Mechanism Of Apolipoprotein E On NF-?B Signaling Pathway And Matrix Metalloproteinase-9 In Astrocytes

Objective To detect the expression of p65/NF-KB and matrix metalloproteinase-9(MMP-9)in brain tissues of ApoE-knockout(ApoE-/-)and wild-type(WT)mice with experimental autoimmune encephalomyelitis.Exploring the possible mechanism by which ApoE affects the secretion of MMP-9 by astrocytes,whether it is regulated by the TNF-?/NF-?B inflammatory pathway.Using bioinformatics techniques,explore the differential genes and key pathways that TNF-? stimulates astrocytes.Methods1.The EAE model was established by immunization-induced wild-type and ApoE knockout mice,and the expression of MMP-9 and p65 in brain tissue of each group was detected by immunohistochemistry,and experimental animals can be divided into 4 groups:W-EAE group,W-Control group,E-EAE group and E-Control group.2.WT and ApoE-/-mouse primary astrocytes were cultured and identified by immunofluorescence;3.Lentiviral packaging siRNA interferes with p65 gene expression in primary astrocytes of WT and ApoE-/-mice,and then these astrocytes can be divided into 6 groups:W-KD group,W-NC group,W-Control group and E-KD group,E-NC group and E-Control group.4.RT-PCR and Western Blot were used to detect the silencing efficiency of p65 gene,and the effect of low expression of p65 on the secretion of MMP-9 and TNF-? by astrocytes was observed.5.After low expression of p65,ELISA and RT-PCR were used to detect the effect of TNF-? intervention on the expression of MMP-9 in WT and ApoE-/-mouse astrocytes.6.GEO database download GSE73022,through KEGG-pathway,protein interaction network and GO analysis,to identify the key pathways of TNF-? for differential gene and enrichment of wild-type mouse astrocytes.Result1.According to Weaver's 15-point scale,the ApoE-/-group score was higher in the chronic phase(Day 35)than in the WT group(P<0.05).2.The results of immunohistochemistry showed that the secretion of p65 and MMP-9 in EAE brain tissue of ApoE-/-mice was higher than that of WT-EAE(P<0.05);3.Immunofluorescence identification of astrocytes The cells were observed by inverted microscope.The purity of the cultured mouse primary astrocytes was>90%,which could be the subsequent experimental cells;4.The mRNA level and protein level test results showed that the lentiviral-packaged siRNA could significantly inhibit p65.Expression in astrocytes in vitro(P<0.05);5.RT-PCR results showed that compared with the negative empty group and the control group,MMP-9 in the p65 gene silencing group showed low expression(P<0.05).The ELISA results showed that the expression levels of MMP-9 in E-KD group and W-KD group were lower than those in normal group(P<0.05).The level of MMP-9 expressed by astrocytes in E-KD group was slightly higher than that in W-KD group(P<0.05).6.After adding 50ng/ml TNF-? for 24 hours,the ELISA results showed that the expression of MMP-9 in NC group and Control group was higher than that before inflammatory factor treatment(P<0.05).The expression level of MMP-9 in astrocytes of T-KD group was slightly higher than that of KD group,but the difference was not statistically significant(P<0.05),but the expression level of ApoE-/-group was higher than that of WT group(P<0.05).7.R language analysis found after TNF-? stimulated astrocytes,it was found that 386 up-regulated genes and 181 down-regulated genes,and the obtained core genes include chemokine CXCL9/10/11,IL-6,Tnfaip3,MYD88,STAT2,etc.;differential gene enrichment pathways include TNF signalingpathway,Toll-like receptor signaling pathway,NF-?B signaling pathway and NOD-like receptor signaling pathway.Conclusion1.ApoE deficiency in the chronic phase will aggravate the EAE condition,and ApoE-/-mouse EAE brain tissue p65,MMP-9 is highly expressed;2.p65 gene is highly expressed in ApoE-/-mouse astrocytes,p65 can regulate TNF-? Secretion affects MMP-9 expression;3.Under the action of pro-inflammatory factor TNF-?,the expression of MMP-9 in astrocytes increased,indicating that TNF-? can positively regulate the expression of MMP-9 in astrocytes;4.After blocking NF-?B pathway,the effect of TNF-? on the expression of MMP-9 in astrocytes was inhibited,indicating that TNF-? can regulate the expression of MMP-9 by astrocytes through NF-?B pathway,and ApoE deficiency causes an increase in the secretion of MMP-9 in astrocytes;5.Bioinformatics study about the effect of TNF-? on astrocytes can provide new ideas for the treatment of central nervous system-related diseases for anti-inflammatory effects.