蛹虫草多糖调节小鼠巨噬细胞RAW264.7免疫活性的分子机制

多种真菌多糖近年来因其免疫调节活性成为保健食品领域研究的热点。本实验以食药用真菌蛹虫草提取物蛹虫草多糖（Cordyceps militaris polysaccharides，CMP）为研究材料，初步探究CMP对小鼠巨噬细胞RAW264.7免疫活性的调节机制。噻唑蓝实验结果显示CMP无细胞毒性，且100、200 μg/mL CMP可以明显增强RAW264.7细胞活性；中性红法、Griess法和酶联免疫吸附检测结果表明25～200 μg/mL的CMP以剂量依赖方式增强RAW264.7细胞吞噬活性并增加一氧化氮（nitric oxide，NO）和白介素-1β（interleukin-1β，IL-1β）分泌，肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）的分泌呈先升高后降低的趋势，在100 μg/mL时达到最高值；抑制剂中和实验结果显示当Toll样受体4（Toll-like receptors 4，TLR4）和甘露糖受体（mannose receptor，MR）受到抑制时，CMP诱导的RAW264.7免疫反应显著降低，这表明TLR4和MR均为CMP激活巨噬细胞的受体；此外，Western blot结果显示丝裂原活化蛋白激酶（mitogen-activated protein kinases，MAPK）信号通路也参与CMP诱导的RAW264.7细胞分泌NO、TNF-α和IL-1β。表明TLR4和MR/MAPK信号传导途径在CMP诱导巨噬细胞RAW264.7产生免疫应答时发挥了重要作用。

Molecular Mechanism of Cordyceps militaris Polysaccharides in Regulating the Immune Function of Macrophage RAW264.7 Cells

In recent years, the immunomodulatory activity of fungal polysaccharides has become a hot spot in the field of health foods. In the present study, Cordyceps militaris polysaccharides (CMP) were used to investigate the mechanism for its immunoregulatory effect on macrophage RAW264.7 cells. Thiazolyl blue assay results showed that CMP had no cytotoxicity and at doses of 100 and 200 μg/mL could significantly promote the proliferation of RAW264.7 cells. The neutral red staining, Griess and ELISA results showed that CMP at a concentration of 25–200 μg/mL increased the phagocytic activity and the secretion of nitric oxide (NO) and interleukin-1β (IL-1β) in RAW264.7 cells in a dose-dependent manner; tumor necrosis factor-α (TNF-α) secretion increased first and then decreased after reaching the highest value at 100 μg/mL. Inhibitor neutralization assay showed that CMP was significantly less able to induce immune response in RAW264.7 cells when Toll-like receptors 4 (TLR4) and mannose receptor (MR) were inhibited, indicating that both TLR4 and MR were CMP-activated macrophage receptors. Western blot results showed MAPK signaling was also involved in CMP-induced secretion of NO, TNF-α and IL-1β in RAW264.7 cells. Therefore, TLR4 and MR/MAPK signal transduction pathways play an important role in CMP-induced immune response in macrophage RAW264.7 cells.