| Background:Ischemia reperfusion injury?IRI?is one of the main reasons for the poor therapeutic effect of ischemic heart diseases and low cardiac output syndrome occurs after open cardiac operation,which may lead to bad prognosis and even death.Exogenous pharmacological intervention is a crucial strategy to counteract myocardial IRI.However,an ideal cardioprotective strategy has not been achieved as the specific mechanisms,source of drugs,side effects and ethical problems remain to be solved.Hence,searching human endogenous anti-IRI substances and studying its role of mechanism has become a hot spot of research in the cardiovascular system.Melatonin?MEL?is an indole hormone secreted by the pineal gland.Previous studies have found that melatonin plays an important role in regulating the rhythm of central nervous system;Recent Studies have found that MEL plays a concrete regulatory role in the physiological function and pathological processes of the cardiovascular system.MEL can also significantly fight against heart,brain,liver,kidney,skeletal muscle and other organs IRI without toxicity and mutagenicity of exogenous drugs,which has become a focus in the research of anti-IRI strategies.The lower extent of myocardial IRI after coronary artery bypass grafting?CABG?is found to be relevant to higher serum MEL levels.Our previous research has manifested that MEL is protective against myocardial IRI and vascular endothelial oxidative stress injury.However,the specific mechanism of MEL fighting against IRI has not been well elucidated,which limits the further development and widespread clinical application.Protein tyrosine kinase 2/signal transducer and activator 3?JAK2/STAT3?signaling pathway is one of the fundamental signaling pathwaysof the original,developmental,pathological and physiological processes of the cardiovascular system.We have found that JAK2/STAT3 plays an important regulatory role in ischemic pretreatment,ischemic post-treatment and protective effects of some drugs.And foreign scholars found that melatonin receptor and JAK/STAT signaling pathway are closely linked.Objective:1.To investigate whether MEL pretreatment can fight against IRI in isolated hearts and myocardial cells.2.To investigate whether the anti-IRI effect of MEL is mediated by JAK2/STAT3 signaling pathway.3.To investigate whether the effect of MEL against mitochondrial oxidative stress caused by IRIis related to JAK2/STAT3 signaling pathways.Methods:?:Investigate the effect and mechanism of MEL fighting against myocardial IRI in isolated hearts.1.Evaluate the effects of MEL and AG490?the specific inhibitor of JAK2/STAT3 pathway?on normal isolated rat hearts.The healthy male adult SD rats were randomly divided into 3 experimental groups with 8 each.1)Group 1?control?:The isolated hearts were perfused for 110 min with KH buffer?n=8?;2)Group 2?MEL treatment?:The isolated hearts were subjected to KH buffer with 5?MMEL for 5 min and then perfused for 105 min with KH buffer?n=8?;3)Group 3?AG490 treatment?:The isolated hearts were subjected to KH buffer with 2?M AG490 for 5 min and then perfused for 105 min with KH buffer?n=8?;2.Evaluate the effects of MEL and AG490 on isolated rat hearts subjected to IRI.The healthy male adult SD rats were randomly divided into 4 experimental groups with 8 each.1)Group 1?IR?:The isolated hearts were perfused with KH buffer for 5 min and then subjected to ischemia for 45 min,followed by 60 min of reperfusion;2)Group 2?MEL+IR?:The isolated hearts were perfused with KHB containing 5?MMEL for 5 min and then subjected to ischemia for 45 min,followed by 60 min of reperfusion;3)Group 3?MEL+AG490+IR?:The isolated hearts were perfused with KH buffer containing 5?M MEL and 2?M AG490 for 5 min and then subjected to ischemia for 45 min,followed by 60 min of reperfusion;4)Group 4?AG490+IR?:The isolated hearts were perfused with KH buffer containing 2?M AG490 for 5 min and then subjected to ischemia for 45 min,followed by 60 min of reperfusion.The isolated heart was retrogradely perfused through the aorta with a noncirculating Langendorff apparatus,which records and analyzes hemodynamic parameters during cardiac perfusion,including heart rate?HR?,left ventricular developed pressure?LVDP?,the highest rate of change of pressure development?