肠道菌群介导的非淀粉多糖对结肠粘液层厚度的影响及其机制

Colonic mucus layer, as a part of the intestinal barrier function, protects the intestinal health of the hosts. Non-starch polysaccharide (NSP), closely related to human nutrition, is able to affect the thickness of colonic mucus layer by mediation of gut microbiota, thus showing a close relationship to the intestinal health or even the health of the hosts. However, the key bacteria affecting the thickness of mucus layer with the intervention of NSP as well as the mechanism within remain unknown. This project aims to identify and verify the key mucosal bacteria by comparing the composition of microbiota in mucus layer and the thickness of mucus layer under the intervention of different NSPs, and by using the methods of microbial community diversity analysis, in vitro fermentation and selective medium culture. This project is also intended to analyze and identify the signal molecules produced from MUC2 decomposition by those key bacteria and their mechanisms on stimulating secretion of mucin in goblet cell by applying metabonomics analysis, in vitro cell culture and the techniques in molecular biology such as western blot. This project will help to clarify the role of key mucosal bacteria in the thickness of mucus layer and illuminate the signal molecules produced by those key bacteria as well as the mechanism on mucin secretion triggered by the signal molecules, showing a high theoretical value and guiding significance to the maintenance of hosts’ intestinal health or even the hosts’ health using the diary regulation.

结肠粘液层是肠道屏障功能的一部分，保护着宿主肠道健康。与人类营养密切相关的非淀粉多糖（non-starch polysaccharide，NSP）能通过肠道菌群影响粘液层厚度，因此与肠道乃至宿主健康息息相关。然而，NSP干预下影响粘液层厚度的关键细菌及其作用机制尚未明了。本项目拟通过动物实验比较不同NSP塑造的粘液层菌群及菌群对粘液层厚度影响的差异，结合微生物群落多样性测序技术、体外发酵实验和选择性培养实验，鉴定影响粘液层厚度的关键细菌；通过代谢组学、体外细胞培养和免疫印迹等技术分析和研究关键细菌菌群刺激杯状细胞分泌粘蛋白的信号分子及其中涉及的机制。本研究有助于阐明NSP干预下定植于粘液层的肠道菌群在影响粘液层厚度中的角色、该菌群作用下产生的信号分子及其影响粘蛋白分泌的机制，对以膳食调节和维持肠道乃至宿主的健康具有重要的理论价值和指导意义。

补充膳食多糖被认为是维持健康的肠道的最佳方法。膳食多糖能够促进肠道屏障功能，比如增加粘液层的厚度。不同的膳食多糖由于结构的差异，对宿主产生的影响会有所不同。果胶和菊粉是两种常见的膳食多糖，两者之间的益生活性差异未见报道。本研究首先分别以AIN-93M标准饮食、果胶饮食和菊粉饮食干预健康Wistar大鼠30天，比较不同饮食的大鼠盲肠菌群和粘液层菌群组成的差异、盲肠内容物中的短链脂肪酸水平和代谢物组成变化，以及结肠内容物中MUC2分泌水平；然后以淀粉、果胶或菊粉为唯一碳源，接种盲肠内容物进行体外模拟发酵后，并用发酵前后的发酵液处理LS174T细胞，比较不同多糖发酵前后对LS174T细胞MUC2分泌的影响。研究结果显示：.（1）在促进MUC2分泌方面，无论从AB-PAS染色的结肠组织或是ELISA检测，结果均显示果胶和菊粉均能增加结肠内容物中MUC2的含量。.（2）在调节菌群方面，果胶和菊粉既能促进潜在的有益菌的生长，也能减少致病菌的丰度。果胶和菊粉促使Christensenellaceae_R-7_group和Lachnospira丰度增加。.（3）在调节代谢物方面，果胶和菊粉在体内外实验中都能够增加乙酸的含量；而且与对照组相比，果胶组和菊粉组中共得到差异代谢物有14种。.（4）MUC2的含量变化与盲肠菌群中的Anaerotruncus和Ruminococcus_1显著负相关，与粘液层菌群无显著相关性，与乙酸、Lucyoside Q、Soyasapogenol B 24-O-b-D-glucoside显著正相关，与25-Acetylvulgaroside、3α-Androstanediol glucuronide和Corosolic acid显著负相关；在体外模拟发酵中，与未经过发酵的培养基和淀粉发酵液相比，发酵24 h后的果胶和菊粉发酵液能够刺激LS174T细胞分泌更多的MUC2。.本研究发现文献中报道的不具有刺激MUC2分泌的果胶，在实际中也能刺激结肠MUC2的分泌，增加粘液层的厚度，因此本研究为果胶被广泛用作功能性食品的原料提供理论依据。此外，本研究找到了潜在的、参与促进MUC2的产生的一些特定的肠道细菌或/和代谢产物，一定程度上揭示了非淀粉多糖增加粘液层厚度的机制的物质基础。