肠道菌群介导的三七皂苷脑保护作用机理及其PK-PD结合研究

Recently, many reports have shown that panax notoginseng saponins (PNS) play an important role in protecting brain injury. The pharmacokinetic characteristics of PNS, low bioavailability, poor permeability, low concentration in brain tissue, were contradict to its central nervous protective effect. Until now, no report was found about the brain damage protection mechanism of PNS. Gut microbiota is an important human "ecological organs ", and has become the target of drugs in the treatment of various diseases. In this project, we will develop a feasible methodology and approach on the pharmacokinetic-pharmacodynamic studies for traditional Chinese medicines. Herein, we will focus on investigating whether the gut microbiota effects on the central nervous system extended to the PNS regulate on the microbiota. In order to explore the mechanism of PNS on the intervention of pathogens and the inflammatory cytokines transferring from intestinal to brain through " gut-brain" axis, and to reveal the real connotation of contradictory phenomena on the pharmacokinetics - pharmacodynamics of PNS, three key steps were involved in this project. Firstly, we will develop a series of suitable and feasible technologies on pharmacokinetic studies for herbal medicines and gut microbiota research. The second step was to look for bio-mark of brain damage by investigating rat intestinal barrier function, blood brain barrier permeability, gut microbiota, inflammatory cytokines. Finally, we will observe the changes of rat behavior, expression of inflammatory cytokines in serum and brain tissue based on germ-free animals and their conventionally colonised counterparts after intragastric administration of PNS, and investigate the contribution of PNS on the treatment of brain damage. Through the research above, we can provide novel and common methods for pharmacokinetic and pharmacodynamic studies for Chinese herbal medicines.

三七皂苷对脑损伤具有明确的保护与治疗作用；然而，三七皂苷生物利用度低、血脑屏障通透性差、在脑组织几乎没有分布；三七皂苷药代-药效不相关使其发挥脑损伤作用的机制尚不明确。肠道菌群是人体重要的"微生态器官"，已成为药物开发与病症治疗的潜在靶标。 本研究拟在建立系统、普适的中药药代动力学、肠道菌群组学研究技术的基础上，通过测定大鼠肠道屏障功能、肠道菌群结构、炎症因子的变化，寻找与脑损伤相关的生物标志物；同时，以肠道菌群为切入点，分别考察清洁级和转染肠道菌群的正常/脑损伤模型大鼠灌胃给予三七皂苷后行为学、血清和脑组织中炎症因子的表达的变化，考察肠道菌群调节在三七皂苷治疗脑损伤过程中的贡献度；基于"脑损伤生物标志物-肠道菌群-炎症传递"探索三七皂苷的脑保护作用机理，揭示三七皂苷的药代-药效相矛盾现象的真正内涵；并为其它中药皂苷类成分"PK-PD不相关"的机制研究提供普适的研究思路与技术体系。

研究背景：三七总皂苷对脑损伤具有明确的保护与治疗作用；然而，三七总皂苷成分复杂、体内生物利用度低、血脑屏障通透性差、在脑内几乎没有分布；三七皂苷药代-药效不相关使其发挥脑损伤作用的机制尚不明确。肠道菌群是人体重要的“微生态器官”，并已成为各种疾病的药物开发与治疗的潜在靶标。.研究内容：在建立普适的中药药代动力学、肠道菌群研究技术的基础上，通过测定大鼠肠道屏障功能、肠道菌群结构、炎症因子，寻找与脑损伤相关的生物标志物；同时，以肠道菌群为切入点，分别考察清洁级和转染肠道菌群的正常/脑损伤大鼠灌胃给予三七皂苷后行为学、血清和脑组织中炎症因子的表达的变化，考察肠道菌群调节对三七皂苷治疗脑损伤的贡献度；基于“脑损伤生物标志物-肠道菌群-炎症传递”探索三七皂苷的脑保护作用机理，揭示三七皂苷的药代-药效相矛盾现象的真正内涵；同时，为其它中药皂苷类成分的PK-PD研究提供普适、可行的研究思路与方法。.重要结果与关键数据：三七皂苷与肠道菌群间存在明显的相互作用：一方面，不同的肠道菌群组成可以显著影响三七皂苷代谢物的种类和暴露量，在正常组和TNBS造模组共鉴定出39种代谢物，在联合抗生素造模组找到35种代谢物；另一方面，三七皂苷可以调节大鼠肠道菌群，发挥脑保护作用。大鼠造急性脑缺血-再灌注（I/R）模型后，肠道菌群组成显著改变，长双歧杆菌显著下调，I/R大鼠连续灌胃6天三七皂苷后，长双歧杆菌显著上调，进而上调海马区GABA-B受体的表达，从而发挥间接的脑保护作用。此外，三七皂苷R1、人参皂苷Rb1和人参皂苷Rg1是三七皂苷的主要的药效物质基础。. 科学意义：建立多特征碎片离子筛选技术，应用于PPD和PPT型皂苷的高通量筛选及其鉴定；并应用于三七皂苷在大鼠体内代谢物的鉴定与归属研究中。建立了中药在阐释三七皂苷PK-PD不相关现象的同时，揭示三七皂苷的药代-药效物质基础，此外，本研究还为其他中药的PK-PD研究提供共性的研究思路与方法。