+dp/dtmax?.Collected coronary effluent within one minute in order to measure the coronary flow.The LDH levels,a commonly used marker for cardiomyocyte damage,in the coronary effluent were determined using the ELISA kit.After perfusion,each treated heart was excised and serially sectioned into slices,followed by triphenyl tetrazolium chloride?TTC?staining to measure the infarct size.Then,the heart samples were analyzed for the expression of p-JAK2,p-STAT3,Bcl2,Bax,cytochrome C,SDH and COX by Western Blot.?:To investigate the effect and mechanism of MEL fighting against myocardial IRI in neonatal rat cardiomyocytes.Primary cultures of neonatal rat cardiomyocytes from 1-to 2-day-old SD rats were prepared.Then,the cardiomyocytes were randomly divided into 4 experimental groups with 8 wells each.1)Group 1?SIR?:The cardiomyocytes were incubated in normal DMEM for 26 hr, subjected to simulated ischemic conditions for 2 hr,and then incubated in normal DMEM to simulate reperfusion for 4 hr;2)Group 2?MEL+SIR?:The cardiomyocytes were incubated in normal DMEM for 24 hr,incubated in DMEM with 2?MMEL for 2 hr,and then subjected to simulated ischemic conditions for 2 hr,and then incubated in normal DMEM to simulate reperfusion for 4 hr;3)Group 3?MEL+JAK2 siRNA+SIR?:The cardiomyocytes were transfected with JAK2 siRNA.After the transfection procedure was completed,the cardiomyocytes were incubated in DMEM with the transfection mixture for 24 hr,incubated in normal DMEM for 2 hr,and then subjected to simulated ischemic conditions for 2 hr,and then incubated in normal DMEM to simulate reperfusion for 4 hr;4)Group 4?JAK2 siRNA+SIR?:The cardiomyocytes were transfected with JAK2 siRNA.After the transfection procedure was completed,the cardiomyocytes were incubated in the DMEM with the transfection mixture for 24 hr,incubated in normal DMEM for 2 hr,and then subjected to simulated ischemic conditions for 2 hr,and then incubated in normal DMEM to simulate reperfusion for 4 hr.The cells were scraped and the apoptosis level of primary cardiomyocytes was analyzed by TUNEL staining.Cardiomyocyte injury was assessed by measuring LDH release into the culture medium using the ELISA kit.Then,the primary cardiomyocytes were analyzed for the expression of p-JAK2,p-STAT3,Bcl2,Bax,cytochrome C,SDH,COX and?-actin by Western Blot.Results:1.Effects of MEL and AG490 on normal isolated rat heartsThe hearts were initially treated with MEL?5?M?or AG490?2?M?for 5 min,followed by KHB perfusion for 105 min.The LVDP decreased significantly compared with the control group in the first 5 min?P<0.01?.However,when MEL or AG490 was removed from the KHB,the experimental HR and LVDP gradually recovered to the control levels during normal KHB reperfusion.In addition,MEL or AG490 treatment had no effect on the infarct size compared with the control group.Compared with the control group,MEL pretreatment significantly increased p-JAK2 and p-STAT3 expressions?P<0.01?,while AG490 pretreatment significantly decreased p-JAK2and p-STAT3expressions?P<0.01?.Compared with the control group,both MEL and AG490 had little effect on the expressions of total JAK2 and total STAT3.2.Effects of MEL and AG490 on isolated rat hearts subjected to IRICompared with the IR group,MEL pretreatment significantly increased the functional