基于小肠M细胞内吞作用探讨醋柴胡多糖吸收机制

Polysaccharides of Chinese Medicine has highly clinical development prospects because of their biological activities such as anti-inflammatory, anti-aging, anti-tumor and immune regulation. Chinese Medicine were triditionally decocted with water and taken orally, so nowadays the develped polysaccharides were always oral fomulations, but the absorption mechanism of those were not clear because of their big molecular weights, complex constructions and detecting difficulties. Microfold cells (M cells) , the intestinal immune cells, play key role on the intestine absorption of macromolecular drugs. M cells used as the target of the research of absorption is the important direction researching the mechanism of absorption of macromolecular drugs. polysccharides were comfirmed to be active ingredients of vinegar-baked Radix Bupleuri, and the immune regulation effect was their main effect. Our previous research showed that Polysccharides of vinegar-baked Radix Bupleuri could form spherical aggregates in water solution, and their particle sizes were suited to be engulfed by M cells which suggested that M cells might mediate the absorption of Polysccharides of vinegar-baked Radix Bupleuri. Thus the polysccharides of vinegar-baked Radix Bupleuri will be studied to determine the active polysccharides by testing the immune regulation effect of polysaccharides with different molecular weight, and to explore the relations between the molecular weight and composition of polysaccharides and their absorption characteristics through in vitro and in vivo methods, which will lay the foundation on the absorption mechanism of polysaccharides and provide the experimental data for production and application of polysaccharides in functional foods and therapeutic agents.

中药多糖因其广泛生物活性而极具开发利用前景，根据中药传统水煎口服的特点，目前研发的多糖多为口服制剂，因其分子量大、结构复杂且不易被跟踪、检测等特殊性质，其体内吸收机制尚不明确，影响了其进一步开发。M细胞是肠道的免疫细胞，同时是生物大分子口服经肠吸收的关键，因此以M细胞为靶点是研究生物大分子口服吸收机制的重要方向。柴胡多糖已证实是柴胡的活性成分之一，免疫调节作用是其主要生理活性，且本课题组前期研究表明醋柴胡多糖在水溶液中形成类似球状的聚集体，其粒径在M细胞吞噬范围内，提示M细胞可能介导醋柴胡多糖的吸收。本项目拟通过筛选不同分子量区间醋柴胡多糖免疫调节活性确定活性多糖部位，进一步分离纯化均一活性多糖，再通过荧光标记活性多糖，采用体内外吸收研究相结合的方法考察醋柴胡多糖的吸收参数，并基于M细胞内吞作用探讨其吸收机制，从而解决醋柴胡多糖吸收机制的科学问题，并为同类口服中药多糖吸收研究提供思路。

中药多糖因其广泛生物活性而极具开发利用前景，根据中药传统水煎口服的特点，目前研发的多糖多为口服制剂，因其分子量大、结构复杂且不易被跟踪、检测等特殊性质，其体内吸收机制尚不明确，影响了其进一步开发。M细胞是肠道的免疫细胞，同时是生物大分子口服经肠吸收的关键，因此以M细胞为靶点是研究生物大分子口服吸收机制的重要方向。柴胡多糖已证实是柴胡的活性成分之一，免疫调节作用是其主要生理活性，且本课题组前期研究表明醋柴胡多糖在水溶液中形成类似球状的聚集体，其粒径在M细胞吞噬范围内，提示M细胞可能介导醋柴胡多糖的吸收。本项目利用膜分离制备了不同分子量的醋柴胡多糖，并通过研究不同醋柴胡多糖部位的免疫活性，筛选出具有较好免疫抑制活性的醋柴胡多糖部位，进一步分离纯化出了均一活性多糖并初步鉴定了其一级结构，再通过荧光标记成功标记了活性多糖，通过建立Caco-2单层细胞模型和M细胞模型探讨荧光标记醋柴胡多糖体外吸收转运特性，通过药代动力学实验和体内肠分布实验探讨体内吸收特性。结果得到了3个不同分子量的活性均一多糖，分别为VBCP1-4(2735.82kd)，VBCP3-4(845.46kd)，VBCP5-1(130.78kd)，3个不同分子量的醋柴胡多糖在 Caco-2 单层细胞模型中均属于吸收不良形，而在M细胞模型中的吸收均能显著增大，且低温会显著抑制其转运，网格蛋白介导的内陷的抑制剂氯丙嗪也能显著抑制其转运，提示醋柴胡多糖可能通过M细胞吸收，且其转运方式为主动转运，以网格蛋白结构介导的内吞途径入胞是其可能的转运机制之一。比较了三种多糖转运结果与结构的关系，提示转运量可能与糖醛酸含量、单糖组成以及糖构型有关。体内药代动力学结果显示醋柴胡多糖能够吸收进入体内，但生物利用度较低。肠吸收分布实验结果显示醋柴胡多糖可能被回肠派氏结上的M细胞内吞进入体内。体内分布结果显示吸收后醋柴胡多糖主要分布在在肾、肝、心和脾中，在肺中较少。本项目的实施为醋柴胡多糖体内吸收研究提供了重要理论数据，并为同类口服中药多糖吸收研究提供思路。