The Protective Role Of CpG Containing Oligodeoxynuceotide On Myocardial Ischemia/Reperfusion Inuried Mice And Associated Mechanisms

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To establish a closed-chest model of myocardial ischemia/reperfusion in miceBackground:Ischemic heart disease,a serious risk of health,is the major cause of death globally.Ischemia reperfusion injury aggravates myocardial damage,which may lead to poor outcome.Therefore,it is important to find a way to prevent ischemic reperfusion injury,in order to improve the prognosis of patients with ischemic heart disease.The aim of this study is to establish a closed-chest model of myocardial ischemia/reperfusion in mice,differed from the open-chest model used before,which may diminish surgical trauma effect on the results of myocardial ischemia reperfusion experiments.Thus made it more credible for experiments to explore therapeutic drugs for ischemic heart disease.Objective:The aim of this study is to establish a closed-chest model of myocardial ischemia/reperfusion in mice.Methods:The C57BL/6(12 weeks,male,WT)mice were intubated and ventilated after anesthetized by isoflurane.Mice underwent a left lateral thoracotomy in the 4th intercostal space,and established the preliminary model of myocardial ischemia/reperfusion microscopically.After a recovery period of 5 days,intravenous anesthesia was conducted for ischemia and reperfusion experiments.Then heart were excised.Infarct size were analyzed by triphenyltetrazolium chloride(TTC)staining at time points 2 h or 24 h of reperfusion following 30 min or 60 min of ischemia,Myocardial mRNA-expression of cytokines was measured using real-time PCR followed 2 h reperfusion.Results:(1)The myocardial infarction was successfully occurred in mice closed-chest model of myocardial ischemia/reperfusion.Following 30 min ischemia and 2 h or 24 h reperfusion,there were myocardial infarction detected by TTC staining,and the infarct size increased following 24h reperfusion.In 30 min and 60 min ischemia,the myocardial infarction can both be detected,but there was no significant differences in the infarct size.(2)The expression of cytokines in heart was significantly increased via myocardial ischemia/reperfusion.Following 30 min ischemia and 2 h reperfusion,there was no significant differences of cytokins including TNF-?,IL-1? and IL-6 between the group of mice undergoing a preliminary model of myocardial ischemia/reperfusion(sham group)and the group of healthy mice(control group).However,the level of IL-1?,IL-6 was higher in myocardial ischemia/reperfusion group(I/R group)than in sham group and control group.Conclusion:It was successfully to establish a close-chest myocardial ischemia/reperfusion mice model.Besides,there were increased cytokines level in myocardial ischemia/reperfusion model,which made it foundation for the follow-up experiment.Part ? The protective role of CpG-ODN post-conditioning in myocardial ischemic-reperfusion injuried miceBackground:Ischemic postconditioning(IPC)refers to repetitive cycles of reperfusion and occlusion of the LAD after ischemic or during at the early stage of reperfusion.A large number of studies demonstrated that,as ischemic preconditioning,IPC could induce activation of the host's innate protective mechanism and exert protective effect in myocardial ischemic-reperfusion injury.CpG-ODN is a ligand of TLR9 and has immune modulatory effect.Previous studies showed that CpG-ODN preconditioning could ameliorate myocardial ischemic-reperfusion injury,reduce infarct size and improve cardial function in mice.However,whether CpG-ODN postconditioning has the same protective effects is still poorly understood.Objective:This part of study aims to explore the protective effects of CpG-ODN postconditioning in ischemic-reperfusion injuried mice.Methods:In order to explore the protective effects of CpG-ODN postconditioning in early stage of ischemic-reperfusion injury in mice,the current study included three groups:1)PBS:250 ?l phosphate buffer solution injected intraperitoneally 5 minutes before reperfusion;2)IPC:Three cycles of twenty-second(20 s)reperfusion and following 20 s occlusion episodes after ischemia;and 3)CpG-ODN:0.2 ?mol/kg of CpG-ODN(dissolved in 250 ?l PBS)injected intraperitoneally 5 minutes before reperfusion.All mice in the 3 groups were subjected to a 30-minute myocardial ischemia first.Then,cardiac tissue was obtained and infarct size was assessed via TTC staining after 2 and 24 h reperfusion.Cardiac function of the mice was evaluated via a left ventricular P-V catheter after 24 h reperfusion.In order to explore the protective effects of CpG-ODN postconditioning in late stage of ischemic-reperfusion injuried mice,the current study included four groups:1)PBS:250 ?l phosphate buffer solution injected intraperitoneally 5 minutes before reperfusion;2)IPC:Three cycles of