through up-regulation of miR-133b-5p.The receptor-interacting protein(RIP)is an important regulator of cellular stress,which interacting with death domain receptor Fas and regulating cardiomyocyte apoptosis and necrosis.In view of the above research background,this study sought to investigate the interaction between ERK pathway and miR-133b-5p targeting regulation of Fas and RIP signals,and the mechanism of MPC in alleviating myocardial I/R injury in rats with heart failure,which providing a new theoretical basis for the study of MPC in myocardial protection in rats with heart failure.Method1.In vivo level Adult male Sprague-Dawley(SD)rats weighing 200-230 g,were injected with 2 mg/kg doxorubicin(DOX)via tail vein,once a week,over a period of 6weeks for a total cumulative dose of 12 mg/kg to establish chronic heart failure(CHF)model.At the end of 8th week,50 rats with CHF were randomly divided into 5 groups(n=10 each): sham operation group(group Sham),ischemia reperfusion group(group I/R),morphine preconditioning group(group MPC),ERK inhbitor PD98059+morphine preconditioning group(group MPD)and PD98059 group(group PD).Myocardial I/R was induced by 30 min occlusion of left anterior descending coronary artery(LAD)followed by 120 min reperfusion in each group except group Sham.In group MPC,the rats were subjected to 3 cycles of 5-min infusion of 0.1 mg/kg morphine via the femoral vein at 5 min interval before isehemia.In MPD group,PD98059 0.5 mg/kg were injected via the femoral vein,respectively,at 10 min before morphine preconditioning.LDH activity was detected in each group at baseline,5 min and 10 min after reperfusion.The animals were sacrificed at 120 min of reperfusion,and the myocardial specimens were obtained for determination of the left ventricular and right ventricular(LV+RV),area at risk(AAR),infarct size(IS),and IS/AAR ratio was calculated.The expression of miR-133b-5p and Fas mRNA were detected by qRT-PCR,in addition,the expression of Fas,ERK,GSK-3? and RIP1/RIP3 protein were detected by western blot.2.In vitro level Primary cardiomyocytes were pretreated with doxorubicin for 24 h and then the cells were randomly divided into 7 groups(n=4 wells each): control group(group Control);hypoxia reoxygenation group(group H/R);morphine preconditioning group(group MPC);mi R-133b-5p agomir+hypoxia/reoxygenation group(group agomiR-133b+H/R);miR-133b-5p agomir-NC+hypoxia/reoxygenation group(group agomiR-NC+H/R);miR-133b-5p antagomir+MPC+H/R(group ant-miR-133b+MPC);miR-133b-5p negative control +MPC+H/R(group ant-mi R-NC+MPC).All groups of cells were subjected to 4 h hypoxia followed by 2 h reoxygenation except control group.The cells were preconditioned with morphine with the final concentration of 1 mmol/L for 10 min before H/R in MPC group.The last four groups were transfected with miR-133b-5p agomir/agomir-NC(final concentration of 50 nmol/L)or miR-133b-5p antagomir/antagomir-NC(final concentration of 100 nmol/L),respectively.After 24 h transfection,the cells were subjected to H/R injury with or without MPC.Cell viability was measured by the cell counting kit-8 assay(CCK-8)and cell injury was detected by LDH kit.Cell apoptosis and necrosis were assessed by FITC Annexin V/PI flow cytometry.The expression of miR-133b-5p and Fas mRNA were detected by qRT-PCR,in addition,the expression of Fas,ERK,GSK-3? and RIP1/RIP3 protein were detected by western blot.Result1.In vivo level There was no significant difference in LV+RV and AAR between the five groups(P>0.05).Compared with group I/R,IS and IS/AAR were significantly decreased,the expression of mi R-133b-5p,p-ERK1/2 and p-GSK-3? protein were significantly increased,while Fas m RNA/protein and RIP1/3 protein expression were dramatically decreased in group MPC(P<0.05).Compared with group MPC,IS and IS/AAR were significantly increased,the expression of miR-133b-5p,p-ERK1/2 and p-GSK-3? protein were significantly decreased,while Fas mRNA/protein and RIP1/3protein expression were dramatically increased in group MPD(P<0.05).2.In vitro level Compared with H/R group,the cell viability,the expression of miR-133b-5p,p-ERK1/2 and p-GSK-3? protein were significantly increased,while the LDH activity,apoptotic rate,necrosis rate,Fas mRNA/protein and RIP1/3 expression were dramatically decreased in group agomiR-133b+H/R and MPC(P<0.05),and no significant change was found in the parameters mentioned above in group agomiR-NC+H/R(P>0.05).Compared with MPC group,the cell viability,theexpression of miR-133b-5p,p-ERK1/2 and p-GSK-3? protein were significantly decreased,while the LDH activity,apoptotic rate,necrosis rate,Fas mRNA/protein and RIP1/3 protein expression were dramatically increased in group ant-miR-133b+MPC(P<0.05),and no significant change was found in the parameters mentioned above in group ant-miR-NC+MPC(P>0.05).Conclusion Morphine preconditioning can attenuate myocardial ischemia-reperfusion injury in rats with heart failure,which may be related to ERK signaling pathway and miR-133b-5p targeted Fas,and there is an interaction between them,acting on downstream RIP signal.