源于太子参抗T2DM均一多糖作用于胃肠道受体多维度吸收/调控机制研究

Is polysaccharides absorbed by the complete molecule orally? because of the difficulties in the lack of chromophore for detection, the problem of structure-activity relationship and oral absorption is not able to decode. Our research had been funded that two active homogeneous polysaccharides isolated from anti type 2 diabetes mellitus (T2DM) polysaccharide extract of Pseudostellaria heterophylla (PHP) were labeled with radioisotopes (99mTc-pertechnetate), performed by SPECT/CT analysis images after oral administration in rats. At post ingestion, about 70% of the radioactivity was observed in the intestine metabolized and is excreted in the urine and stools. Thus, we propose the hypothesis that polysaccharide could been taken in full molecule or to regulate intestinal physiological function..This project intends to study 5 different structures PHP. The nuclear 99mTc-PHPn probe will been prepared, 99mTc-PHPn were oral administered to rats, MRI&PET/CT imaging in vivo were collected, processed, and present information about biodistribution and into blood. To prepare 2-AB/FITC fluorescence probe and establish gel permeation chromatography with fluorescence detector (HPGPC-FLD) detection method, so as to Judging the integrity of polysaccharides absorption molecules. Caco-2 cells model in vitro, transport of uptake pathways were clear. Quantitative measurements of serous fluid PHP by everted rats intestinal sac to determine the intestinal absorption area. The tight junction (TJ) of IEC-6 cells were observed in Laser confocal scanning microscope (LCSM), the protein level, the expression of mRNA of Occludin, ZO-1, Clathrin and JAM as well as the glucose transporters of intestinal tract SGLT-1, GLUT-2 and GLUT-5 were by western blot and RT-PCR assay in order to determine the intestinal epithelial tight junction polysaccharide targets. And the inhibitory effect of PHP on intestinal digestive enzyme was tested with ELISA . We will put research emphasis on the mechanism of interaction between PHP and target gastrointestinal receptors by surface plasmon resonance technology (SPR) and computer virtual screening method. To explore the intrinsic law of oral absorption and structure of polysaccharides and their intestinal biological behavior from a multidimensional perspective.

多糖缺乏生色团体内检测困难，其是否以完整分子口服吸收？难题至今未解。前期研究表明，大鼠口服太子参抗T2DM活性均一多糖(PHP)，大部分滞留胃肠道代谢，少量分布心肝肾，未见大肠膀胱排出。由此提出多糖作用于胃肠道受体以完整分子吸收或生理调控假说。本项目拟研究5个PHP，制备多糖-锝核素探针，大鼠灌胃活体成像，获PHP体内分布和入血信息。以多糖-荧光探针，建立高效凝胶色谱-示踪分析方法，可判断多糖吸收分子的完整性。Caco-2细胞预测PHP转运方式及吸收途径；大鼠外翻肠囊浆膜液PHP定量，确定肠道靶向吸收区域；LCSM观察肠上皮细胞IEC-6紧密连接(TJ)超微结构，ELISA、WB和RT-PCR法测PHP对单糖水解酶抑制活性、肠道葡萄糖转运体和TJ蛋白水平及mRNA表达的影响；SPR和VS技术进一步明确PHP与靶标作用机理。以多维视角探索多糖口服吸收奥秘及结构与其肠道生物学行为内在本质规律。

多糖缺乏生色团体内检测困难，其是否以完整分子口服吸收产生活性，一直是中医药领域研究的难点和关注的焦点；中药多糖分离纯化，化学结构的研究也是中药物质基础研究的难点。.本研究从太子参抗糖尿病活性部位中，首次分离获得了杂多糖、直链葡聚糖和支链葡聚糖等5个均一多糖（PHP），并表征其化学结构；经抗糖尿病药物体外细胞模型和动物体内药效学验证，证实其具有药理活性。.本研究制备PHP-荧光探针(FITC/2-AB)，建立高效凝胶色谱-示踪分析方法，以Caco-2细胞模型考察多糖分子吸收的完整性和转运方式；太子参多糖灌胃进入大鼠体内，以PHP-锝核素探针(99mTc)，SPECT/CT示踪，证实多糖以完整分子通过肠粘膜吸收进入体循环。太子参多糖促进大鼠肠上皮细胞IEC-6紧密连接蛋白的表达，以改善细胞通透性；太子参多糖抑制肠道葡萄糖转运体SGLT-1、GLUT-2和GLUT-5基因和蛋白的表达，通过计算机模拟对接方法，分析三种多糖（直链葡聚糖PHP1、支链葡聚糖PHP2、杂多糖PHP3与GLUT2、GLUT5、α-糖苷酶GAA结合的分子构象，推测可能的亚结构，探讨其降血糖的作用机制，揭示太子参多糖口服吸收与化学结构及生理活性之间的内在关系。.本项目以闽产道地药材太子参为研究对象，针对太子参标志性活性成分不明确，药典标准不完善的问题，围绕其质量控制难点，创新完成了对太子参多糖特异性大分子化学成分结构及药理活性表征，阐明药效物质基础，为太子参质量标准制定，以及质量管理体系的建立，提供了理论依据和技术支撑。