The Effect And Mechanism Of Glibenclamide On The Post-Cardiac Arrest Brain Injury Via Targeting NLRP3 Inflammasome Activation In Microglia

BackgroundBrain injury is the leading cause of death and disability in survivors of cardiac arrest/cardiopulmonary resuscitation(CA/CPR),where the excessive activation of microglia is believed to play a pivotal role in the inflammatory brain injury after CA/CPR.The nod-like receptor family protein 3(NLRP3)in the brain is mainly expressed in microglia,and involved in a variety of neurological diseases in the form of NLRP3 inflammasome.We have found that the target of glibenclamide(GLB),sulfonylurea receptor 1-transient receptor potential M4(SUR1-TRPM4),is up-regulated in microglia after CA/CPR and correlated with neuroinflammation.Besides reducing brain edema,recent studies have suggested a strong anti-inflammatory effect of GLB,via inhibiting NLRP3 inflammasome activation.In view of our previous finding that GLB improves the neurological outcome of post-CA/CPR rats,we speculate that GLB inhibits the NLRP3 inflammasome activation in microglia through blocking SUR1-TRPM4,and thereby alleviates brain injury after CA/CPR.ObjectiveThe aims of this study are:to evaluate whether NLRP3 inflammasome activation exists in microglia after CA/CPR,and whether inhibiting NLRP3 inflammasome activation can play a neuroprotective role after CA/CPR;to evaluate whether GLB alleviates brain injury after CA/CPR by blocking SUR1-TRPM4-mediated NLRP3 inflammasome activation in microglia,and explore the potential mechanisms.MethodsIn this study,Sprague-Dawley rats underwent asphyxial CA/CPR or sham-operation.Flow cytometry,western blotting(WB),and quantitative real-time polymerase chain reaction(qRT-PCR)were used to evaluate microglia activation,myeloid leukocyte infiltration,and microglial NLRP3 inflammasome activation after CA/CPR.To further evaluate the role of NLRP3 inflammasome activation in brain injury after CA/CPR,we used MCC950 and Ac-YVAD-cmk to block NLRP3 and caspase-1,respectively.Besides,SUR1-TRPM4 channels were blocked by injection of Trpm4 siRNA or GLB,And WB,qRT-PCR were used to evaluate the role of GLB in preventing NLRP3-mediated neuroinflammation through inhibiting SUR1-TRPM4,and corresponding neuroprotective effect.In order to explore the potential mechanisms underlying SUR1-TRPM4-mediated NLRP3 inflammasome activation,BV2 cells were subjected to lipopolysaccharides(LPS)or oxygen-glucose deprivation/reperfusion(OGD/R).ResultsOur results showed that there was significant microglia activation and myeloid leukocytes infiltration in CA/CPR rats.NLRP3 inflammasome activation in activated microglia mediated microglial pyroptosis and neuroinflammatory response.MCC950 and Ac-YVAD-cmk treatment significantly prevented the NLRP3 inflammasome activation in microglia after CA/CPR,and alleviated post-CA/CPR brain injury.Besides,GLB significantly alleviated neurocognitive deficit via the inhibition of microglial NLRP3 inflammasome activation by blocking SUR1-TRPM4.In vitro under circumstance of eliminating distractions from brain edema,SUR1-TRPM4 and NLRP3 inflammasome were also activated in BV2 cells subjected to LPS,or OGD/R,which could be blocked by GLB.Importantly,activation of SUR1-TRPM4 in BV2 cells required the P2X7 receptor(P2X7R)-mediated Ca2+influx,which in turn magnified the K+efflux via the Na+ influx-driven opening of K+channels,leading to the NLRP3 inflammasome activation.ConclusionGLB alleviates brain injury after CA/CPR through the direct anti-inflammatory neuroprotective effect,which may be attributed to the blockage of SUR1-TRPM4-mediated K+efflux and consequent microglial NLRP3 inflammasome activation.