基于Toll样受体通路的干制龙眼多糖免疫调节活性增效机制研究

We found that polysaccharides from dried longan pulp showed stronger immunomodulatory activity than that from fresh longan pulp in previous studies, and the mechanism may be related to the TLRs-signaling pathway. The main active polysaccharide fractions of dried longan pulp have been isolated and the structure of polysaccharide of fresh pulp has been characterized. To elucidate the molecular mechanisms of the difference in immunomodulatory activity between dried and fresh longan polysaccharides, we will carry out experiments: 1) the spatial structure of the main polysaccharides fractions from dried longan pulp will be characterized using NMR and molecular simulation; 2) TLRs types activated by polysaccharides from dried and fresh longan pulp will be explored by TLRs deletion macrophage model, and effects of the hydrolysis fragments of polysaccharides on the phagocytic ability of macrophages will be compared in order to study TLRs binding domain structure activated by polysaccharides; 3) The immune-related gene pathways affected by longan pulp polysaccharides will be explored using gene chip. The changes in gene expression and protein levels of the critical point of TLRs downstream pathway will be analyzed. The molecular mechanism of activating TLRs-signaling pathway and the structure-activity relationships will be clarified. The research will enrich the theoretical basis of health-promoting effects of longan, and provide the scientific basis for the development and application of active polysaccharides form plant.

项目前期的研究表明，龙眼干果多糖的免疫调节活性显著高于其鲜果多糖，其机制可能与Toll样受体（TLRs）-信号有关。为明确龙眼干果多糖免疫调节活性增效的机制，本项目拟在前期对龙眼主活性级分多糖分离和已明确其鲜果多糖结构的基础上，采用NMR等技术和分子模拟手段表征龙眼干果主活性级分多糖的空间结构；通过TLRs缺失细胞模型，明确龙眼鲜、干果多糖活化TLRs的类型与生理作用，分析多糖水解片段对巨噬细胞吞噬能力的影响，进而探明龙眼多糖活化TLRs的结合域结构；利用基因芯片分析龙眼多糖对细胞免疫相关基因通路的影响，结合对TLRs下游通路关键分支点的基因表达与蛋白水平变化的分析，明确龙眼鲜、干果多糖活化TLRs-信号通路的分子途径，探讨其免疫调节活性的构效关系与机制，从而揭示龙眼干果多糖免疫调节活性增效的分子机制。研究结果将丰富龙眼保健功效的理论基础，为特色植物活性多糖的开发应用提供科学依据。

分离纯化获得6种龙眼活性多糖，采用GC/MS、NMR等手段对多糖的结构进行了表征，确定了干、鲜龙眼的主活性多糖及其结构。阐明龙眼多糖通过TLR-2和TLR-4介导的MyD88/Cxcl-s/Mapk9/NF-κB途径激活巨噬细胞的信号通路。发现龙眼多糖活化TLRs的结合域主要包括(1→4)-β-Glc和(1→6)-β-Man组合结构以及(1→6)-β-Glc结构。揭示了龙眼干果多糖免疫调节活性增效的分子机制，发现干、鲜龙眼多糖调节TLRs、NOD样受体、TNF和NF-κB信号通路中Ccl5，Nfkbia，Ticam1，Cd14，Tnfaip3，Stat5a关键基因表达的差异导致了其免疫活性的差异；龙眼多糖调节活性的差异可能与其TLRs结合域单位数量有关。同时发现干制龙眼多糖可以通过调节巨噬细胞和肠系膜淋巴结细胞功能以及肠道微生物生态，改善应激条件下的机体免疫功能。项目的研究结果可以丰富龙眼保健功效的理论基础，为特色植物活性多糖的开发应用提供科学依据。