四君子汤多糖的肠道菌群介导免疫作用发挥机制及物质基础研究

Based on progress in the interaction between polysaccharides and intestinal microbiota, a comprehensive efficacy evaluation indicator system including immune function (organ, cell and molecular level), intestinal microbiota (changes in the structure of the flora) and polysaccharide metabolites (short chain fatty acids)，will be explored to study the immunological polysaccharides of Si-Jun-Zi decoction mediated by intestinal microbiota. The immunological polysaccharides determined by cellular and humoral models will be subjected to animal models of spleen deficiency, intestinal bacteria disorder and germ free animals for further study on the immunological mechanism of oral polysaccharides medicated by intestinal microbiota. Then, the effective polysaccharides will be compared with those of crude materials in the prescription reported in literature or parallel isolation and purification of each herb in the prescription to confirm the origin resource. The interaction between effective polysaccharides and intestinal microbiota will be explored by applying the established technology of human gastrointestinal metabolism in vitro to figure out the metabolic process of the effective purified polysaccharides in gastrointestinal environment and the polysaccharides induced intestinal bacteria change. Thus, combined with previous results about group of small molecules in the prescription, effective substances and mechanism of strengthening the spleen and replenishing Qi after oral administration of Si-Jun-Zi decoction will be explained. This study will lay the foundation for the rationality of traditional Chinese medicine prescriptions and provide new idea for researches on immunological function of polysaccharides.

基于多糖、肠道菌群相互作用调节宿主免疫功能新进展，本项目引入免疫功能（器官、细胞、分子水平）、肠道菌群和多糖代谢物（短链脂肪酸） "三联效应"指标的药效评价体系，研究四君子汤复方汤剂多糖在肠道菌群介导下的免疫调节作用物质基础，确定活性单一多糖；采用致敏模型从细胞和体液免疫两方面验证单一多糖的免疫活性，进一步比较其在脾虚模型、肠道菌紊乱和无菌动物模型中免疫功能差异，探讨多糖口服后经肠道微生物介导发挥免疫作用的机制。对各组方药水煎剂进行活性单一多糖的来源追踪，明确单味药对复方的贡献；采用已建立的体外模拟人胃肠道代谢技术，分析活性单一多糖在胃肠道中的代谢进程及多糖对肠道菌群的作用，验证肠道菌与多糖的互利作用。综合前期复方小分子活性物质的研究结果，阐明四君子汤口服后发挥"健脾益气"功效的物质基础和免疫作用机制。本研究对中医复方配伍理论的科学阐述奠定基础，为中药多糖调节免疫功能研究提供新的思路。

基于多糖、肠道菌群相互作用调节宿主免疫功能新进展，本项目引入免疫功能（器官、细胞、分子水平）、肠道菌群和多糖代谢物（短链脂肪酸，SCFAs） “三联效应”指标的药效评价体系，筛选四君子汤复方水煎液的活性多糖组份；对活性多糖组份进一步分离纯化，采用不同细胞模型对四君子汤复方总多糖、活性多糖组份、活性均一多糖进行免疫活性验证；在脾虚动物模型的基础上，增设肠道菌紊乱模型以及伪无菌动物模型进行活性多糖的免疫作用及机制探讨；采用已建立的体外模拟人胃肠道代谢研究技术，考察活性均一多糖在人工胃、肠液以及人源肠道菌的代谢过程；对活性均一多糖进行一级结构表征；依据其结构特性，对四君子汤组成单味药水煎液进行活性多糖溯源，明确活性多糖的来源以及四味组成单味药对复方汤剂的贡献。结果证明，复方四君子汤多糖免疫作用的物质基础为多糖组分S-3；进一步分离纯化得到的活性均一多糖S-3-1/S-3-AG具有较强免疫调节作用；活性均一多糖在人工胃/肠液、人源肠道菌中均发生代谢产生还原糖，能够促进人源肠道菌中有益菌的生长繁殖，提高SCFAs水平。经口服后的免疫活性多糖组分S-3通过调节肠道菌群，改善脾虚证大鼠肠道菌群紊乱状态，调节SCFAs含量，刺激肠上皮T淋巴细胞分泌，对脾虚模型大鼠发挥肠道免疫保护作用。活性多糖的溯源实验证明，其与复方组成的四味药中人参和白术水煎剂中均一多糖高度相关，推测其可能产生于复方煎煮过程。本研究建立的免疫-肠道微生物-多糖代谢物“三联效应”综合评价筛选策略，符合中药特点，对深入探讨肠道微生物在中药发挥免疫功能中的介导作用具有积极意义，为探究中医药理论的实质、中药免疫调节作用物质基础以及基于经典方的新药研发提供了新的思路。