Vasorin过表达对巨噬细胞极化及急性炎症反应的影响

Effects of Vasorin overexpression on macrophage polarization and acute inflammatory response

摘要

目的：探讨Vasorin（Vasn）在小鼠单核巨噬细胞白血病细胞（RAW 264.7）极化中的表达和功能，为多种炎症性疾病的机制研究提供理论基础。方法：分别使用脂多糖（lipopolysaccharide，LPS）和白细胞介素（IL）-4诱导巨噬细胞M1/M2型极化并检测Vasn的表达；利用慢病毒技术构建 Vasn过表达细胞株，设置空白对照组（control组）、过表达对照组（NC组）和过表达组（OE组），并设立未添加以及添加LPS和IL-4的处理组，检测细胞中一氧化氮（NO）含量，采用实时荧光定量聚合酶链反应（RT-qPCR）、酶联免疫吸附试验（ELISA）及蛋白免疫印迹法（western blotting，WB）等方法，检测极化标志物、炎症因子表达。此外，通过转录组测序筛选差异表达基因，并利用GO功能和KEGG通路富集分析锁定核心信号通路，最后通过WB实验对通路中关键蛋白的磷酸化水平进行验证。结果： Vasn的mRNA及Vasn的蛋白表达在LPS刺激下显著下调，在IL-4刺激下显著上调（均 P＜0.05）；与NC组相比，OE组中 Vasn的mRNA及Vasn的蛋白表达显著上调（均 P＜0.05）；添加LPS后，细胞NO浓度、炎症因子中小鼠肿瘤坏死因子-α（TNF-α）、IL-6、IL-1β分泌水平及其mRNA表达、M1型极化标志蛋白分化簇86（CD86）、诱导型一氧化氮合酶（iNOS）显著上调，而OE组中M1型极化因子的分泌水平、标志基因与蛋白表达均显著低于NC组（均 P＜0.05）；GO功能分析和KEGG通路富集到NF-κB等重要信号通路，OE组中，NF-κB通路关键蛋白p65磷酸化水平显著下调（P＜0.05），与测序结果一致；添加IL-4后，IL-10的水平及M2型极化标志蛋白精氨酸酶1（ARG1）、甘露糖受体（CD206），M2型极化基因 ARG1、几丁质酶3样分子（YM1）、 IL-10的mRNA表达显著升高，而OE组中的M2型极化因子的分泌水平、标志基因与蛋白表达显著高于NC组（均 P＜0.05）；GO功能分析及KEGG通路富集到JAK-STAT等重要信号通路，OE组中JAK-STAT信号通路中关键蛋白STAT6磷酸化水平显著增强（P＜0.05），与测序结果一致。结论：Vasn不仅通过调控NF-κB通路，抑制LPS诱导的巨噬细胞M1型极化，从而缓解炎症反应，而且还通过促进STAT6磷酸化，增强IL-4诱导的M2型极化及抗炎反应。

Abstract

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 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 inflammatory response, but also enhances IL-4-induced M2 polarization and anti-inflammatory responses by promoting STAT6 phosphorylation.

关键词

Vasorin / 巨噬细胞极化 / 炎症反应 / 脂多糖 / 白细胞介素-4 / 小鼠单核巨噬细胞白血病细胞 / NF-κB信号通路 / JAK-STAT信号通路

Key words

Vasorin / macrophage polarization / inflammatory response / lipopolysaccharide / interleukin-4 / mouse monocyte-macrophage leukemia cells / NF-κB signaling pathway / JAK-STAT signaling pathway

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基金资助

国家自然科学基金资助项目(32500451)

广西自然科学基金资助项目(2025GXNSFAA069124)

中国-东盟实验动物科创中心科学基金资助项目(KCZX2024004)