| Objective: An in vitro model of ischemic stroke was established by oxygenglucose deprivation(OGD)of HT22 cells to investigate the effects of M2 microgliaderived exosomes(M2-EXO)on HT22 cells after OGD,and bioinformatics was used to predict the molecular mechanism by which M2-EXO exerts neuroprotective effects after ischemic stroke,providing data reference for basic research on ischemic stroke and the development of neuroprotective drugs.Methods: 1.CCK-8 assayed the viability of HT22 cells after OGD 1,2,3 and 6h to screen the optimal modeling time.BV2 cells were treated with interleukin-4(IL-4)for 24 h,and the expression of M2 phenotypic markers CD206 and Arg-1 were detected by Western Blot.BV2 cell-derived exosomes were extracted and identified,exosome membranes were labeled with PKH26,and the uptake of BV2 cell-derived exosomes by HT22 cells was observed under fluorescence microscope.The CCK-8 was used to assess the viability of OGD HT22 cells before and after intervention with different volumes(1μL,5μL and 10μL)of M2 BV2 cell-derived exosomes and to screen the effect concentration of exosomes.The expression levels of LDH and NO in the supernatant of HT22 cells before and after exosome intervention were measured using LDH and NO kits,and the effects of exosomes on the expression levels of Bax and Bcl-2 in HT22 cells after OGD were determined by Western Blot.2.Primary microglia were treated with IL-4 for 12 h and 24 h,and the expression of CD206 was identified by immunofluorescence,and the expression of M2 phenotypic markers CD206 and Arg-1 were determined by Western Blot.M2 primary microglia conditioned medium(M2-CM)was collected,and the expression of pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2 in OGD HT22 cells before and after M2-CM intervention were detected by Western Blot.3.The up-regulated differentially expressed mi RNAs in M2-EXO or M2-CM were obtained by searching databases and literature,and the mi RNA target genes were predicted.Brain tissue differentially expressed genes(DEGs)in mice after middle cerebral artery occlusion/re-perfusion(MCAO/R)were retrieved from database and analyzed using online tool.The mi RNA target genes were intersected with the upregulated DEGs in the brain tissue of mice after MCAO/R.GO functional annotation and KEGG pathway analysis were performed on the intersecting genes,and the proteinprotein interaction(PPI)network was constructed.Visualized PPI networks and screened key genes;and constructed mi RNA-m RNA regulatory networks.Results: 1.Cell viability of HT22 cells was reduced by OGD for 2,3 and 6h,3h was selected as the optimal modeling time(P<0.0001).Expression of CD206(P<0.05)and Arg-1(P<0.01)was upregulated in BV2 cells after 24 h of IL-4 treatment.Exosomes derived from M0 and M2 BV2 cells were extracted successfully,and it was observed that both exosomes could be taken up by HT22 cells.M2 BV2 cell-derived exosomes(5μL and 10μL)treatment significantly increased the cell viability of HT22 cells after OGD,and 10μL M2 BV2 cell-derived exosomes(0.11μg/μL)intervention had the best effect(P<0.0001).After OGD,the expression levels of LDH(P<0.01)and NO(P<0.01)in the supernatant of HT22 cells were significantly increased,and M2BV2 cell-derived exosomes(0.11μg/μL)intervention reversed the above effects(P<0.05).Western Blot result showed that M2 BV2 cell-derived exosomes treatment downregulated Bax expression(P<0.05)and upregulated Bcl-2 expression(P<0.01)in HT22 cells after OGD,attenuating neuronal apoptosis.2.Immunofluorescence identification results showed that the expression level of CD206 in primary microglia cells was elevated after 12 h intervention with IL-4(P<0.01),and Western Blot results further showed that the expressions of CD206(P<0.05)and Arg-1(P<0.001)were increased after 12 h intervention with IL-4,which successfully induced M2 phenotype primary microglia.M2-CM treatment downregulated Bax expression(P<0.05)and upregulated Bcl-2 expression(P<0.01)in HT22 cells after OGD.3.6 literatures meeting inclusion criteria were retrieved through Pub Med and Web of Science,and 9 up-regulated mi RNAs that were differentially expressed and validated in M2-EXO were obtained.The mi RNA target genes were intersected with the upregulated DEGs after mice MCAO/R,and there were 153 intersected genes.The results of GO functional annotation and KEGG pathway analysis predicted that these genes could be enriched in apoptosis and PI3K-AKT signaling pathway.14 key genes were screened by constructing PPI network of intersecting genes,and 8 genes could be involved in apoptosis by regulating PI3K-AKT signaling pathway.They were respectively Itgav,Itga5,Itga6,Spp1,Col1a1,Col1a2,Col4a1 and Lamc1.The mi RNA-m RNA regulatory network showed that these 8 genes were regulated by mi R-124,mi R-186,mi R-137,mi R-218 and mi R-26a-5p.Conclusion: M2 BV2 cell-derived exosomes increased the viability of HT22 cells after OGD,decreased LDH and NO levels in cell supernatants,and reduced neuronal apoptosis by regulating the expression of apoptosis-associated proteins Bax and Bcl-2.It was further verified by primary microglia that M2-CM could downregulate Bax expression and upregulate Bcl-2 expression in HT22 cells after OGD,thus attenuating apoptosis.The results of bioinformatics analysis predicted that M2 microglia might regulate target gene expression through mi R-124,mi R-186,mi R-137,mi R-218 and mi R-26a-5p in exosomes,thereby affecting PI3K-AKT signaling pathway,alleviating neuronal apoptosis,and providing reference for further studies to follow. |
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