Effects And Mechanisms Of Microglia-Mediated Neuronal Loss In Peri-Infarct Area Based On SUR1-TRPM4 Channel

BackgroundIn the past few decades,stroke has become a leading cause of death and disability worldwide,of which ischemic stroke（IS）accounting for approximately 70%.There are two key regions in the pathophysiological process of IS:infarct core and peri-infarct area.It has been acknowledged that neurons in the peri-infarct area are salvageable with timely vascular recanalization.However,microglia has been validated to act as a critical secondary injury factor that damages neural cells in IS especially in the peri-infarct area.Therefore,inhibition of microglia-mediated neuronal loss in the peri-infarct area may be a promising target for rescuing damaged neurons and improving the prognosis of patients after ischemia and recanalization.After IS,all cells in the neurovascular unit in ischemic area are induced to express a newly-formed sulfonylurea receptor 1-transient receptor potential M4（SUR1-TRPM4）channel,engaged in inducing brain edema and blood brain barrier disruption.Trpm4^-/-and glibenclamide（a SUR1 inhibitor）can reduce the infarct volume and inhibit the formation of brain edema.In various types of acute brain injury models with or without brain edema,SUR1-TRPM4 channel is also up-regulated in microglia with the application of Trpm4^-/-and glibenclamide capable of inhibiting the activation of microglia and secondary inflammatory response.Therefore,SUR1-TRPM4 channel in microglia may play a direct role in regulating inflammatory response.However,the specific mechanisms remain to be explored.Based on this,we hypothesized that blocking SUR1-TRPM4 channel of microglia could alleviate neuronal loss in the peri-infarct area.Methods1.Transient middle cerebral artery occlusion（tMCAO）animal model was used to verify the protective effects of glibenclamide and Trpm4^-/-on neurons in the peri-infarct area.2.RNA-seq was used to analyze the differentially expressed genes between the wild-type mice and Trpm4^-/-mice.3.Two types of BV-2 microglia models（OGD and LPS）were constructed to evaluate the expression of SUR1 and TRPM4 after modeling,and to explore the relative signaling pathway.4.The apoptosis of SH-SY5Y neurons induced by the condition medium collected from BV-2 microglia models were evaluated to detect the signal transmission process between microglia and neurons.Results1.After tMCAO,abundant neuronal apoptosis appeared in the peri-infarct area,which could be inhibited by glibenclamide and Trpm4^-/-.SUR1-TRPM4 channel was significantly up-regulated with the majority of which colocalized with microglia in the peri-infarct area.2.RNA-seq suggested that TGFa and its relative pathway were up-regulated in Trpm4^-/-mice.Application of glibenclaimide and Trpm4^-/-induced the up-regulation of TGFa in microglia in the peri-infarct area.3.Glibenclamide and Trpm4^-/-up-regulated the expression of TGFa in microglia.4.In the tMCAO mouse model,intracerebroventricular injection of TGFa significantly alleviated the neuronal apoptosis in the peri-infarct area.5.In BV-2 cell models,SUR1-TRPM4 channel was up-regulated after modeling.Blocking SUR1-TRPM4 with glibenclamide and 9-phenanthrol up-regulated the expression and release of TGFa by activating CaMKII/CREB pathway.6.BV-2 microglia condition medium after OGD and LPS could induce apoptosis of SH-SY5Y neurons,which could be inhibited by glibenclamide and 9-phenanthrol applied to microglia.7.Application of TGFa alleviated SH-SY5Y neuronal apoptosis mediated by BV-2 microglia condition medium.Conclusions1.After ischemia and reperfusion,SUR1-TRPM4 channel is up-regulated in microglia in the peri-infarct area.2.Blocking SUR1-TRPM4 channel can induce the up-regulation and the release of TGFa and various anti-inflammatory cytokines in the peri-infarct area,thus reducing neuroinflammation and alleviating neuronal apoptosis.