Role Of KEAP1/NRF2/ARE Signaling Pathway In Regulating Mitophagy In Oxygen-glucose Deprivation Reperfusion Injury Of SH-SY5Y Cells

ObjectiveRegulation of mitochondrial fission,mitochondrial fusion and mitophagy play a key role in maintaining mitochondrial homeostasis,which is the key to maintaining cell function and reducing cerebral ischemia-reperfusion injury.The purpose of this study was to investigate the activation of KEAP1 / NRF2 / ARE signaling pathway during cerebral ischemia-reperfusion and its role in regulating mitophagy.MethodsWe used the human neuroblastoma cells SH-SY5 Y oxygen glucose deprivation model to simulate cerebral ischemia-reperfusion injury,and randomly divided the cells into different groups:We used human neuroblastoma cell SH-SY5 Y oxygen glucose deprivation model to simulate cerebral ischemia-reperfusion injury.After the cells were cultured to a good growth state,the density was about 70%,and they were randomly divided into four groups:(1)Control group(group C): normal culture,without any special treatment;(2)Ischemia/reperfusion group(I/R group): After deprivation of oxygen-glucose for 4 h,the oxygen-glucose supply was restored for 18 h,that is,the ischemia/reperfusion model was successfully constructed;(3)Brusatol +Ischemia/reperfusion group(Bru+I/R group): KEAP1/NRF2/ARE signaling pathway inhibitor Brusatol(Bru)100nM was added 4 h before ischemia,followed by ischemia and reperfusion procedure;(4)Vehicle group(Veh+I/R group): dimethyl sulfoxide(DMSO)was added 4 h before ischemia,and the final concentration was controlled to <0.1% due to its cytotoxicity.Then,the process of ischemia and reperfusion is performed.Transmission electron microscopy was used to observe the mitochondrial ultrastructure.Western blot was used to detect the expression of the pathway-related proteins KEAP1,NRF2,the mitochondrial fission-related proteins Drp1,Fis1,the mitochondrial apoptosis-related proteins BAX,BCL-2 and the expression of mitophagy-related proteins LC3?/LC3?,Beclin1,Tom20 and P62.Fluorescence microscope was used to observe the translocation of NRF2 protein from cytoplasm to nucleus.Live cell / dead cell double staining and Cell Counting Kit-8 were used to detect cell viability.Apoptosis rate was determined by flow cytometry.Results1.Compared with group C,the expression level of Beclin1 and LC3?/LC3? protein in group I / R was increased,while the expression level of p62 and Tom20 protein was decreased(P < 0.05);the autophagic bodies surrounded by the bilayer membrane were observed by transmission electron microscopy,and mitophagy was activated.2.Compared with group C,the NRF2 in the nucleus was increased in the I/R group (P<0.05),and the NRF2 was translocated into the nucleus by the immunofluorescence observation.Therefore,the KEAP1/NRF2/ARE pathway is activated.3.In the Bru+I/R group,the pathway inhibitor Brusatol was added.The level of NRF2 protein in the nucleus was significantly decreased by western blot(P<0.05).The translocation of NRF2 into the nucleus was not observed in immunofluorescence,and the cytoplasmic level was also decreased,the pathway is suppressed.Compared with the I/R group,the expression levels of mitochondrial autophagy-related proteins Beclin1 and LC3?/LC3? decreased,the expression levels of P62 and Tom20 increased in the Bru+I/R group(P<0.05).The mitophagy structure was not observed,mitophagy was inhibited.These have verified that the KEAP1/NRF2/ARE pathway regulates mitophagy.4.Compared with the I / R group,the Bru + I / R group inhibits the KEAP1/NRF2/ARE pathway and mitophagy,the expression levels of mitochondrial fission-related proteins DRP1 and FIS1 increased,the levels of pro-apoptotic proteins BAX increased,and apoptosis-inhibitory proteins BCL-2 expression decreased,the number of living cells decreased,and the apoptosis rate increased by flow cytometry.It was proved that the mitochondria divided increased,the apoptosis rate increased,and the cell damage increased.ConclusionDuring cerebral ischemia-reperfusion,the KEAP1 / NRF2 / ARE signaling pathway can activate mitophagy to clear damaged mitochondria and maintain mitochondrial homeostasis,thereby reducing apoptosis and exerting brain protective effects.