Vasorin过表达对巨噬细胞极化及急性炎症反应的影响OA

Objective:To investigate the expression and function of Vasorin(Vasn)in the polarization of mouse monocyte-macrophage leukemia cells(RAW 264.7),providing a foundation for the mechanistic study of various inflammatory diseases.Methods:Macrophages were induced to undergo M1/M2 polarization using lipopolysac-charide(LPS)and interleukin(IL)-4 respectively,and the expression of Vasn was detected.A stable Vasn-overexpressing cell line was constructed using lentiviral technology.Experimental groups included a control(con-trol)group,a negative control(NC)group,and an overexpression(OE)group.Additionally,each of these groups was further subdivided into untreated,LPS-treated,and IL-4-treated subgroups.Expressions of polarization mark-ers and inflammatory factors were assessed using nitric oxide(NO)detection,reverse transcription-quantitative polymerase chain reaction(RT-qPCR),enzyme-linked immunosorbent assay(ELISA),and western blotting(WB).In addition,differentially expressed genes were screened through transcriptome sequencing,and the core signaling pathways were identified by GO functional and KEGG pathway enrichment analysis.Finally,the phos-phorylation levels of key proteins in the pathways were verified by WB experiments.Results:The expressions of Vasn mRNA and Vasn protein were significantly downregulated following LPS stimulation,and were signifi-cantly upregulated following IL-4 stimulation(all P＜0.05).Compared with those in the NC group,the Vasn mRNA and Vasn protein expression levels in the OE group were significantly elevated(all P＜0.05).After LPS stimulation,the cellular NO concentration,secretion levels and mRNA expressions of inflammatory factors in-cluding mouse tumor necrosis factor-alpha(TNF-α),IL-6 and IL-1β,as well as the M1 polarization marker pro-teins cluster of differentiation 86(CD86)and inducible nitric oxide synthase(iNOS),were significantly upregu-lated.In contrast,the secretion levels,marker gene expression,and protein expressions of M1 polarization factors in the OE group were significantly lower than those in the NC group(all P＜0.05).GO functional enrichment analysis and KEGG pathway enrichment were used to identify important signaling pathways including NF-κB.In the OE group,the phosphorylation level of p65,a key protein in the NF-κB pathway,was significantly down-regulated(P＜0.05),which was consistent with the sequencing results.After IL-4 stimulation,the levels of IL-10,the M2 polarization marker proteins arginase-1(ARG1)and mannose receptor(CD206),as well as the mRNA ex-pressions of the M2 polarization genes ARG1,chitinase 3-like protein 1(YM1),and IL-10 were significantly el-evated.Moreover,the secretion levels,marker gene expression,and protein expression of M2 polarization factors in the OE group were significantly higher than those in the NC group(all P＜0.05).GO functional enrichment analysis and KEGG pathway enrichment were uesd to identify important signaling pathways including JAK-STAT.In the OE group,the phosphorylation level of STAT6,a key protein in the JAK-STAT signaling pathway,was significantly enhanced(P＜0.05),which was consistent with the sequencing results.Conclusion:Vasn not only inhibits LPS-induced M1 polarization of macrophages by regulating the NF-κB pathway,alleviating the in-flammatory response,but also enhances IL-4-induced M2 polarization and anti-inflammatory responses by pro-moting STAT6 phosphorylation.