| Objective:Intracranial neurological ischemic stroke is the most common cerebrovascular disease worldwide today,and it places a heavy burden on modern social health care systems and individual families because of its high morbidity and mortality.After restoration of blood flow in the ischemic area of the brain,further damage to the ischemic tissue,cerebral ischemia-reperfusion injury(CIRI),is caused by the deregulation of cellular ion homeostasis,accumulation of reactive oxygen species,and inflammatory response.Nuclear factor E2-related factor 2(Nrf2),a transcription factor that regulates endogenous antioxidant defense,has been shown to be involved in the defense mechanism of the brain to protect itself from ischemia-reperfusion injury.NLRP3 inflammasome is a newly discovered protein complex involved in the process of brain ischemia/reperfusion(I/R)induced injury.In this study,we discuss the role of Nrf2 transcription factor in the regulation of NLRP3 inflammasomes during oxygen glucose deprivation and reoxygenation in simulated endothelial cells in vitro.Methods:1.bEnd.3 cells(mouse brain microvascular endothelial cells)were cultured in vitro and treated with oxygen glucose deprivation and reoxygenation(OGDR),and the CCK-8 method was used to detect the cell viability at each time point of OGD2 h R0h、R6h、R12h、R24h、OGD4h R0h、R6h、R12h、R24h、OGD6h R0h、R6h、R12h、R24h,find the point in time when the cell survival rate is 50%.According to the experimental actually requiring,we have to choose the following methods which m RNA and protein expression levels of Nuclear factor E2-related factor 2 and NLRP3 at OGD6 h R0h,R6 h,R12h and R24 h were detected by RT-qPCR and Western blot.Combined with the time points selected by CCK-8 test,the time points with low Nuclear factor E2-related factor 2 expression and high NLRP3 expression and the time points with high Nuclear factor E2-related factor 2 expression and low NLRP3 expression were selected as the right time points.2.bEnd.3 cells were pretreated with tBHQ(an Nrf2-specific agonist)and ML385(an Nrf2-specific inhibitor),Nrf2 protein expression was detected by Western blot,cell viability was detected by CCK-8 method,and the effect concentrations of tBHQ and ML385 were selected,followed by OGDR stimulation.Experiment vichels as Western blot was used to detect the expression level of Nuclear factor E2-related factor 2,NLRP3 and caspase-1,and ELISA was used to detect IL-18 and IL-1 β Cell immunofluorescence method was used to observe the transfer of Nuclear factor E2-related factor 2 to the nucleus.The actual exist condition level of reactive oxygen species(ROS)in vascular endothelial cells was found by taken fluorescent probe.Results:1.CCK-8 showed that at the same oxygen and glucose deprivation time,the cell viability decreased with the prolongation of reoxygenation time,and at the same reoxygenation time,the cell viability decreased with the prolongation of oxygen and glucose deprivation time.Based on the expression levels of Nrf2 and NLRP3 detected by Western blot and RT-qPCR,OGD6 h R6h and OGD6 h R24h were selected as the time points for subsequent experiments.2.The experiment test vichel Western blot results manifeste that after tBHQ pretreatment,the expression of Nuclear factor E2-related factor 2 protein increased,the expression of NLRP3 and caspase-1 protein decreased,and the concentration of IL-18 and IL-1β decreased;After ML385 pretreatment,Nuclear factor E2-related factor 2 protein expression decreased,NLRP3 and caspase-1 protein expression increased,IL-18 and IL-1 β The concentration increases.3.Cellular immunofluorescence showed that Nrf2 nuclear translocation increased after OGDR stimulation,and tBHQ pretreatment enhanced the nuclear entry trend of Nrf2,while ML385 inhibited the nuclear entry trend;loading fluorescent probes showed that ROS production increased significantly after OGDR stimulation,while Nrf2 could inhibit the production of ROS.Conclusion:1.Nrf2 into the nucleus of endothelial cells increased after OGDR stimulation.2.Nrf2 can decrease ROS production.3.Nrf2 negatively regulates NLRP3 inflammasome in OGDR-induced endothelial cells. |
