肠道网络屏障影响白头翁五环三萜皂苷抗肿瘤活性成分群PPTS吸收机制的研究

In our previous research we discovered that five Pentacyclic Triterpene Saponins (PPTS, we named them B3, BD, B7, B10 and B11) separated from Chinese Pulsatilla extraction had antitumor activity, and pharmacokinetic research demonstrated that their absorption was extremely poor and various biotransformations occurred in intestinal tract. A favorite absorption profile is the precondition of therapeutic effects of bioactive components in TCMs (Traditional Chinese Medicines), and malabsorption is the most important factor limiting their efficacy. It is necessary to investigate the key factors and their links which impact PPTS’s therapeutic effects. Intestinal absorption barrier network is composed by three different aspects---intestinal microflora, drug-metablising enzymes and drug transporters in intestinal epithelial cells. This study is intended to establish the research models and technology platform which suit for exploringTCM’s intestinal absorption barrier network function, and permeable mathematical evaluable method which suit for multiple components feature of TCM. Particularly we focus on the effects of intestinal microflora, drug-metablising enzymes and drug transporters on the absorption of PPTS to illuminate the interaction among intestinal barrier factors which influence drug’s oral absorption, and to clarify the key area, stage and mechanism which impact the antitumor effects via intestinal absorption barrier network, then analyse the influence of intestinal microenvironment on drug active component’s absorption and bioavailability, and finally improve drug’s efficacy and security.

课题组前期研究发现5种白头翁皂苷提取物白头翁五环三萜皂苷（PPTS，即B3，BD，B7，B10和B11）具有抗肿瘤活性，药代动力学特征研究表明其吸收极差，在肠道发生了广泛的生物转化。中药活性成分的体内吸收是发挥作用的前提，吸收障碍是制约其发挥药效的重要因素。研究影响五环三萜类皂苷发挥药效的重要因素和关键环节，即从肠道菌群和肠上皮细胞的代谢酶和药物转运蛋白这三层次所构成的肠道网络屏障进行探索是必要的。本课题旨在建立适宜于中药特点的肠吸收屏障网络的研究模型与技术平台，探索建立符合中药多成分特点的渗透性评价数理模型，重点研究PPTS胃肠渗透的相关菌群及酶系和药物转运体对其吸收机制的影响, 从而阐明口服给药途径吸收的肠道屏障的相互协同作用，从肠吸收屏障网络角度阐明影响PPTS发挥药效的关键区域、阶段及机制，分析肠道微环境对中药活性成分吸收的作用及其生物利用度的影响，提高药物的有效性和安全性。

本课题研究影响五环三萜类皂苷发挥药效的重要因素和关键环节，即肠道菌群和肠上皮细胞的代谢酶和药物转运蛋白这三层次所构成的肠道网络屏障。建立适宜于中药特点的肠吸收屏障网络的研究模型与技术平台，探索建立符合中药多成分特点的渗透性评价数理模型。.经过实验，得出结论：（1）白头翁皂苷上调了P-gp蛋白和MDR1水平，白头翁皂苷可以诱导P-gp功能活性，并有可能提高P-gp/mdr1 mRNA的表达。（2）CCK8 检测显示高剂量的PPTS 有一定细胞毒性；流式细胞周期显示PPTS对细胞周期有一定影响，可能是阻滞S期；细胞侵袭与迁移结果显示PPTS抑制了 SW480细胞的侵袭与迁移能力；ELISA 检测无明显差异，结果表明PPTS对TGF-β1诱导的SW480 细胞有明显的抑制作用，并促进其凋亡。（3）白头翁皂苷可能一元羧酸转运体和有机阴离子转运体的底物。肠细胞上的一元羧酸转运体（MCTs） 和有机阴离子转运体（OATP）可将白头翁皂苷吸收进入到细胞内，与 P-gp 的外排作用形成相互制衡的作用力，共同影响白头翁皂苷的生物利用度。（4）白头翁皂苷增加大鼠肠道菌群部分有益菌的丰度，降低肠道菌群部分有害菌的丰度， 从而影响大鼠肠道菌群的组成及比例。通过一系列实验，发表(或接收)文章7篇，培养研究生2名。从肠吸收屏障网络角度阐明影响PPTS发挥药效的关键区域、阶段及机制，分析了肠道微环境对中药活性成分吸收的作用及其生物利用度的影响，提高药物的有效性和安全性。