红毛五加多糖AHP-II对巨噬细胞的激活作用及分子机制研究

Acanthopanax giraldii Harms as a traditional medicine is known to have the ability to enhance immune response. Acanthopanax giraldii Harms Polysaccharide(AHP) is thought to be the major bioactive component exhibiting its immunomodulatory funtions. However, there has no report focusing on any homogenous AHP fractions and their immune regulatory activities. In a recent research, we successfully purified three homogenous AHP fractions, named AHP-I, AHP-II and AHP- III. Our experiment proved that the homogenous AHP-II is more competent in activating murine peritoneal macrophages. Treatment of peritoneal macrophages in vitro with AHP-II significantly promoted macrophage phagocytosis. AHP-II induced markedly increased level of NO, inflammatory cytokines (such as TNFa, IL-6) production from macrophages. In a Cyclophosphamide indueced immunosurpressed murine model, AHP-II increased spleenocyte proliferation, and macrophage phogocytosis, and serum IL-6, TNFa and IgM level. These data indicated clearly that the newly prepared homogenous AHP-II has the potential to stimulate macrophage activation, but the molecular mechanims are not known. Here in the present study, we are aiming to reveal the detailed biological activities and molecular mechanisms of AHP-II on macrophage activation. Firstly, we will demonstrate how AHP-II acts on macrophages. These include effects of AHP-II on macrophage phagocytosis, activation/cytotoxicity, cytokine secretion, and co-stimulation. In the subsequent experiment, we will try to identify major membrane pattern recognition receptor TLRs ( mainly TLR2, TLR4) on macrphages invovled in interacting with AHP-II and are responsible for AHP-II stimulated macrophage activation by using TLR knock-down. Finally, we will continitue to elucidate critical signaling pathways induced by AHP-II in macrophges. We will combine results from western blotting, EMSA and diverse signaling pathway inhibition to elustrate if the AHP-II triggered signal transduction is adaptor protein MyD88 dependent or independent. We still want to identify downstream signaling molecules, (include TRIF, TRAF-6, NF-кB, p38MAPK) that helps to deliver MyD88 dependent or independent signaling, and will try to associate important downstream effective genes such as NO, IL-6, TNFa with different signaling pathways. Our attemp and resutls here will provide very fundermental proofs for future utilization fo AHP-II as a novel immunomodulating agent in clinical trials.

传统药材红毛五加具有增强免疫的功效，红毛五加多糖（Acanthopanax giraldii Harms Polysaccharide, AHP）是其主要活性成分之一，但对AHP单一组分免疫激活及分子机制的研究很少。我们前期分离到3种AHP单一组分，其中组分Ⅱ（AHP-Ⅱ）对巨噬细胞有较强激活作用。在此基础上，本课题拟通过检测巨噬细胞代谢关键酶活性，NO、细胞因子分泌及相关基因表达的变化，研究AHP-Ⅱ对巨噬细胞的激活作用。将以TLR2、4作为候选受体，通过受体阻断等方法确定AHP-Ⅱ激活巨噬细胞的模式受体，利用WB、EMSA等技术分析AHP-Ⅱ作用于巨噬细胞的信号通路如MyD88、TRAF-6、NF-кB等，阐明AHP-Ⅱ激活巨噬细胞的分子机制，为AHP-II的深入开发利用提供研究依据，也为新型的免疫调节剂开发提供新选择。

传统药材红毛五加是五加科植物红毛五加的地上茎皮。现代药理学研究证实，红毛五加中的主要有效成分红毛五加多糖（Acanthopanax giraldii Harms Polysaccharide, AHP）具有调节机体免疫系统等功效，但其作用机制尚不完全清楚，且以往研究多采用多糖粗提物，成分相对复杂。巨噬细胞作为机体固有免疫系统的重要成员，广泛参与机体特异性和非特异性免疫应答， 在机体抗感染、 抗肿瘤和组织修复等方面发挥重要作用。本课题旨在研究红毛五加多糖单一组分AHP-II对巨噬细胞的激活作用，探讨AHP-II激活巨噬细胞可能的信号转导通路，阐明其作用机制。本研究首先从细胞代谢关键酶活性变化、细胞因子释放等方面研究了AHP-II对不同来源的巨噬细胞的激活作用，结果显示，AHP-II在实验浓度范围内（25~100μg/ml）对THP-1源巨噬细胞、小鼠腹腔巨噬细胞、巨噬细胞RAW264.7均具有激活作用。然后利用荧光标记的AHP-II（AHP-II-FITC），观察多糖与小鼠腹腔巨噬细胞的结合情况，以及TLR2、4阻断型抗体竞争性抑制作用，实验的结果显示AHP-II-FITC可特异性结合在小鼠腹腔巨噬细胞表面，并初步证实TLR4是其发挥作用的主要靶标分子。在比较AHP-II对不同基因缺陷型小鼠（TLR4-/-，TLR2-/-）腹腔巨噬细胞的激活作用的实验中，进一步证实了上述结论。在上述实验基础上，采用Real time PCR、Western blotting方法对TLR4下游信号转导通路活化情况进行研究，发现AHP-II作用后巨噬细胞内MyD88 mRNA表达增加，JNK和NFκB磷酸化比例增高，且在特异性阻断剂对通路阻断后，AHP-II作用下巨噬细胞的细胞因子释放量下降，表明AHP-II可通过MyD88-JNK/NF-κB信号通路激活巨噬细胞。同时，课题组还针对红毛五加多糖提取效率低问题，建立优化了红毛五加多糖超声波辅助提取方法。本课题的研究结果初步阐明AHP-II激活巨噬细胞的分子机制，为AHP-II的进一步开发利用奠定理论基础，也为新型的免疫调节剂（BRM）提供新的选择。