| Background global myocardial ischemia reperfusion injury often occurs in the cardiac or vascular surgeries under cardiopulmonary bypass,and cardiopulmonary resuscitation after heart attack,myocardial injury will be aggravated when the ischemic myocardium restore the blood supply,which is life threatened to patients.Many research suggest that ischemia preconditioning is able to significantly reduce this ischemia reperfusion injury,while whether morphine preconditioning can also confer this protection effect and its mechanism against global IRI remain unclear.Cardiomyocytes death is the common characteristics of heart disease,including necrosis,apoptosis and autophagy.The necrosis induced cell death is much more than that caused by apoptosis during myocardial ischemia reperfusion.LDH is also an indicating index for myocardial injury.Necroptosis is a new fashion of cell death,which has a similar cell morphology with that of necrosis while can be regulated by signaling pathway as apoptosis.Recent study showed that necroptosis plays an important role in the IRI,and receptor interacting protein is a key factor in necroptosis.Extracellular signal-regulated kinase is an important pro-survival kinase which can promote the cardiomyocytes survival in the early stage of reperfusion in activated fashion to decrease the myocardial infarct size.Previous study in our lab showed that MPC reduce the IRI via activating ERK signaling pathway,and the present study was designed to further investigate whether MPC-activated ERK pathway is able to affect the expression of RIP(RIP1 and RIP3),and in turn confer a cardio-protective effect in the global IRIMethod1.Groups and global IRI establishment Adult male Sprague-Dawley rats,weighing 200-250 g,were randomly distributed into 8 groups(n=10 each)using a random number table: control group(group CON),ischemia-reperfusion group(group I/R),ischemia preconditioning group(group IPC),morphine preconditioning group at three concentration levels(0.3 mmol/L,1 mmol/L and 3 mmol/L),MPC+ERK inhibitor PD98059 group(group MPD),PD98059 alone group(group PD).The hearts in each group were perfused in the Langendorff apparatus and subjected to 40 min of global ischemia followed by 90 min of reperfusion to establish the I/R injury model except for the group CON.In group I/R,the hearts were perfused with K-H solution for 45 min before ischemia.In group IPC,the hearts were perfused with K-H solution for 15 min followed by 5 min of ischemia and 5 min of normal perfusion for 3 cycles in total before the prolonged ischemia.In the three groups of MPC,the hearts were perfused with K-H solution containing morphine at three concentration levels of 0.3,1 and 3 mmol/L,respectively,for 5 min and then intermitted with K-H solution for 5 min(3 cycles in total)before ischemia;In addition,in group MPD,the hearts were perfused with PD-contained K-H solution 10 min before MPC until the start of ischemia while the hearts in group PD were perfused with PD-contained K-H solution for 40 min prior to the prolonged ischemia.At 15 min of equilibration(baseline)and 5 and 10 min of reperfusion,the coronary effluent was collected to detect the activity of lactate dehydrogenase(LDH).A water-filled balloon was inserted into the left ventricular through the left atrium,the heart rate(HR)and left ventricular developed pressure(LVDP)were recorded continuously during the whole perfusion.2.Study of the role of ERK signaling pathway regulating RIP in MPC induced cardio-protection the coronary effluent was collected for detection of LDH activity,the heart was removed to extract protein at 10 min of reperfusion,the level of ERK protein and RIP in the heart were measured by Western blot(n=4),at the end of reperfusion,the infarct size(IS)and the area at risk(AAR)as well as the ratio of IS/AAR were calculated using TTC staining(n=6).Results1.Successful establishment of global IRI the HR and LVDP as well as the coronary effluent in the I/R group were significantly reduced comparing with that in CON group(P<0.05),whereas the IS and the ratio of IS/AAR of I/R group were markedly increased as compared to the CON group.2.Mechanism of ERK pathway regulating RIP in MPC-induced cardio-protection against global IRI2.1 Effect of MPC at 3 levels of concentration on the global IRI the global IRI was significantly reduced by MPC at 0.3 and 1 Um as evidenced by the decreased infarct size and LDH activity after reperfusion,as well as the improved cardiac function.However,MPC at 3 mM did not confer a protective effect against the global IRI.2.2 Effect of MPC on the phosphorylated ERK and RIP the phosphorylation of ERK was up-regulated while the level of RIP was inhibited by MPC at 0.3 and 1 mM,however,there was no significant change in the level of phosphorylated ERK and RIP in the MPC at 3 mM.2.3 Effect of PD98059 on MPC induced cardioprotection against global IRI Treatment with PD98059 alleviated the MPC induced protective effect on the global IRI as indicated by the increased infarct size and the elevated LDH activityafter reperfusion,as well as the depressed LVDP during the reperfusion period,which may suggest MPC confer this protective effect against global IRI via activating ERK signaling pathway.2.4 Effect of PD98059 on the MPC induced phosphorylation of ERK protein and expression of RIP PD98059 inhibited the MPC-induced phosphorylation of ERK,disinhibited the depressed expression of RIP.Conclusion The protection of MPC against global IRI may be mediated by inhibition of RIP via ERK signaling pathway... |
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