Role Of P2X7-mediated PANoptosis In Myocardial Ischemia-reperfusion Injury In Diabetic Mice

BackgroundThe incidence of diabetes in China is increasing year by year,the treatment and control rates remains low,which adding the burden of medical care significantly.Cardiovascular events are the leading cause of death in diabetic patients.With the development of coronary intervention,patients with myocardial infarction are effectively treated,however,diabetic patients still have a lower survival rate,which is associated with aggravated myocardial ischemia reperfusion injury(MIRI).The mechanisms of the exacerbation in MIRI are not fully elucidated.Studies have shown that hyperactivation of innate immune can amplify inflammation and promote myocardial injury.Purinergic P2X7 receptor has been widely demonstrated to be involved in inflammatory diseases,and its most significant downstream effect is to promote the assembly and activation of NLRP3 inflammasome.PANoptosis is a unique form of programmed cell death,its biological effects overlap between pyroptosis,apoptosis and necroptosis,which may explain the crosstalk effect in cell death pathway.At present,the correlation between innate immune and P2X7 in diabetic mice with MIRI is unclear,and the role of P2X7 and PANoptosis has not been reported.Exploring the mechanism of P2X7 may provide new and effective therapeutic targets in MIRI.Objective1.To identify the characteristics of MIRI in diabetic mice.2.To elucidate the relationship between myocardial P2X7 and aggravated MIRI in diabetic mice.3.To explore whether P2X7 mediates PANoptosis to exacerbate MIRI in diabetic mice.MethodsChapter 1 Characteristics of MIRI in diabetic miceMIRI models of diabetic and non-diabetic mice were constructed to compare myocardial infarction area,left ventricular systolic function,serum CK-MB concentration,myocardial apoptosis,immune cell infiltration and cell types 24 hours after reperfusion.Chapter 2 Up-regulation of P2X7 was associated with MIRIExpression of P2X7,CD39,NLRP3,caspase1,and IL-1β were compared by Western blots 24 hours after myocardial ischemia reperfusion.Identification of P2X7 in innate immune cells by flow cytometry.Chapter 3 Pharmacology blockade of P2X7 inhibited the expression of related molecules in PANoptosisThe role of P2X7 in MIRI was further elucidated with the P2X7 agonist BzATP and the inhibitor BBG.Expression of PANoptosis related molecules(ZBP1,caspase-8,NLRP3,caspase-1,GSDMD,IL-1β,Caspase-3,RIPK3,MLKL)was analyzed by Western blots.Chapter 4 BBG decreases the heart rate after reperfusion may be related to inhibition of sympathetic germinationMice were monitored ECG 24 hours after reperfusion using BIOPAC MP160 and analyzed using AcqKnowledge software.Myocardial tyrosine hydroxylase and NOX2 expression were analyzed by Western blots.Results1.Diabetic mice showed that increased infarct area,deteriorating cardiac function,increased serum CK-MB,increased apoptosis,and monocyte macrophages were significantly infiltrated.2.Myocardial P2X7 was upregulated after induction of diabetes and further increased 24 hours after ischemic reperfusion.In MIRI,P2X7 was mainly expressed in monocyte macrophages and NLRP3-Caspasel-IL-1β signals was enhanced.3.BBG reduced the infarct area,improved cardiac function and inhibited apoptosis in diabetic mice.BzATP eliminated differences in myocardial injury between non-diabetic and diabetic mice.BBG inhibited the expression of related molecules in PANoptosis 24 hours after reperfusion in diabetic mice.4.BBG decreased heart rate after MIRI in diabetic mice,this may be associated with inhibited sympathetic germination.ConclusionMyocardial P2X7 expression was upregulated in diabetic mice by recruiting innate immune cells 24 hours after myocardial ischemia reperfusion.Pharmacological blockade of P2X7 inhibited the expression of PANoptosis-related molecules,thereby alleviating MIRI in diabetic mice.