Study Of The Relationship Between OX40/OX40L-NFAT Signaling Pathway In The Immune Damage Of Kawasaki Disease

Part Ⅰ Analysis of Kawasaki disease’s related genes and construction of NFAT signaling pathway by systems biologyObjective:To analysis Kawasaki disease’s related genes by systems biology method and construct the possible signal pathway for further study of the pathogenesis of immune injury of KD.Methods:Using related retrieval language,just like "Kawasaki disease","Kawasaki syndrome","lymph node syndrome","mucocutaneous lymph node syndrome" combined with "polymorphism","gene","genetic","allele" "genotype"etc.to retrieve literature of KD related genes from database of Pubmed,Ovid,Google Scholar and extract statistically genes.To annotate genes functionally via gene function classification and information pathways etc.by data analysis software-DAVID,to do enriched analysis and establish pathways of biological information by GeneGo MetaCore and to analysis the relation of these gene code’s protein by STRING.Results:Genes with statistic significance of Kawasaki disease are mainly involve with KD susceptibility,coronary artery damage,incomplete Kawasaki disease,non-response of IVIG treatment,KD delayed diagnosis etc.,of which 65 are alternate genes and 18 are from all genome.The functions of these genes indicated by DAVID software are focused on ’injury and defensive reactions,inflammation reactions(enrichment score = 15.91);intracellular calcium ion balance homeostasis,cell chemotaxis(enrichment score = 3.75),regulating of the immune system(enrichment score = 3.58).GO Process shows that the genes function are concentrated in the immune response,damage and defense response,stress response etc.,which is consistent with DAVID.GO Disease illustrates that these genes are involved in pathological physiological processes of the immune inflammatory diseases like KD,inflammatory bowel disease,skin diseases,inflammatory vascular diseases,arthritis etc.GO Pathway shows that these enriched genes pathways are concentrated in the immune pathway,such as HSP60 and HSP70/TLF signaling pathway,Inflammasome in inflammatory response,Plasmin signaling,NFAT signal and leukocyte interactions,etc.In addition to the immune pathway,Cell adhesion pathway,Development pathway,Apoptosis and survival pathway were also enriched.STRING shows that the protein coded by those genes associated with KD are closely related and form a network,with TNF-α in its central part.Conclusion:1.Genes having statistically significant association with KD are closely interrelated,form numerous immune cell adhesion,proliferation,apoptosis and survival signaling pathway and jointly participate in the immune injury process of KD.2.NEAT signal plays a vital role in the immune injury of KD.Part Ⅱ The expression of OX40,OX40L,NFAT in PBMC of KD and regulation mechanism of OX40-OX40L on NFATObjective:To analysis the expression and clinical significance of OX40,OX40L,NFAT in the acute phage of KD and to probe the regulation mechanism between OX40,OX40L and NFAT.Methods:1.55 KD patients are involved to be samples,in which 12 are with coronary artery lesion while 43 not.23 cases with fever and healthy children as the control group.Real-time RT-PCR,western blot was used respectively to measure the gene and protein expression level of OX40,OX40L,NFAT1,NFAT2 in peripheral blood mononuclear cells(PBMC)of KD cases before and after treatment.2.Anti-OX40 monoclonal antibody was used to stimulate OX40-OX40L axis in normal PBMC.Anti-OX40L monoclonal antibody was used to inhibit OX40-OX40L axis in PBMC of KD.The gene and protein expression level of NFAT1,NFAT2 in PBMC were measured by Real-time RT-PCR and westernblot respectively.Results:1.NEAT1,NFAT2 mRNA and protein expression in PBMC of KD group before treatment are distinctively higher than the matched group’s;while NFAT1,NFAT2mRNA and protein expression coronary injury in sub-group is higher than that of non-coronary injury sub-group,but there is no statistical significance(P>0.01).The mRNA and protein expression in PBMC of NFAT1,NFAT2 is distinctively decreased(P<0.01)after treatment.2.OX40、OX40L mRNA and protein expression in PBMC of KD group before treatment are distinctively higher than those of the matched group’s(P<0.01);while OX40、OX40L mRNA and protein expression coronary injury in sub-group is higher than that of non-coronary injury sub-group,but there is no statistical significance(P>0.01).The level of OX40、OX40LmRNA and protein decreased significantly after treatment(P<0.01).3.The expression of protein OX40,OX40L in PBMC at acute stage of KD have a positive correlation with the one of both NFAT1 and NFAT2(P<0.01).4.After the anti-OX40 monoclonal antibody stimulated the normal PBMC with 40 ug/ml for 48h,the expression of NFAT1,NFAT2 mRNA and protein is significantly higher than the non-stimulated group’s(p<0.01).After the anti-OX40L monoclonal antibody stimulated the KD PBMC with 20 ug/ml for 24h,the expression of NFAT1,NFAT2 mRNA and protein is significantly suppressed than the KD group’s(p<0.01).Conclusion:1.NFAT1,NFAT2,OX40 and OX40L participate in immune response process in the acute stage of KD.2.OX40-OX40L to be upstream regulates the expression of NFAT.3.To interpose and block OX40/OX40L axis may probably to be a new target spot to prevent the process of KD immune injury.