EZH2 Aggravates Ischemic Brain Injury By Promoting Pro-Inflammatory Microglial Activation

Background:Stroke has become a major cause of death and disability worldwide.The latest epidemiological studies have found that 10.3 million new stroke patients occur globally every year.Stroke imposes a high burden on individuals.and society,especially in low-and middle-income countries.By 2020,the burden of stroke will rise to 61 million disability-adjusted life years.EZH2(Enhancer of zeste homolog-2),a histone methyltransferase of the catalytic component of the polycomb Repressor(PCR2),has been widely reported to play a vital role in immune regulation in recent years,especially in tumor-related immune and autoimmune diseases.However,its role in microglial activation and ischemic brain injury after ischemic stroke remains to be further explored.The aim of this study was to explore the role of EZH2 in microglial activation-related inflammatory response after ischemic stroke,and to further determine the effect of EZH2 inhibition on ischemic brain injury,in order to reveal novel therapeutic targets for ischemic stroke.Methods:In vivo,we constructed a middle cerebral artery occlusion model(MCAO)in 8 weeks-old male C57BL/6(B6)mice.EZH2 inhibitor DZNep(0.05 mg/kg)was given intravenously 24 hours before MCAO and once a day after MCAO.The volume of cerebral infarction and neurological deficit were assessed by TTC staining,mNSSs score,rotarod test and grip strength.In vitro,we used primary microglia to construct oxygen-glucose deprivation model(OGD)and also treated with EZH2 inhibitor DZNep(10uM).The expression levels of EZH2,STAT3 and P-STAT3 were detected by immunofluorescence staining(IF)and Western blotting.Microglial activation phenotype was evaluated by IF staining and flow cytometry in vivo and in vitro.The expression levels of various inflammatory factors were detected by RT-qPCR.Results:(1)Both in vivo ischemia/reperfusion injury(I/R)and in vitro OGD/reperfusion injury(OGD/R)induced significant up-regulation of EZH2 expression in microglia.(2)DZNep,an inhibitor of EZH2,can improve the neurological impairment and reduce the volume of cerebral infarction in mice after MCAO.(3)Inhibiting EZH2 can block the activation of CD86+microglia induced by MCAO and OGD,and increase the percentage of CD206+microglia.(4)EZH2 Inhibition can reduce the high expression of inflammatory cytokines induced by MCAO and OGD,such as IL-1β,IL-6,TNF-α and CXCL10.(5)The phosphorylation level of STAT3 in microglia increased significantly after MCAO and OGD.EZH2 Inhibition decreased the phosphorylation level of STAT3.Conclusions:(1)We confirmed that of EZH2 was significantly up-regulated in microglia and positively correlated with pro-inflammatory microglial activation after ischemia/reperfusion injury.EZH2 Inhibition can significantly alleviate the pro-inflammatory response induced by I/R and OGD/R in vivo or in vitro,thus improving behavioral performance and reducing infarct size.(2)We demonstrated that EZH2 may regulate the pro-inflammatory response of microglia after I/R injury by activating STAT3.