| Objective:Propofol is a sedative commonly used in emergency treatment,clinical anesthesia and intensive treatment.In addition to sedation,it also has brain protective effects.However,its specific signaling pathway has not been fully elucidated.The purpose of this study was to investigate the role of Keap1/Nrf2 signal pathway in the protective effect of propofol on oxygen-glucose deprivation-reoxygenation(OGD/R)injury in rat neurons.Methods:The rat primary hippocampal neuron oxygen-glucose deprivation(OGD6h/R20h)model was used to simulate cerebral ischemia/reperfusion injury.The experiment was divided into four parts.The first part is divided into four groups: Control group(group C),vehicle group(group V),oxygen sugar deprivation reperfusion group(OGD/R group)and Propofol group(group P).Control group had no special treatment.The vehicle group was immediately added to DMSO after reoxygenation and glycogen,the final concentration was less than 0.1%,and no other treatment was given.Neurons in the OGD reperfusion group and the propofol group were placed in glucose free medium EBSS at37 ℃ in a three-gas incubator for 6 h to prepare OGD model.Then the non-sugar medium was replaced by Neurobasal medium containing neurobasal-A,B27,sodium pyruvate and glutamine,and cultured for 20 h under normal oxygen condition for reoxygenation and glycosylation.In the propofol group,propofol was added immediately after reoxygenation and glycogen,and the final concentration was 100 nm.The LC3Ⅰ、LC3Ⅱ、Beclin、P62 and TOMM20 expression of autophagy protein were detected by Western Blot.MRFP-GFP-LC3(autophagy flow)was used to detect the changes in the number of red fluorescent protein and green fluorescent protein and to determine the changes in the number of autophagosome and lysosome.The changes of mitochondria and autophagosomes were observed by transmission electron microscopy.The second part explores the protective effect of mitochondrial autophagy induction on the brain during oxygen-glucose deprivation and reoxygen-glucose reoxygenation.It can be divided into control group(group C),Vehicle group(group V),oxygen sugar deprivation reperfusion group(OGD/R group),propofol group(group P),rapamycin group(mitochondrial autophagy activator,RAPA group),propofol and rapamycin group(P+ RAPA group);Control group had no special treatment.The vehicle group was immediately added to DMSO after reoxygenation and glycogen,the final concentration was less than 0.1%,and no other treatment was given.Neurons in the OGD reperfusion group,the propofol group,the rapamycin group and the propofol plus rapamycin group were placed in glucose free medium EBSS at 37 ℃ in a three-gas incubator for 6 h to prepare OGD model.Then the non-sugar medium was replaced by Neurobasal medium containing neurobasal-A,B27,sodium pyruvate and glutamine,and cultured for 20 h under normal oxygen condition for reoxygenation and glycosylation.In the propofol group,propofol was added immediately to the reoxygenated retose with a final concentration of 100 nm.In the rapamycin group,rapamycin was added immediately to the reoxygenated retose with a final concentration of 30μm.The expression of apoptotic protein Bax、Bcl-2 was detected by Western Blot.JC-1 Mitochondrial Membrane Potential Detection Kit was used to detect the level of early apoptosis.And the apoptosis rate was determined by flow cytometry.The third part explores the effect of propofol on Keap1/Nrf2 pathway activation.They were divided into control group(group C),Vehicle group(group V),oxygen sugar deprivation reperfusion group(OGD/R group),propofol group(group P),brusatol group(Keap1/Nrf2 pathway inhibitor,Bru group);Control group had no special treatment.The vehicle group was immediately added to DMSO after reoxygenation and glycogen,the final concentration was less than 0.1%,and no other treatment was given.Neurons in the OGD reperfusion group,the propofol group and the brusatol group wwere placed in glucose free medium EBSS at 37 ℃ in a three-gas incubator for 6 h to prepare OGD model.Then,the sugar-free medium was replaced by neurobasal-A,B27,sodium pyruvate and glutamine.The medium was cultured for 20 h under normal oxygen condition for reoxygenation and glycosification.Propofol was added to the group immediately after reoxygenation and glycogen,and the final concentration was 100 nm.In brusatol group,brusatol was added immediately after reoxygenation and glycogen,and the final concentration was100 nm.Western blot method was used to detect the expression of Nrf2 protein,and immunofluorescence method was used to detect whether nuclear translocation occurred during OGD reperfusion of Nrf2.The forth part explores the role of mitochondrial autophagy in Keap1/Nrf2 pathway induction.And it was divided into Control group(group C),Vehicle group(group V),Oxygen sugar deprivation reperfusion group(OGD/R group),propofol group(group P),TBHQ group(Keap1/Nrf2 pathway activator),Propofol plus TBHQ group(P+ TBHQ group);Control group had no special treatment.The vehicle group was immediately added to DMSO after reoxygenation and glycogen,the final concentration was less than 0.1%,and no other treatment was given.Neurons in the OGD reperfusion group,the propofol group,the TBHQ group and the propofol plus TBHQ group were placed in glucose free medium EBSS at 37 ℃ in a three-gas incubator for 6 h to prepare OGD model.Then,the sugar-free medium was replaced by neurobasal-A,B27,sodium pyruvate and glutamine.The medium was cultured for 20 h under normal oxygen condition for reoxygenation and glycosification.Propofol was added to the group immediately after reoxygenation and glycogen,and the final concentration was 100 nm.In the TBHQ group,TBHQ was added immediately after reoxygenation and glycogen,and the final concentration was 5 μM.The expression of autophagy protein LC3Ⅰ、LC3Ⅱ、Beclin、P62、TOMM20 and pathway protein Nrf2 was detected by Western Blot.MRFP-GFP-LC3(autophagy flow)was used to detect the changes in the number of red fluorescent protein and green fluorescent protein and to determine the changes in the number of autophagosome and lysosome.The changes of mitochondria and autophagosomes were observed by transmission electron microscopy.Results:1.The expression of LC3-II and Beclin protein in P group was increased compared with that in the OGD/R group,while the expression of p62 and TOMM20 was decreased(0.05),the difference was statistically significant.m RFP-GFP-LC3 results showed that the amount and intensity of red fluorescence(LC3 expression)in P groups were high.The complete mitochondria and autophagosomes appeared in P group and the number of autophagosomes was higher than that in OGD/R group.2.The expression of Bcl-2 protein in RAPA group was up-regulated and the expression of BAX decreased(P<0.05),the difference was statistically significant.The number of red fluorescent cells in the RAPA group was significantly higher than that in the OGD/R group.The effect of RAPA on apoptosis was detected by flow cytometry,which indicated that rapamycin could significantly inhibit apoptosis.3.Compared with OGD/R group,the expression of Nrf2 in P group was significantly up-regulated(P < 0.05).At the same time,the results of immunofluorescence detection showed that the Nrf2 protein in the cytoplasm had obvious nuclear translocation in this process,that is,the Nrf2 protein moved from the cytoplasm to the nucleus.4.Compared with OGD/R group,the expression of C3-II and Beclin protein increased,while the expression of p62 and TOMM20 decreased in TBHQ group.MRFP-GFP-LC3 results showed that the number and intensity of red fluorescence(LC3 expression)were high in TBHQ group.Transmission electron microscope showed that intact mitochondria and autophagosomes were found in TBHQ group,and the number of autophagosomes was higher than that in OGD/R group. |
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