Role Of P38 MAPK Signaling Pathway In Morphine Preconditioning-Mediated Cardioprotection In Rats With Heart Failure And Its Mechanisms

Background and Objective Ischemia-reperfusion injury(IRI)probably occurs in the patients with heart failure undergoing cardiac surgery,which makes great threatens to anesthesia and patients’ life.However,ischemia preconditioning(IPC)does not appear to work to deliver protective effect against myocardial IRI in the patients with hyperglycemia,hyperlipidemia and hypertension as well as heart failure.Thus,it is of vital importance to investigate the effect of morphine precondition(MPC)on failing heart and the possible signaling mechanism.Our previous studies suggested that MPC reduced IRI in the intact and the isolated failing heart,while the mechanism is still unknown.p38 signaling pathway is a member of the mitogen activated protein kinase(MAPK),the role of activation of this signaling pathway in myocardial IRI still remains controversial.Thus,the present study aims to investigate the role of p38 pathway in morphine preconditioning-mediated cardioprotection against ischemia reperfusion injury in rats with heart failure and its mechanism.Methods Adult male Sprague-Dawley rats,weighing 200~240 g,were injected with doxorubicin 2 mg/kg via the tail vein once a week for 6 consecutive weeks,12 mg/kg in total,to induce chronic heart failure.At the end of 8th week,30 rats with chronic heart failure were randomly divided into 5 groups(n=10 each)using a random number table:sham operation group(group Sham),ischemia reperfusion group(IR group),morphine preconditioning group(group MPC),SB203580(p38 MAPK inhibitor)+ morphine preconditioning group(group MSB),and SB203580 group(group SB).The heartswere quickly excised and passively perfused on a Langendorff apparatus and the left anterior descending coronary artery(LAD)was ligated to establish the model of I/R model.All rat hearts except for the Sham group were subjected to LAD ligation to induce 30 min ischemia followed by 120 min reperfusion.MPC was performed through5 min infusion of 1 mM morphine hydrochloride and 5 min drug-free infusion,three cycles in total before I/R.SB203580 was perfused for 10 min before MPC until 5 min after the end of MPC followed by I/R in the MSB group.In SB group,the inhibitor was perfused alone for 40 min until the start of ischemia without MPC.The coronary effluent were collected at the end of equilibrium,5 and 10 min of reperfusion,respectively,to detect the level of lactate dehydrogenase(LDH).Myocardial infarct size(IS)was measured as the percentage of area at risk(IS/AAR)by TTC staining after the end of reperfusion.In addition,at 10 min of reperfusion,the ventricular tissue was acquired for assessing the expression of p-p38 and p38 in myocardium by Western blot.In another series experiments,the primary myocardial cells of SD rats were isolated and cultured.After 24 h of doxorubicin treatment,they were divided into 5 groups(n=4each): control group(group CON),hypoxia and re-oxygenation group(group H/R),MPC group,MSB group and SB group.The cardiomyocytes in all groups were incubated in hypoxia circumstances for 4 h in a hypoxia chamber flushed with hypoxic gas mixture of 5% carbon dioxide and 95% nitrogen followed by 2 h normoxia incubation to induce hypoxia re-oxygenation(H/R)injury,except that the cells in control group were cultured normally.The cells in MPC group were pretreated with morphine(1 mM)for 10 min followed by 30 min drug-free incubation before H/R injury.SB203580(5 mmol/L)was added in the culture medium 10 min before MPC in the MSB group.In SB group,the inhibitor was added alone 20 min prior to hypoxia without MPC.Cell viability was detected by CCK-8 and the activity of lactate dehydrogenase(LDH)was measured by microplate method while cell apoptosis was assessed by Annexin-V-FITC/PI flow cytometry,in addition,the expression of p-p38/p38,p-GSK-3β/GSK-3β,p-Cx43/Cx43 in cardiomyocytes were observed by western blot,and the expression of Bax,Bcl-2 mRNA were detected by qRT-PCR.Results In isolated failure rat hearts,LDH activity was raised during reperfusion,and IS/AAR was increased following myocardial IR injury,MPC protected the failure hearts against IR injury indicated by the significant decrease in the LDH activity and the IS/AAR ratio,up-regulation of the expression of p-p38,while these protective effects were blocked by SB203580.In doxorubicin-treated cardiomyocytes,HR decreased the cell vitality and elevated the LDH activity as well as the cardiomyocyte apoptosis,while these H/R-induced injuries were attenuated by MPC evidenced by the promotion in the cell viability,and the repression in the LDH activity as well as the apoptosis rate in cardiomyocytes.However,the MPC-mediated protection on cardiomyocytes were inhibited by SB.What’s more,the MPC-conferred up-regulation in the expressions of p-p38,p-GSK-3β and p-Cx43,and the ratio of Bcl-2/Bax were all suppressed by SB203580.Conclusion The activation of p38 MAPK signaling pathway may be involved in morphine preconditioning-mediated cardioprotection against myocardial ischemia-reperfusion injury via regulating the expressions of the downstream signaling and apoptosis gene in rats with heart failure.