Department of Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou 310058, China.
Instrumental Analysis Center of Zhejiang Gongshang University, Hangzhou 310058, China.
Biomed Pharmacother. 2022 Aug;152:113258. doi: 10.1016/j.biopha.2022.113258. Epub 2022 Jun 13.
The present study was to investigate the molecular mechanisms underlying macrophage inflammatory response to polysaccharides from Peucedanum praeruptorum Dunn (PPDs) and elucidate the receptors and signaling pathways associated with PPDs-mediated macrophage activation. MTT and Griess method were performed to investigate the effects of PPDs on cell viability and NO production. Neutral red and FITC-dextran were used to determine the pinocytic and phagocytic activity. RT-qPCR and ELISA were employed to analyze the mRNA expression of inflammatory factors and production of cytokines and chemokines. RNA-seq and bioinformatics analysis were conducted to determine the underlying molecules, regulators and pathways, which were further validated by pathway inhibition and neutralization assays. The results indicated that PPDs significantly enhanced pinocytic and phagocytic activity, promoted the expression and secretion of inflammatory factors and chemokines, and boosted the expression of accessory and costimulatory molecules. RNA-Seq analysis identified 1343 DEGs, 405 GO terms and 91 KEGG pathways. IL6 and TNF were identified as hubs of connectivity in PPDs-mediated macrophage activation. "Cytokine-cytokine receptor interaction", "TNF signaling pathway", "NF-kappa B signaling pathway", "JAK-STAT signaling pathway" and "MAPK signaling pathway" were the most significant pathways. The pathway inhibition assay revealed that MAPK and NF-κB pathways were essential to macrophage activation by PPDs. TLR2 and TLR4 were uncovered to be the functional receptors and involved in recognition of PPDs. These results indicated that PPDs modulated macrophage inflammatory response mainly through TLR2/TLR4-dependent MAPK and NF-κB pathways.
本研究旨在探讨白芷多糖（PPDs）诱导巨噬细胞炎症反应的分子机制，并阐明与 PPDs 介导的巨噬细胞激活相关的受体和信号通路。MTT 和 Griess 法用于研究 PPDs 对细胞活力和 NO 产生的影响。中性红和 FITC-葡聚糖用于测定吞噬作用和吞噬作用。RT-qPCR 和 ELISA 用于分析炎症因子的 mRNA 表达和细胞因子和趋化因子的产生。RNA-seq 和生物信息学分析用于确定潜在分子、调节剂和途径，进一步通过途径抑制和中和测定进行验证。结果表明，PPDs 显著增强了吞噬作用和吞噬作用，促进了炎症因子和趋化因子的表达和分泌，并增强了辅助和共刺激分子的表达。RNA-Seq 分析鉴定出 1343 个差异表达基因、405 个 GO 术语和 91 个 KEGG 途径。IL6 和 TNF 被鉴定为 PPDs 介导的巨噬细胞激活中连接的枢纽。“细胞因子-细胞因子受体相互作用”、“TNF 信号通路”、“NF-κB 信号通路”、“JAK-STAT 信号通路”和“MAPK 信号通路”是最重要的通路。通路抑制试验表明，MAPK 和 NF-κB 通路是 PPDs 激活巨噬细胞所必需的。TLR2 和 TLR4 被揭示为功能性受体，参与 PPDs 的识别。这些结果表明，PPDs 主要通过 TLR2/TLR4 依赖的 MAPK 和 NF-κB 通路调节巨噬细胞炎症反应。
