Abstract：
Our previous investigations have successfully identified the repeating structural units of EPS53, an exopolysaccharide derived from Streptococcus thermophilus XJ53 fermented milk, and substantiated its potential immunomodulatory properties. The present study further elucidated the structural characteristics of EPS53 and investigated the underlying mechanisms governing its in vitro immunoreactivity as well as its in vivo immunoreactivity. The results obtained from multi-detector high performance gel filtration chromatography revealed that EPS53 adopted a rigid rod conformation in aqueous solution, with the weight-average molecular weight of 1464 kDa, the number-average molecular weight of 694 kDa, and the polydispersity index of 2.11. Congo red experiment confirmed the absence of a triple helix conformation. Scanning electron microscopy showed that EPS53 displayed a three-dimensional fibrous structure covered with flakes. The in vitro findings indicated that EPS53 enhanced phagocytosis ability, reactive oxygen species (ROS) production, and cytokine levels of macrophages via the TLR4-mediated NF-κB/MAPK signaling pathways as confirmed by immunofluorescence staining experiments, inhibition blocking experiments, and Western blot assay. Additionally, the in vivo experiments demonstrated that EPS53 significantly increased macrophage and neutrophil number while enhancing NO and ROS levels in zebrafish larvae; thus, providing further evidence for the immunomodulatory efficacy of EPS53.
摘要：
我们以前的研究已经成功地确定了EPS53的重复结构单元，EPS53是一种源自嗜热链球菌XJ53发酵乳的胞外多糖，并证实了其潜在的免疫调节特性。本研究进一步阐明了EPS53的结构特征，并研究了其体外免疫反应性以及体内免疫反应性的潜在机制。从多检测器高效凝胶过滤色谱获得的结果表明，EPS53在水溶液中采用了刚性棒构象，重均分子量为1464kDa，数均分子量为694kDa，和2.11的多分散指数。刚果红实验证实不存在三螺旋构象。扫描电子显微镜显示EPS53显示出覆盖有薄片的三维纤维结构。体外研究结果表明，EPS53增强了吞噬能力，活性氧（ROS）的产生，和细胞因子水平的巨噬细胞通过TLR4介导的NF-κB/MAPK信号通路如免疫荧光染色实验证实，抑制阻断实验，和蛋白质印迹分析。此外,体内实验表明，EPS53显著增加斑马鱼幼虫的巨噬细胞和中性粒细胞数量，同时增加NO和ROS水平；为EPS53的免疫调节功效提供进一步的证据。
