Role And Mechanism Of PIAS1 Regulating PPARγ/NF-κB Signaling Pathway In Myocardial Ischemia Reperfusion Injury

Background: Myocardial ischemia-reperfusion injury(IRI)is one of the serious complications after myocardial reperfusion therapy,and its mechanism is complicated.In the course of its development,it is associated with inflammation,calcium overload,oxidative stress,cytokine release and infiltration of neutrophil,etc.,there are still many problems need to be resolved.Recent studies have shown that SUMO modification is involved in many aspects of myocardial IRI,and PPARγ,which is an inflammatory suppressor and has been gradually arose great attentions in recent years,is a protein that can be modified by SUMO.Our previous studies have also confirmed that PIAS1,one of the SUMO ligases,has a significant expression change during myocardial IRI.Our project aims to explore the role and mechanism of PIAS1 and PPARγ in myocardial IRI,as well as seek a new target for more effective treatment and prevention of myocardial IRI.Materials and Methods:1.The stability of the left ventricular myocardium IRI model and the hypoxia/reoxygenation(H/R)injury model of cardiomyocyte cell line H9C2 was confirmed by H-E staining and cell morphology observation.2.The expression of PIAS1 was detected by immunohistochemistry and Western blot before and after IRI treatment.3.The SUMO modification of exogenous PPARγ and the interaction between PPARγ and PIAS1 were detected by plasmid transfection and immunoprecipitation.The SUMO modification of endogenous PPARγ was detected by nuclear separation and immunoprecipitation.4.The SUMO-modified site of PPARγ was found by the method of genemutation,and it was confirmed that PIAS1 was the SUMO ligase ofPPARγ via immunoprecipitation.5.The effect of SUMOylation of PPARγ and the effect of PIAS1 on theapoptosis were confirmed by TUNEL staining.6.The effect of SUMOylation of PPARγ and the effect of PIAS1 onactivation of NF-κB pathway were confirmed by immunofluorescencestaining.Results: 1.The model of Myocardial IRI and H9C2 H/R injury were stable andeffective,it could be observed significant damage effects bypathological sections and morphology.2.The change of PIAS1 expression was the most obvious among SUMOligases after myocardial IRI.3.PPARγ could be modificated by SUMO in vitro experiments,thismodification could occur within the cardiomyocytes,as well.Themodification sites were 77 and 365.4.PIAS1 was the SUMO ligase of PPARγ and it could bind to PPARγ.5.The SUMOylation of PPARγ had protective effects on myocardial IRI,these effects involved in NF-κB pathway.6.PIAS1 had protective effects on myocardial IRI and NF-κB pathwayparticipated in these effects.Conclusion: In this study,we found a new regulatory mechanism in myocardial IRI——PIAS1-mediated SUMOylation of PPARγ,which was the first time in myocardium studies.This biochemical process played important role in inflammatory injury,apoptosis and NF-κB pathway activation during myocardial IRI.This study broadened the scope of myocardial IRI research,and further promoted the combination of SUMOylation theory and IRI,which provided a new theoretical basis for the prevention and treatment for myocardial IRI.