衰老相关炎症通路的生物信息学分析和实验验证OA

Objective To systematically analyze the expression profiles of inflammation-related genes and signaling pathways in the context of normal aging to provide bioinformatics evidence for elucidating the molecular mechanisms of age-related inflammation,followed by experimental validation.Methods The GSE11882 human brain tissue transcriptomic dataset was obtained from the Gene Expression Omnibus(GEO).The limuma package was used to screen for aging-associated differentially expressed genes(DEGs);inflammation-related targets were identified via GeneCards,online Mendelian inheritance in man(OMIM)and therapeutic target database(TTD),and their intersection with the DEGs was calculated to obtain aging-associated inflammatory genes;a protein-protein interaction(PPI)network was constructed using the search tool for the retrieval of interacting gene/proteins(STRING)database,and core targets were selected using Cytoscape software;Genetic Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses were performed on the core targets using the DAVID database to investigate their biological functions and regulatory pathways.Finally,a lipopolysaccharide(LPS)-induced microglial senescence and inflammation model was established,comprising control group(conventional culture in complete medium),model group(1 μg·mL-1 LPS)and experimental group[10 μg·mL-1 platelet factor 4(PF4)+1 μg·mL-1LPS].Western blotting(WB)was used to detect the cyclin-dependent kinase inhibitor 2A(CDKN2A)encoding p16 protien and CDKN1A encoding p21 protein using Western blotting.Changes in the levels of the inflammatory factors interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and nitric oxide(NO)in microglia were detected using an enzyme-linked immunosorbent assay(ELISA).Results A total of 134 aging-associated differentially expressed genes(DEGs)were identified,including 82 up-regulated and 52 down-regulated genes;after intersecting with inflammation-related targets,22 aging-associated inflammatory genes were identified.The top five core targets were somatostatin(SST),glial fibrillary acidic protein(GFAP),adenosine A3 receptor(ADORA3),hemoglobin beta subunit(HBB),and myeloid trigger receptor 2(TREM2),all of which are closely associated with neuroimmune regulation.GO enrichment analysis revealed that the core targets are primarily involved in biological processes such as inflammatory responses;KEGG pathway enrichment analysis indicated that the core targets are mainly enriched in pathways related to inflammation and neural signaling regulation,such as neuroactive ligand-receptor interactions.The relative expression levels of p16 in the control,model and experimental groups were 0.80±0.04,1.63±0.08 and 0.46±0.06,respectively;the relative expression levels of p21 were 0.84±0.13,1.41±0.07 and 0.67±0.10,respectively;the IL-6 levels were 0.20±0.01,428.50±22.07 and 265.20±23.89,respectively;TNF-α levels were 0.14±0.05,28.50±2.17 and 17.16±0.90,respectively;IL-1 β levels were 182.50±87.42,3 015.00±192.10 and 1 842.00±132.10,respectively;NO levels were 6.40±0.44,20.82±0.81 and 12.09±0.17,respectively.Comparisons of the above indicators between the two groups all revealed statistically significant differences(P＜0.001,P＜0.0001).Conclusion The interaction between inflammatory responses and neural signaling pathways may be a key mechanism mediating age-related neuroinflammation.PF4 may exert its potential anti-aging effects by inhibiting LPS-mediated neuroinflammation and improving cellular senescence phenotypes,thereby providing both theoretical and experimental evidence for the mechanisms underlying age-related neuroinflammation and potential targets for disease intervention.