肠道菌群降解坛紫菜多糖形成的低聚糖调控紧密连接蛋白的机制

The integrity of tight junction proteins is essential for intestinal barrier function. Studies have revealed that dietary polysaccharides can promote the repair of intestinal barrier function by regulating the expression of tight junction proteins. However, when dietary polysaccharides enter the large intestine, they will be degraded by gut microbiota. At present, the effects of the oligosaccharides degraded from polysaccharides by gut microbiota on tight junction proteins as well as the regulatory mechanism have not been studied systematically. Our previous studies found that polysaccharides from Porphyra haitanensis could improve diarrhoea, and can be degraded by gut microbiota to produce a variety of oligosaccharides. In order to further study the effect of P. haitanensis polysaccharides on the repair of intestinal barrier function, this proposed project intends to isolate and purify oligosaccharides derived from P. haitanensis polysaccharide upon in vitro stimulation by gut microbiota. The structural characteristics of the purified oligosaccharides will then be analyzed using several methods. The project will also include the studies on in vivo and in vitro models as well as immune-histochemical analysis, RT-PCR, Western-blot, and other techniques would be used to explore the effects of the oligosaccharides on tight junction proteins and the mechanisms involved. The results from the proposed study would give us a better insight and understanding of the degradation process of P. haitanensis polysaccharides in human intestinal tract by gut microbiota, as well as the role and regulatory mechanisms of the resultant oligosaccharides in the repair of tight junction proteins. This project will provide important theoretical basis for understanding the role and regulation mechanism of P. haitanensis polysaccharides in the repair of intestinal barrier function and in promoting the health of the host.

紧密连接蛋白的完整性对于肠道屏障功能至关重要。现有研究证实膳食多糖能通过调控紧密连接蛋白的表达促进肠道屏障功能的修复，但膳食多糖进入大肠会被肠道菌群降解，目前对于多糖的降解产物低聚糖对紧密连接蛋白的影响及调控机制还缺乏系统的研究。本课题组前期研究发现，坛紫菜多糖可以改善腹泻且能被肠道菌群降解产生多种低聚糖，为了深入研究坛紫菜多糖对肠道屏障的修复作用，本项目拟通过对坛紫菜多糖体外模拟肠道菌群降解得到的低聚糖进行分离纯化并解析其结构特征；通过实验动物模型、细胞模型、免疫组化、RT-PCR、Western-blot等技术探究降解产物低聚糖对紧密连接蛋白表达的影响，最终阐明其作用机制。这些结果将有助于更好地理解坛紫菜多糖在人体肠道中被肠道菌群的降解过程，及其产物低聚糖在紧密连接蛋白修复中扮演的角色及调控机制，为坛紫菜多糖修复肠道屏障功能乃至促进宿主健康提供重要理论依据。

肠道健康的关键在于维持肠道屏障完整性与平衡肠道微生态。本项目以坛紫菜为原材料，经提取分离纯化获得坛紫菜纯多糖，并通过模拟酵解和可控部分酸水解得到坛紫菜低聚糖，联合多元色谱和光谱技术理清了坛紫菜多糖及低聚糖的理化性质、结构和构象。本研究从坛紫菜多糖和低聚糖在体外模拟酵解过程中结构和构象变化情况以及对肠道菌群的影响方面探讨了坛紫菜多糖和低聚糖的代谢特征和益生活性，研究发现坛紫菜多糖与低聚糖能有效调节肠道菌群，并被肠道菌群酵解利用，尤其是提高了拟杆菌门中多形拟杆菌的丰度，且产生了多种功能性代谢分子短链脂肪酸。在体外细胞水平上，本项目研究了坛紫菜多糖对IEC-6细胞的划痕损伤修复情况，结果发现坛紫菜多糖可以有效加快划痕损伤的愈合和细胞迁移，其分子机理为上调了IEC-6的Cdc-42和Rac-1、PKCβⅡ、Paxillin和FAK蛋白的表达。在体内动物模型中，研究发现坛紫菜多糖具有改善小鼠溃疡性结肠炎的作用，能够修复肠道屏障功能，其修复作用相关机理包括上调抑炎因子、促进IgA分泌的相关蛋白表达、上调与肠道屏障完整性相关的紧密连接蛋白与粘蛋白的表达，进而促进肠道健康。这些结果对坛紫菜多糖及其低聚糖作为益生元的有效开发和利用提供可靠的理论依据，也对南粤特色经济海藻研究的继续深化，尤其对坛紫菜的营养宣传与深加工具有重要的理论价值和指导意义。