Effect Of Apolipoprotein E On Autophagy Of Astrocytes Under Oxygen Glucose Deprivation

Objective:Ischemic stroke refers to the brain tissue ischemia,hypoxic necrosis,encephalomalacia,resulting in interruption of blood supply to the brain,insufficient collateral circulation,leading to neurological dysfunction.Autophagy is a highly conserved lysosome dependent degradation pathway that widely exists in eukaryotic cells.Through this pathway,long-lived proteins,damaged organelles and misfolded proteins are degraded and recycled to maintain cell homeostasis and normal cell function.Astrocytes are the most abundant cell type in the central nervous system.Autophagy of astrocytes can be over activated after cerebral ischemia,which can change their survival status and function,thus affecting the recovery of neural function after cerebral ischemia.Therefore,it is very important to reduce the damage of astrocytes after ischemic stroke for the prognosis of neural function after stroke.Apolipoprotein E(ApoE)is a kind of glycoprotein which is mainly secreted by astrocytes in the central nervous system and has the biological functions of neuroprotection,neurotrophic,cleaning metabolites,regulating immunity and so on.Some studies have shown that in Alzheimer’s disease,apoE4 can down regulate the transcription of autophagy gene products,thereby down regulating the autophagy level of astrocytes and leading to the decline of the ability of amyloid plaque clearance.However,there are few studies on the effect of ApoE on autophagy and survival of astrocytes after ischemic injury.Therefore,we verified the changes of autophagy activity of astrocytes after ischemic brain injury by using in vitro glucose and oxygen deprivation(OGD)model of astrocytes,explored the effect of autophagy on the survival of astrocytes after ischemic brain injury,and tried to explore the effect of ApoE on the autophagy and survival of astrocytes after glucose and oxygen deprivation,so as to provide a new theory for the treatment of ischemic stroke basis.Methods:The cerebral cortex of neonatal rats was extracted and astrocytes of C57BL/6J apoE knockout(ApoE-/-)and wild-type(WT)neonatal rats were cultured in vitro.The experiment was divided into wt control group,ApoE-/-control group,WT OGD group and ApoE-/-OGD group.The astrocytes in the control group had no intervention,while the astrocytes in the OGD group were cultured in glucose free serum-free medium for 4 hours to establish OGD cell model.The purity of astrocytes was determined by immunofluorescence;the survival rate and OGD duration of astrocytes were determined by CCK8;the apoptotic rate of astrocytes in WT control group,ApoE-/-control group,WT OGD group and ApoE-/-OGD group was detected by flow cytometry;the apoptotic rate of astrocytes in WT control group,ApoE-/-control group,WT OGD group and ApoE-/-OGD group was detected by RT qPCR-/-Western blotting was used to detect the protein expression levels of autophagy related proteins lc3b,p62 and Beclinl in astrocytes of WT control group,ApoE-/control group,WT OGD group and ApoE-/-OGD group,so as to determine the effect of ApoE on autophagy and survival of astrocytes after oxygen glucose deprivation.Results:1.The proportion of astrocytes reached more than 95%,which met the experimental requirements.2.Cell survival and functional status:Cell survival was reduced in the OGD group compared to the control group(P<0.05),apoptosis rate increased(p<0.05);In the control group,compared with WT group,the apoptosis rate of ApoE-/-group showed an increasing trend,but there was no statistical difference(p>0.05);In the OGD group,cell survival rate of WT OGD group was significantly higher than that of ApoE-/-OGD group(p<0.05),apoptosis rate in WT OGD group was significantly lower than that in ApoE-/-OGD group(p<0.05),APOE significantly improved the survival and functional status of cells after OGD.3.Changes in autophagy-related genes:mRNA expression of LC3B was increased in the OGD group compared with the control group(p<0.05);In the control group,compared with the WT group,LC3b mRNA expression level was slightly higher in the ApoE-/-group,but there was no statistical difference(p>0.05).In the OGD group,the mRNA expression level of LC3B in the ApoE-/OGD group was higher than that in the WT OGD group(p<0.05),APOE inhibited the over-autophagy of astrocytes after OGD.4.Changes in autophagy related proteins:Beclinl expression and LC3b Ⅱ/LC3b Ⅰ ratio were significantly increased in OGD group compared with the control group(p<0.05),the expression level of P62 was decreased(p<0.05);In the control group,there were no significant differences in P62 and Beclinl expression levels and LC3b Ⅱ/LC3b I ratio between WT and ApoE-/-groups(p>0.05).In the OGD group,LC3b Ⅱ/LC3b I expression level of Beclinl autophagy marker in WT OGD group was lower than that in ApoE-/-OGD group(p<0.05),the expression level of P62 in WT OGD group was higher than that in ApoE-/-OGD group(p<0.05),APOE inhibited the over-autophagy of astrocytes after OGD.Conclusion:1.Autophagy of astrocytes is activated after glucose deficiency and hypoxia.2.Apolipoprotein E can inhibit the autophagy of astrocytes after glucose oxygenation deprivation,thereby reducing the damage of astrocytes caused by glucose deficiency and hypoxia.