灰树花硒多糖的吸收变化及其参与免疫调节分子机制研究

Selenium (Se) is an essential trace element. Low Se can cause suppressed immunity. Se-polysaccharide contain both Se and polysaccharide activities, which is one of the main existing forms of Se in Se-enriched Grifola frondosa. Our previous study showed that the Se-polysaccharide purified from Grifola frondosa (Se-GP11)could significantly improve the activity of macrophages when compared to the polysaccharide from Grifola frondosa (GP11), which suggest that Se-GP11 has better regulatory role in immune than GP11. In this study, we further explored the primary and advance structures as well as immunomodulatory activity of Se-GP11. Stimulated gastro-intestinal fluid, rat intestinal flora, Caco-2 cells and low Se mouse models were employed to investigate the in vitro and vivo absorption pattern of Se-GP11.Modern instrumental analytical methods were applied to verify the absorption changes and the products. The effect of Se-GP11 and its products of absorption changes on expression of p38MAPK and p-p38 proteins, MEF2, EIK-1, ATF-2 and CREB transcription factors, c-fos and c-jun genes studied was based on the common immunomodulatory signaling pathway p38MAPK of Se and polysaccharide. The molecular mechanism of the Se-GP11 was clarified and this provided the basis for application of Se-enriched Grifola frondosa, Se-polysaccharide and the dietary structure adjustment of people with low Se intakes.

硒是人体必需的微量元素，人体低硒会引起免疫力下降。硒多糖为富硒灰树花中硒的主要赋存形式之一，兼具硒和多糖两者的活性，前期研究发现，与未富硒多糖相比，硒多糖可显著提高巨噬细胞活性，提示其具有更好的参与免疫调节作用。本项目将进一步探明硒多糖的一级结构和高级结构及其参与免疫调节活性；采用模拟胃肠液、鼠肠道菌群试液共孵法、Caco-2细胞和低硒小鼠模型，探讨其在体外、体内的吸收规律，并运用现代仪器分析手段研究硒多糖在吸收过程中的变化及其产物；基于p38MAPK是硒和多糖免疫活性调节的共同作用通路，在分子和基因水平层次探明硒多糖及其吸收变化产物对p38MAPK及p-p38MAPK蛋白表达，MEF2、EIK-1、ATF-2和CREB等转录因子表达以及即早基因（c-fos和c-jun）表达的影响，阐明其参与免疫调节的分子机制，为富硒灰树花及其硒多糖的应用提供基础，为低硒摄入人群膳食结构调整提供依据。

硒是人体必需的微量元素，人体低硒会引起免疫力下降。硒多糖为富硒灰树花中硒的主要赋存形式之一，兼具硒和多糖两者的活性。本项目研究了灰树花硒多糖的吸收变化及其参与免疫调节分子机制。采用DEAE-52纤维素柱和SephacrylTM S-400葡聚糖凝胶柱从富硒灰树花粗提物中分离纯化得到富硒灰树花硒多糖纯化组分（Se-GFP-22），结构鉴定确定了Se-GFP-22的一级结构重复单元。Se-GFP-22分子中存在硒酸酯的特征吸收峰；不具有三螺旋结构，为无规线团；在水溶液中排列紧凑，分子间相互缠绕；其固体为不规则纤维网状结构。采用环磷酰胺（CTX）致免疫抑制小鼠模型，研究了Se-GFP-22对CTX致免疫抑制小鼠的免疫调节作用，结果表明，Se-GFP-22可显著提高免疫抑制小鼠的免疫器官指数、吞噬指数、淋巴细胞增殖活性、耳肿胀度、溶血素水平、免疫球蛋白含量、细胞因子分泌量及mRNA表达量和抗氧化酶活力；可通过MAPKs信号通路激活脾淋巴细胞。采用低硒小鼠模型，研究了Se-GFP-22对低硒小鼠的免疫调节作用和和体内吸收变化，结果表明，Se-GFP-22可显著提高低硒小鼠免疫器官指数、吞噬指数、淋巴细胞增殖活性、耳肿胀度、免疫球蛋白含量、细胞因子分泌量、含硒酶活力、血清和组织硒含量。采用RAW264.7巨噬细胞模型，研究了Se-GFP-22参与免疫调节的作用机制，结果表明，Se-GFP-22可显著提高巨噬细胞吞噬能力、细胞因子分泌量及mRNA表达量、ROS水平和SOD活力；可通过调节MAPKs信号通路相关蛋白（JNK、ERK和p38，MEF2、EIK-1、ATF-2和CREB）的表达，增强巨噬细胞的免疫活性。可见，Se-GFP-22对CTX致免疫抑制小鼠和低硒小鼠具有较好的免疫调节作用，可有效补充低硒小鼠体内硒含量，该研究为富硒灰树花及其硒多糖的应用提供了基础，为低硒摄入人群膳食结构调整提供了依据。