| Objective:Current therapies for ischemia/reperfusion are insufficient because of our poor understanding of the mechanisms of brain injury after ischemic stroke.As a vital component of the innate immune system,NLRP3 inflammasome contributes to ischemic brain injury;however,a detailed understanding of their molecular mechanisms is unknown.This study was designed to investigate the effect of nuclear factor E2-related factor-2(Nrf2)on NLRP3 inflammasome.Methods:1.To investigate whether Nrf2 was involved in OGDR induced cell injury,BV2 microglia cells were cultured in vitro.After OGDR stimulation,the expression of Nrf2 after OGDR 1、2、4、8、16、24h were tested by Western Blot.2.To investigate whether Nrf2 was involved in the regulation of NLRP3 inflammasome in BV2 cells after OGDR exposure,t BHQ and Nrf2 CRISPR plasmid were pretreated before OGDR exposure,respectively.BV2 microglia cells randomly divided into six groups : Control group;OGDR group;OGDR+t BHQ group;OGDR+DMSO group;OGDR+ CRISPR plasmid group;OGDR+PBS group.The expressions of Nrf2,NQO1,NLRP3 inflammasome and IL-1βwere tested by Western Blot.Immunofluorescence was used to test Nrf2 nuclear transfer.The production of ROS was tested by ROS kit.3.To investigate whether ROS was involved in the inhibitory regulation of Nrf2/ARE pathway on NLRP3 inflammasome,NAC was pretreated before OGDR exposure.BV2 microglia cells randomly divided into four groups :Control group;OGDR group;OGDR+NAC group;OGDR+PBS group.The production of ROS was tested by ROS kit.The expressions of Nrf2,NQO1,NLRP3 inflammasome and IL-1βwere tested by Western Blot.Results:1.The expression of Nrf2 was higher in BV2 microglia cells with OGDRexposure for 1、2、4、8、16、24h when compared to BV2 cells without OGDR exposure.2.tBHQ pretreatment significantly increased the expression of Nrf2 and NQO1 but reduced the expression of NLRP3 inflammasome and IL-1β;whereas CRISPR plasmid pretreatment reduced the expression of Nrf2 and NQO1 but increased the expression of NLRP3 inflammasome and IL-1β.3.Nrf2 nuclear transfer increased after OGDR exposure,and tBHQ enhanced the trend of nuclear transfer,while crispr plasmid inhibited the trend.After OGDR stimulation,intracellular ROS significantly increased and Nrf2 activation could inhibit the production of ROS.3.NAC pretreatment inhibited the production of ROS,which then reduced the expressions of NLRP3 inflammasome and IL-1β.Conclusions:1.Nrf2 could inhibit the activation of NLRP3 inflammasome and ROS in BV2 microglial cells after OGDR exposure.2.Nrf2/ARE pathway inhibited ROS induced NLRP3 inflammasome activation in BV2 microglial cells after OGDR exposure. |
