Effect Of Apolipoprotein E On Astrocytes After Oxygen And Glucose Deprivation And Its Related Mechanism

Objective:To explore the effect of apolipoprotein E（apo E）on astrocytes under oxygen glucose deprivation and related mechanisms.Methods:In vitro cμlture of wild-type（wt）and apo E knock-out(apo E^-/-)astrocytes under the background of C57BL/6J mice.Use anti-glial fibrillary acidic protein（GFAP）antibody to identify the purity of astrocytes.The CCK-8 method was used to determine the duration of oxygen glucose deprivation（OGD）of astrocytes.The wt and apo E^-/-astrocytes were divided into wt control group,wt OGD4h group,apo E^-/-control group,and apo E^-/-OGD4h group.There was no intervention in the control group.Astrocytes in the OGD4h group were cμltured in a sugar-free serum-free medium and hypoxia chamber for 4h.Flow cytometry was used to determine the apoptosis of astrocytes in the wt control group,wt OGD4h group,apo E^-/-control group,and apo E^-/-OGD4h group.The transcript levels of GFAP and vimentin,markers of reactive proliferation,as well as inflammation related molecules TNF-α,IL-1β,IL-6,and Arg-1,of astrocytes in wt control group,wt OGD4h group,apo E-/-control group,and apo E-/-OGD4h group were determined by real-time PCR.The expression of Vimentin protein in astrocytes in wt control group,wt OGD4h group,apo E^-/-control group,and apo E^-/-OGD4h group was determined by immunofluorescence method.The protein expression levels of GFAP,Vimentin and Arg-1 in astrocytes of wt control group,wt OGD4h group,apo E^-/-control group,apo E^-/-OGD4h group were determined by Western blotting to determine the astrocyte OGD The subsequent state changes and the effect of apo E on astrocytes in the OGD process.P<0.05 was considered statistically significant.Result:1.The purity of astrocytes reached more than 96%,which met the requirements of subsequent experiments.2.Astrocytes OGD 2h,4h,6h,cell viability decreased,and the apo E^-/-astrocytes than wt astrocytes activity decreased more obvioushe viability(P_2h=0.004,P_4h=0.009,P_6h=0.033),after OGD 6h,astrocytes vitality gradually rise,wt astrocyte cell vitality in OGD 8h to return to the control group,while the apo E^-/-astrocytes in OGD 10h cell vitality to return to the control level.After 4 hours of OGD,the activity of wt astrocytes was about 65%,and that of apo E^-/-astrocytes was about 35%,which better reflected the damage of cell viability after OGD.3.After astrocyte OGD,the apoptosis rate of apo E^-/-group and wt group both increased(P_{apo E-/-}<0.000 and P_wt=0.007),and the apoptosis of apo E^-/-group was more obvious than that of wt group（P=0.003）.4.Compared with the wt group,the m RNA and protein expression levels of GFAP and Vimentin in the wt OGD4h group increased(P_{GFAP m RNA}=0.046,P_{GFAP protein}=0.000,P_{Vimentin m RNA}=0.01,P_{Vimentin protein}<0.000);Compared with the apo E^-/-group,the m RNA and protein expression levels of GFAP and Vimentin were not significantly different in the apo E^-/-OGD4h group(P_{GFAP m RNA}=0.608,P_{GFAP protein}=0.982,P_{Vimentin m RNA}=0.299,P_{Vimentin protein}=0.281);compared with wt OGD4h group,GFAP m RNA and protein expression levels in apo E^-/-OGD4h group were lower(P_{GFAP m RNA}<0.000,P_{GFAP protein}=0.007),and the m RNA expression levels of Vimentin in the apo E^-/-OGD4h group were not significantly different(P_{Vimentin m RNA}=0.088),but the protein expression levels were low(P_{Vimentin protein}=0.040).5.After astrocyte OGD 4h,the m RNA expression levels of IL-1βand TNF-αin the wt OGD4h group and apo E^-/-OGD4h group increased(P_{wt TNF-α}=0.025,Pwt _IL-1β=0.001 and P_{apo E-/-TNF-α}=0.000,P_{apo E-/-IL-1β}=0.000),and the m RNA expression level of IL-6 in the wt OGD4h group decreased（P=0.041）,the m RNA expression level of IL-6 in the apo E^-/-OGD4h group increased（P=0.034）,and the m RNA and protein expression levels of Arg-1 in the wt OGD4h group and apo E^-/-OGD4h group decreased(P_{wt m RNA}=0.019,P_{wt protein}=0.001,P _{apo E-/-m RNA}=0.016,P _{apo E-/-protein}=0.004),The m RNA expression level of Notch1 in the wt OGD4h group increased（P=0.001）,and the m RNA expression level of Notch1 in the apo E^-/-OGD4h group did not increase significantly（P=0.265）;Compared with the wt OGD4h group,the m RNA expression levels of IL-1β,TNF-αand IL-6 in the apo E^-/-OGD4h group were high(P_TNF-α=0.017,P_IL-1β=0.001,P_IL-6=0.01),the m RNA and protein expression levels of Arg-1 were not significantly different(P _{Arg-1 m RNA}=0.723,P _{Arg-1 protein}=0.924),and the m RNA expression level of Notch1was decreased（P=0.007）.Conclusion:The lack of apo E causes astrocytes to show lower cell viability in the case of oxygen and glucose deprivation,cell proliferation slows down,cell apoptosis increases,and at the same time presents a stronger inflammatory state.The effects of apo E on astrocytes may be mediated by regulation of Notch1expression.These findings elucidate the role of apo E in the ischemic hypoxic environment from a new perspective and provide a new theoretical basis for the treatment of ischemic stroke.