黑灵芝多糖通过Notch信号通路调控巨噬细胞极化及其作用机制

Ganderma atrum, a natural ingredient, is commonly used for health benefits. Currently, a highly purified polysaccharide derived from Ganderma atrum have been obtained in our laboratory. Its primary structural feature was characterized, and was demonstrated to possess immunomodulatory effects via macrophage activation. However, little is known about the mechanism for immune response of Ganderma atrum polysaccharide. It is well known that macrophage plays an important role in immunity for the regulation of macrophage polarization and becomes a new hot spot. Recently, it is suggested that Notch signaling plays a vital role in the regulation of macrophage polarization and provides a new idea for the research of macrophage polarization. The aim of this study is to investigate the effect of Ganderma atrum polysaccharide on macrophage polarization and Notch pathway. Whether the activation of Notch signaling contribute to modulate TLR pathway as a result of controling immune response is further determined. On the other hand, we try to figure out the relationship between Notch pathway and TLR signaling so as to exploring a new mechanism of Ganoderma polysaccharide exerting immune function. Taken together, this research will be of great value in explaining the intrinsic molecular mechanisms of Ganoderma atrum polysaccharide, indicating a new target for the research of the bioactivities of polysaccharide. It provides a theoretical evidence for further development and application of Ganoderma atrum. Meanwhile, it also develops a new sight for other bioactive compounds obtained from natural food products in terms of immune mechanism.

黑灵芝是一种对人体健康具有重要作用的天然食材。本团队前期已纯化出黑灵芝多糖并解析了一级结构，证实该多糖能调控巨噬细胞功能，发挥较好的免疫调节活性，但其具体机制不明。 巨噬细胞是机体重要免疫细胞，其极化的调控对维系健康十分重要，相关研究已成为新热点。研究表明，Notch 信号通路对巨噬细胞极化有重要调控作用，为极化研究提供了新思路。本项目拟从整体、离体、细胞水平，观察巨噬细胞极化对黑灵芝多糖免疫调节活性的影响；进一步基于Notch介导的极化相关信号通路研究黑灵芝多糖对巨噬细胞极化调控的分子机制；在此基础上，以Toll样受体介导信号通路为切入点，探讨基于Notch信号通路介导的巨噬细胞极化作用机制，揭示黑灵芝多糖免疫调控新机制。本项目的完成有利于阐明黑灵芝多糖免疫调控的内在分子机制，为多糖生物活性研究提供新的分子靶点，丰富天然食材免疫调控机制，也为其他类似功能的食源性活性组分研究拓展新的思路。

本实验室在前期已经研究了黑灵芝多糖（PSG-1）对巨噬细胞的免疫调节活性及其作用机制。为了进一步研究PSG-1基于Notch信号通路对巨噬细胞极化的调控作用机制，本研究拟在本实验室前期工作基础上，分别从体内和体外两种方式系统地研究PSG-1对M1/M2型巨噬细胞的调控作用及针对Notch信号通路研究其分子机制。运用细胞和分子生物学技术，分别从整体、细胞、分子水平，深入探讨PSG-1通过Notch通路对M1/M2亚型巨噬细胞的调控作用及其机制研究。具体结论如下：.1、PSG-1可降低M1型巨噬细胞CD80的表达、吞噬能力和ROS水平。同时PSG-1对M1型巨噬细胞的NO的释放有明显的抑制作用，这种抑制效果呈剂量依赖性。PSG-1可降低促炎因子TNF-α和IL-6的产生，并且促进抗炎因子IL-4和IL-10的分泌。.2、PSG-1可增强M2型巨噬细胞CD23的表达、吞噬能力和ROS水平。同时PSG-1可促进促炎细胞因子IL-1β、TNF-α、IL-6的分泌以及NO的释放，抑制抗炎因子IL-4的分泌。提示PSG-1对调控巨噬细胞向M1/M2极化的内稳态平衡有一定的作用。.3、PSG-1可增加巨噬细胞表面MR的表达量，降低其吞噬功能和ROS生成水平。同时，PSG-1可减少促炎因子TNF-α的含量。实验证实PSG-1具有抗炎作用，可部分抑制炎症模型小鼠体内的炎症，其机理与PSG-1促进小鼠腹腔巨噬细胞表面MR的表达，抑制巨噬细胞向M1型极化有关。.4、阻断Notch信号后，巨噬细胞CD80的表达量减少，细胞的吞噬能力增强，其ROS的生成水平降低。同时，阻断Notch信号后，NO的含量、IL-1β、IL-6和IL-10的分泌水平显著降低。.综上所述，本研究证实PSG-1对LPS+IFN-γ/IL-4诱导剂分别诱导巨噬细胞的M1和M2型的巨噬细胞极化，具有调控作用。同时PSG-1在体内具有一定的抗炎作用，其作用机制可能与PSG-1抑制巨噬细胞向M1型极化，促进细胞向M2方向极化有关；此外，PSG-1抑制LPS+IFN-γ介导的巨噬细胞向M1型极化的作用机理与Notch信号通路紧密相关。