Immunomodulatory activity of enzymatically synthesized glycogen and its digested metabolite in a co-culture system consisting of differentiated Caco-2 cells and RAW264.7 macrophages.
Immunomodulatory activity of enzymatically synthesized glycogen and its digested metabolite in a co-culture system consisting of differentiated Caco-2 cells and RAW264.7 macrophages.
复制标题
在由分化的 Caco-2 细胞和 RAW264.7 巨噬细胞组成的共培养系统中，酶促合成的糖原及其消化的代谢物的免疫调节活性。
DOI：
10.1039/c3fo60035a
复制
发表时间：
2013
期刊：
影响因子：
--
通讯作者：
Ashida H.
中科院分区：
文献类型：
--
作者：
Yasuda M;Furuyashiki T;Nakamura T;Kakutani R;Takata H;Ashida H.
以前，我们开发了酶促合成糖原（ESG）从淀粉，并显示其免疫调节和膳食纤维样活性。在这项研究中，我们研究了ESG的代谢及其免疫调节活性，使用分化的Caco-2细胞作为肠屏障的模型。在由分化的Caco-2细胞和RAW264.7巨噬细胞组成的共培养系统中，在用5 mg ml−1 ESG处理24 h顶端后，Caco-2细胞中IL-6、IL-8、IL-1β和BAFF细胞因子的mRNA表达上调，基底外侧培养基中的IL-8产生被诱导。在RAW264.7巨噬细胞中iNOS mRNA水平也明显上调。在表征抗糖原单克隆抗体（IV 58 B6和ESG 1A 9）与ESG及其消化代谢产物抗性糖原（RG）的结合后，开发了用于定量ESG和RG的酶联免疫吸附测定（ELISA）系统。使用该系统，我们研究了ESG在分化的Caco-2细胞中的代谢。当将ESG（7000 kDa，5 mg ml−1）添加到Caco-2单层的顶侧时，在24 h孵育期间，ESG消失，RG（约3000 kDa，3.5 mg ml−1）出现在顶侧溶液中。在基底侧溶液中未检测到ESG和RG。此外，ESG和RG都与Caco-2细胞中的TLR 2结合。总之，我们认为ESG在肠道中代谢为RG样结构，并且这种代谢物通过刺激肠上皮激活免疫系统，尽管在我们的实验条件下ESG及其代谢物都不能渗透肠细胞。这些结果为ESG作为食品成分的有益功能提供了证据。
Previously, we developed enzymatically synthesized glycogen (ESG) from starch, and showed its immunomodulatory and dietary fiber-like activities. In this study, we investigated the metabolism of ESG and its immunomodulatory activity using differentiated Caco-2 cells as a model of the intestinal barrier. In a co-culture system consisting of differentiated Caco-2 cells and RAW264.7 macrophages, mRNA expression of IL-6, IL-8, IL-1β and BAFF cytokines was up-regulated in Caco-2 cells and IL-8 production in basolateral medium was induced after 24 h apical treatment with 5 mg ml−1 of ESG. The mRNA level of iNOS was also up-regulated in RAW264.7 macrophages. After characterization of the binding of anti-glycogen monoclonal antibodies (IV58B6 and ESG1A9) to ESG and its digested metabolite resistant glycogen (RG), an enzyme-linked immunosorbent assay (ELISA) system was developed to quantify ESG and RG. Using this system, we investigated the metabolism of ESG in differentiated Caco-2 cells. When ESG (7000 kDa, 5 mg ml−1) was added to the apical side of Caco-2 monolayers, ESG disappeared and RG (about 3000 kDa, 3.5 mg ml−1) appeared in the apical solution during a 24 h incubation. Neither ESG nor RG was detected in the basolateral solution. In addition, both ESG and RG were bound to TLR2 in Caco-2 cells. In conclusion, we suggest that ESG is metabolized to a RG-like structure in the intestine, and this metabolite activates the immune system via stimulation of the intestinal epithelium, although neither ESG nor its metabolite could permeate the intestinal cells under our experimental conditions. These results provide evidence for the beneficial function of ESG as a food ingredient.