The Protective Role And Molecular Mechanism Of TIPE2 In Myocardial Ischemia/Reperfusion Injury And Chronic Heart Failure

Background:Myocardial ischemia-reperfusion injury and chronic heart failure are the phenomenon of increasing the metabolic dysfunction and structural damage when blood supply returns to the myocardium tissue after a period of ischemia.Myocardial ischemia-reperfusion injury had complex mechanism;currently its main mechanism including the oxygen free radicals,energy Cetabolism,Ca2+ overload,cell apoptosis,and a variety of inflammatory cell and factor-mediated inflammatory reaction.And innate immunity is the indispensable part of the inflammatory response.The innate immune system is the first line of defense against pathogens and tissue injury,which induce tissue in a state of’ continuous injury,and is recognized highly conserved PAMPs or DAMPs by pattern-recognition receptors(PRRs).Activation of PRRs induces the recruitment of innate immune cells,initiates tissue repair processes,and results in adaptive immune activation.Abnormalities in any of PRR mediated processes lead to excessive inflammation.Despite the pathogenesis of ischemic cardiovascular disease is complex,studies have highlighted the importance of PRRs,particularly the membrane-bound Toll-like receptors(TLRs)and the intracellular NOD-like receptors(NLRs)to ischemic injury.So far,researchers have currently discovered 22 NLR family members,and the most representative NLRs are NOD1 and NOD2.The activation of cardiac NODI was demonstrated to induce cardiac dysfunction simultaneously with cell apoptosis and cardiac fibrosis in the murine heart,which resulted from the activation of the NF-κB and TGF-β pathways.Crohn’s disease susceptibility and Blau syndrome are thought to be associated with NOD2 gene mutations,a member of the family of NLR,and researchers have underscored the importance of NOD2 in inflammatory homeostasis.Although the existing research have showed that NOD2 is associated with many diseases,such as diabetes mellitus,Alzheimer disease and atherosclerosis,the role of NOD2 and the mechanisms by which NOD2 mediates inflammatory responses in I/R-induced myocardial injury is still unclear.Tumor necrosis factor(TNF)-a-induced protein 8-like 2(TIPE2),belonging to TNFAIP8 family,is a newly identified negative immune regulator and was identified in the inflamed spinal cord of mice with experimental autoimmune encephalomyelitis(EAE).TIPE2 is a cytoplasmic protein and normally predominantly expressed in immune cells of the myeloid and lymphoid lineages,especially highly expressed in T lymphocytes and monocytes or macrophages.It has been found that TIPE2 contains a particular DED-like domain and depletion of TIPE2 causes severe inflammatory diseases.Moreover,TIPE2 play an important in maintaining immune homeostasis.TIPE2 regulates TCR and TLR signaling by targeting signaling complexes that contain caspase-8.It binds to caspase-8 and inhibits activating protein-1(AP-1)and nuclear factor-B(NF-B)activation while promoting Fas-induced apoptosis.TIPE2-deficient cells are hyper-responsive to TCR and TLR activation and produce significantly increased levels of inflammatory cytokines.In humans,the abnormal expression of TIPE2 is associated with systemic autoimmunity,diabetic nephropathy,and hepatitis B.But,TIPE2 role and whether involved in negative regulation of NOD2-mediated pathway in myocardial ischemia-reperfusion injury has not yet been reported.Objective:First,we detect the expression of NOD2 in myocardial ischemia-reperfusion injury animal model,and reveale the association of NOD2 withinflammatory pathogenesis.Second,we explore the possible expression of TIPE2 in myocardial cells and cardiac fibroblasts in vitro,under the stimulation of pathogens in diabetic nephropathy.Third,we investigate if the activation of TIPE2 can induce the activation of MAPKs and NF-κB signalings,and be associated with the inflammatory of myocardial ischemia-reperfusion injury,and explore the relationship with NOD2.Methods:Studies in vitroIn vitro,We cultured Myocardial cells,cardiac fibroblasts,macrophages and neutrophils and detected the level of TIPE2 by western blot(WB)in myocardial cells and cardiac fibroblasts under stress conditions such as TNF-a or hypoxia/reoxygenation.MDP activate NOD2 in macrophages,we observe the expression changes of TIPE2,and further confirm the relationship between the NOD2 and TIPE2 by co-immunoprecipitation.We treated macrophages and neutrophils with MDP and the levels of NOD2 and TIPE2 examed by immunofluorescence,WB and real time RT-PCR.After silence of NOD2,we detect the express level of NOD2 and TIPE2.In macrophages NOD2 was activated by MDP,then,we examined the activation of NF-κB and MAPKs signalings by western blotting,including phospho-p38,phospho-ERK 1/2,phospho-IκBa,phospho-p65 and phospho-JNK.After overexpression of TIPE2.we detect the phosphorylation level of ERK1/2,p38,JNK,IκBα and p65,and the nuclear translocation level of p65 by immunofluorescence.In addition,we exam the levels of TNF-α,IL-1β,IL-6 and MCP-1 in macrophages treated with MDP by enzyme-linked immune sorbent assay(ELISA),and the change of proinflammatory factors after overexpression of TIPE2.Animal studiesMale C57BL/6J mice and male NOD2-/-mice(25-30g)were randomly divided into two groups,operated group and sham operated group.In this study,mice were subjected to myocardial ischemia/reperfusion injury induced by occluding the left anterior descending artery for 30 min followed by reperfusion at different time points.We collected myocardium at 12h,24h,48h and 72 h after reperfusion,and detected the relative level of NOD2 mRNA and protein