twenty-second(20 s)reperfusion and following 20 s occlusion episodes after ischemia;3)CpG-ODN:0.2 ?mol/kg of CpG-ODN(dissolved in 250 ?l PBS)injected intraperitoneally 5 minutes before reperfusion and 4)sham:pre-ischemic-reperfusion model without any ischemic-reperfusion interventions.Except for the sham group,mice in the other 3 groups were subjected to a 30-minute myocardial ischemia first.Then,cardiac tissue was obtained and detected after a certain period of reperfusion.Myocardial collagen fibrils deposit as well as myocardial fibrosis were assessed via Sirius-Red staining after 7 and 28 days reperfusion.Myocardial macrophage infiltration was measured via immunohistochemistry method after 3 and 7 days reperfusion.Cardiac function of the mice was evaluated via a left ventricular P-V catheter after 3,7 and 28 days reperfusion.Results:(1)Compared to PBS treatment,postconditioning of CpG-ODN as well as IPC could remarkably reduce infarct size in mice after 2 and 24 h reperfusion.(2)Compared to PBS treatment,postconditioning of CpG-ODN could remarkably enhance mice' left cardiac function after 24 h reperfusion,which was indicated by elevated ejection fraction(EF)and preload recruitable stroke work(PRSW).(3)Compared to PBS treatment,postconditioning of CpG-ODN as well as IPC could significantly lower the level of myocardial collagen fibrils deposit after 7 and 28 days reperfusion.(4)Compared to PBS treatment,postconditioning of CpG-ODN could significantly alleviate myocardial macrophage infiltration after 3 and 7 days reperfusion.IPC could only reduce myocardial macrophage infiltration after 3 days reperfusion.(5)Compared to PBS treatment,postconditioning of CpG-ODN could markedly improved mice' EF and PRSW after 3,7 and 28 days reperfusion,which indicated an enhanced cardiac function.IPC could only improved mice' EF and PRSW after 3 days reperfusion.Conclusion:CpG-ODN postconditioning could reduce infarct size and improve early cardiac function in a mice model of ischemic-reperfusion injury.Moreover,this treatment could also alleviate myocardial collagen fibrils deposit and macrophage infiltration as well as improve long-term cardiac function in mice.Part ? The Mechanisms of the protective role of CpG-ODN postconditioning in myocardial ischemic-reperfusion injuried miceBackground:Reperfusion injury salvage kinases(RISK),including PI3K/Akt,MAPKs and ERK1/2,are thought to be key molecular pathways that improve myocardial function after ischemic-reperfusion injury.CpG-ODN is a synthetic oligodeoxynucleotide containing motifs mimic the immunostimulatory effects of bacterial DNA.Via its ligand,Toll like receptor 9(TLR9),CpG-ODN exerts modulatory effects in host immune-inflammatory response.Recent studies showed that,through the activation of PI3K/Akt pathway and modulation of IL-10,CpG-ODN preconditioning could reduce infarct size and improve cardiac function in mice assaulted by ischemic-reperfusion injury.However,the molecular mechanisms of the protective role of CpG-ODN postconditioning in myocardial ischemic-reperfusion injuried mice are still not well understood.Objective:The current study was aimed to explore the molecular mechanisms how CpG-ODN postconditioning could alleviate myocardial ischemic-reperfusion injury in mice.Methods:The current study comprised:1)PBS:250 ?l phosphate buffer solution injected intraperitoneally 5 minutes before reperfusion;2)IPC:Three cycles of twenty-second reperfusion(20 s)and following 20 s occlusion episodes after ischemia;3)CpG-ODN:0.2 ?mol/kg of CpG-ODN(dissolved in 250 ?l PBS)injected intraperitoneally 5 minutes before reperfusion.All mice in the 3 groups were subjected to a 30-minute myocardial ischemia and then a 2-hour reperfusion.After that,cardiac tissue was obtained and inflammatory cytokines were detected using Real-Time PCR.Furthermore,male C57BL/6 mice(WT)as well as their TLR9 knockout mice(TLR9-D)were used in the study.Some of the WT mice were intervened by wortmannin,a PI3K/Akt pathway inhibitor,before a 15-minute of reperfusion and then a 2-hour reperfusion.After that,cardiac tissue was obtained and infarct size was assessed via TTC staining.Results:(1)Compared to IPC or PBS treatment,CpG-ODN postconditioning remarkably induced the IL-10 expression level in mice myocardial tissue after 2 h reperfusion.(2)In WT mice,CpG-ODN postconditioning could significantly reduce the infarct size.(3)In DMSO treated controls,both CpG-ODN postconditioning and IPC could notably reduce the infarct size in mice.However,in wortmannin pretreated mice,neither CpG-ODN postconditioning nor IPC improved the infarct size.Conclusion:CpG-ODN postconditioning could significantly induce the expression of the anti-inflammatory cytokine IL-10.The protective effects of CpG-ODN postconditioning in myocardial ischemic-reperfusion injury could be antagonized by TLR9 knockout or inhibitor of PI3K/Akt pathway.