Effects of Vasorin overexpression on macrophage polarization and acute inflammatory response

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 lipopolysaccharide (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 (control) 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 markers 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 phosphorylation 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 significantly 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 including mouse tumor necrosis factor-alpha (TNF-α), IL-6 and IL-1β, as well as the M1 polarization marker proteins cluster of differentiation 86 (CD86) and inducible nitric oxide synthase (iNOS), were significantly upregulated. 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 downregulated (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 expressions of the M2 polarization genes ARG1, chitinase 3-like protein 1(YM1), and IL-10 were significantly elevated. 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 JAKSTAT. 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 inflammatory response, but also enhances IL-4-induced M2 polarization and anti-inflammatory responses by promoting STAT6 phosphorylation.