by real time RT-PCR and western blot at different time points.To further investigate the role of NOD2 in I/R injury,we built myocardial I/R injury model in NOD2-/-mouse were used to establish.By echocardiography we detected heart function and left ventricular internal diameter;the degree of myocardial injury and cell apoptosis was observed by HE staining and Tunel staining in WT and NOD2-/-mice;ELISA measured tissue proinflammatory factor levels;flow cytometry detected the number of inflammatory cells in the myocardial tissue.We analyzed the expression of TNFAIP8 by western blotting in I/R injury.To further investigate the role of TIPE2 in I/R injury,TIPE2 was silenced by pGLV3-shRNA2-TIPE2 delivered into NOD2-1-ischemic mice.By echocardiography we detected heart function and left ventricular internal diameter;transfection efficiency was tested by immunofluorescence;proinflammatory factor levels were measured by ELISA;myocardial risk and infarct area were distinguished by EB and TTC staining.In addition,we overexpressed TIPE2 by LV-TIPE2 delivering into mice;and cardiac function,proinflammatory factor levels and myocardial risk and infarct area were tested by echocardiography,ELISA,and EB and TTC staining.Results:In this study.mice were subjected to myocardial ischemia/reperfusion injuryinduced by occluding the left anterior descending artery for 30 min followed by reperfusion at different time points.NOD2 levels were significantly increased in ischemic myocardium.The lack of NOD2 led to decreasing inflammatory mediators including tumor necrosis factor-α(TNF-α),interleukin-β(IL-1β),IL-6,and monocyte chemoattractant protein-1(MCP-1)and the numbers of infiltrated inflammatory cells including CD45+ leukocyte,neutrophils,macrophages,and T cells.This ameliorated the inflammatory responses and myocardial injury and protected heart function.We found that among the TNFAIP8 family,TIPE2 levels were markedly enhanced in ischemic myocardium after myocardial I/R injury in mice.TIPE2 was not expressed in either cardiac fibroblasts or myocardial cells even under stress conditions such as TNF-a or hypoxia,but in immune cells such as macrophages and neutrophils.Upon stimulation with MDP.the level of NOD2 expression was increased and TIPE2 expression was significantly reduced in vitro.In response to hypoxia/reoxygenation,the level of NOD2 expression was increased and TIPE2 expression was significantly decreased,which was prevented by gene silencing of NOD2 from western blot and immunofluorescence analysis.The result from the coimmunoprecipitation shows an interaction between TIPE2 and NOD2.Our results indicated that NOD2 negatively regulates TIPE2 expression.Considering that NOD2 initiates inflammatory responses largely dependent on MAPK and NF-κB activation,we further detected the effect of TIPE2 on MAPK and NF-κB signaling pathways.We also found that TIPE2 overexpression inhibited MAPKs and NF-κB activation as documented by the decreased levels of phospho-ERK-1/2.phospho-p38,phospho-JNK,phospho-IκBα,phospho-p65and the nuclear translocation of p65.and attenuated inflammation injury in vitro.The silence of TIPE2 reduced percent fractional shorting and increased infarct area,the diastolic and systolic LV dimensions and production of pro-inflammatory mediators after myocardial I/R in NOD2-/-mice.In other words,the silence of TIPE2 counteracted that NOD2 gene knockout bring the protection of myocardial injury in ischemic/reperfusion.In addition,the increasing TIPE2 can reduce production of pro-inflammatory mediators,infarct area and inflammatoryinjury and protect cardiac function in WT I/R mice.Conclusion:NOD2 levels were significantly increased in ischemic myocardium.NOD2 deficiency reduced inflammatory injury and protected cardiac function in ischemic myocardium.We found that among the TNFAIP8 family,TIPE2 levels were markedly enhanced in ischemic myocardium after myocardial I/R injury in mice,and TIPE2 was not expressed in either cardiac fibroblasts or myocardial cells.Our results indicated that NOD2 negatively regulates TIPE2 expression.Upon stimulation with MDP,in vitro,the level of NOD2 expression was increased and TIPE2 expression was significantly reduced,increased the levels of phospho-ERK-1/2,phospho-p38,phospho-JNK.phospho-IκBαI and phospho-p65,promoted the activation of MAPK and NF-κB signaling pathways and aggravated inflammatory responses.When macrophages were transfected by pRK5-TIPE2,TIPE2 expression was increased,reduced the phosphorylation and the nuclear translocation level of p65,inhibited the activation of MAPK and NF-κB signaling pathways and alleviated inflammatory damage.The silence of TIPE2 counteracted that NOD2 gene knockout bring the protection of myocardial injury in ischemic/reperfusion mice.In addition,the increasing TIPE2 can reduce production of pro-inflammatory mediators,infarct area and inflammatory injury and protect cardiac function in I/R mice.To make a long story short,myocardial ischemic/reperfusion led to increasing the level of NOD2,reducing TIPE2 expression,increasing the levels of phospho-ERK-1/2.phospho-p38.phospho-JNK,phospho-IκBα,phospho-p65 and the nuclear translocation level of p65,promoting the activation of MAPK and NF-κB signaling pathways,elevating the production of pro-inflammatory mediators(TNF-α,IL-1β,IL-6 and MCP-1)and accelerating inflammatory cells(macrophages and neutrophils)gather and infiltration;inflammatory cells elevated the production of inflammatory mediators and aggravated inflammatory injury;but,TIPE2 overexpression inhibited MAPKs and NF-κB activation,reduced the production of pro-inflammatory mediators and alleviated myocardial inflammatory injury.