recovery of the postischemic hearts,which was demonstrated by a significantlyhigher HR?IR,222.1±7.9;MEL+IR,251.6±7.5;P<0.01?,LVDP?IR,30.9±7.7;MEL+IR,56.4±7.5;P<0.01?,+dP/dt max?IR,607.0±101.0;MEL+IR,1173.0±89.0;P<0.01?,and CF?IR,7.42±0.80;MEL+IR,9.82±0.78;P<0.01?throughout thereperfusion period.However,AG490 can eliminate the cardioprotective effect of MEL.Compared with the MEL+IR group,the MEL+AG490+IR group attenuated the functional recovery of the postischemic hearts and significantly reduced the HR,LVDP,+dP/dt max and CF?P<0.01?.The myocardial infarct size?MEL+IR,10.25±3.44%;MEL+AG490+IR,35.64±4.08%;P<0.01?,apoptotic index?MEL+IR,15.18±3.41%;MEL+AG490+IR,42.06±3.50%;P<0.01?,and LDH release?MEL+IR,36.88±4.72IU/g;MEL+AG490+IR,72.45±4.37IU/g;P<0.01?in the MEL+AG490+IRgroup increased significantly.3.Effects of MEL and AG490 on myocardial mitochondria of isolated rat hearts subjected toIRICompared with the IR group,the MEL+IR group significantly increased the mitochondrial SOD activity?P<0.01?and reduced levels of mitochondrial malonaldehyde?MDA?and H2O2?P<0.01?.Compared with the MEL+IR group,MEL+AG490+IR group significantly reduced the mitochondrial SOD activity?P<0.01?and increased levels of mitochondrial MDA and H2O2?P<0.01?.Compared with the IR group,MEL+IR significantly reduced the mitochondrial Eh.However,AG490 treatment almost abolished the effect of MEL pretreatment.4.Effects of MEL and AG490 on myocardial JAK2/STAT3 and apoptosis-related signaling pathways in isolated rat hearts subjected to IRICompared to the IR group,the MEL+IR group significantly increased the expressions of p-JAK2,p-STAT3,Bcl2,SDH and COX?P<0.01?,and decreased that of Bax and cytosolic cytochrome C?P<0.01?.However,the effect of MEL pretreatment on the expressions of these proteins was abolished by AG490.Compared with the IR group,AG490+IR treatment significantly decreased p-JAK2 and p-STAT3 expressions.However,AG490+IR also had little effect on the expressions of total JAK2 and total STAT3compared with the IR group.5.Effects of MEL and JAK2 siRNA on the cardiomyocytes of neonatal rats subjected to SIRCompared with the SIR group,the MEL+SIR group markedly increased cell viability?P<0.01?.As observed by microscopy,the MEL+SIR group resulted in improvedcell shrinkage and an increased rate of cellular attachment compared with the SIR group.The TUNEL immunofluorescent staining showed that the MEL+SIR group significantly decreased the apoptotic index compared with the SIR group?P<0.01?.However,the protective effect of MEL pretreatment was abolished by JAK2 siRNA compared with the MEL+SIR group?P<0.01?.Significantly reduced levels of LDH and mitochondrial MDA were observed in the MEL+SIR group compared with the SIR group?P<0.01?.In addition,as Fig.7C shows,MEL pretreatment significantly increased mitochondrial SOD activity compared with the SIR group.However,JAK2 siRNA pretreatment almost abolished the protective effect mediated by MEL pretreatment compared with the MEL+SIR group?P<0.01?.Compared with the SIR group,the MEL+SIR group significantly increased the expressions of p-JAK2,p-STAT3,Bcl2,SDH and COX?P<0.01?,and decreased that of Bax and cytosolic cytochrome C?P<0.01?.However,compared with the MEL+SIR group,the effect of MEL pretreatment on the expressions of these proteins was abolished by JAK2 siRNA?P<0.01?.Compared with the SIR group,JAK2 siRNA+SIR treatment significantly decreased the expressions of p-JAK2,p-STAT3,total-JAK2,and total-STAT3?P<0.01?.Conclusions:Melatonin can fight against IRI in isolated hearts and myocardial cells.This protection seems to be largely due to the activation of JAK2/STAT3 by melatonin attenuates myocardial IR-induced mitochondrial oxidative damage and apoptosis in myocardial cells. |
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