基于转运体的泻心汤效应组分肝脏浓集机理研究

The study on the intracorporal process and related mechanisms of the effective constituents is important to explore the scientific intension of Traditional Chinese Medicine (TCM) formula. It is also important to both inherit and promote the modernization of TCM theories. TCM formulas with clearing heat and detoxicating effects (such as Xiexin decoction according to our previous studies) show significant effects on preventing hepatic fibrosis. Based on our previous studies, we aimed to study the transporter mediated liver distribution of the effective constituents of Xiexin decoction. The rat model of hepatic fibrosis, isolated rat livers, primary cultured liver cells, and cells expressed specific transporters would be built; LC-MS/MS would be used to detect the concentrations of the effective constituents as well as the related metabolites of Xiexin decoction in biological samples; the relationship between the dynamic change of the concentrations of the constituents and the express levels of particular transporters would be analyzed; the effects of some specific inhibitors of the transporters on the concentrations of the constituents would be investigated. Hopefully, the transporter mediated liver distribution of the effective constituents of Xiexin decoction could be explained from in vivo, organ, tissue, cellular, and molecular levels. This project was anticipated to promote the studies on both the act mechanism and the law of the compatibility of Xiexin decoction on hepatic fibrosis. It was helpful to develop new anti-hepatic fibrosis drugs from TCM formula. It was also meaningful to explore the efficient methods in studying the interactions between TCM formula and drug transporters.

方剂药效组分体内过程规律及机理研究，对于诠释方剂的科学内涵、促进中医药理论传承和发展具有重要意义。清热解毒中药在防治肝纤维化中具有明显优势，课题组前期研究表明经典名方泻心汤具有抗肝纤维化作用。在已阐明泻心汤抗肝纤维化效应组分基础上，本项目采用LC-MS/MS方法测定泻心汤效应组分及代谢物在肝纤维化模型大鼠肝脏、离体肝脏、原代肝细胞和表达特定转运体细胞中的浓度，分析浓度动态变化与转运体表达水平及特异性转运体抑制剂影响的关系，从整体、器官、组织、细胞、分子水平研究泻心汤抗肝纤维化效应组分与转运体的相互作用，阐明效应组分在肝脏浓集的机理。研究结果将促进泻心汤抗肝纤维作用机理及配伍规律的研究，对经典方剂的传承及创新有重要意义，可为创制防治肝纤维化复方中药新药提供基础，并有助于对方剂与转运体相互作用研究方法进行探索。

方剂药效组分体内过程规律及机理研究，对于诠释方剂科学内涵具有重要意义。在前期阐明泻心汤抗肝纤维化效应组分基础上，本项目针对大黄蒽醌、黄芩黄酮和黄连生物碱成分，采用大鼠和小鼠整体动物、离体肝脏、原代肝细胞和转运体转染细胞、膜囊和分子对接模型，研究介导效应组分肝脏摄取和外排的转运体,阐明效应组分肝脏浓集机理。结果：①蒽醌类：大黄酸(RA)在肝切片和原代肝细胞主动摄取可被利福平和丙磺舒抑制；RA可被HEK-OAT2，-OATP1B3，-OATP2B1摄取，被BCRP泵出；利福平和丙磺舒使大鼠RA肝脏浓度降低，泮托拉唑使RA肝脏浓度升高。表明OAT2,OATP1B3和OATP2B1介导了RA摄取，BCRP介导其外排。②黄酮类：口服黄芩素(B)和汉黄芩素(W),B及代谢物(黄芩苷BG, BG’等)总暴露显著高于W及代谢物(汉黄芩苷WG等)总暴露；肠和肝中B以葡萄糖醛酸物为主，W以原形存在, B和W浓度高于血浓；WG为MRP1、2、3和4的底物，而BG为MRP4底物；BG和WG为OATP2B1、1B3底物；在RIS9、RLS9中WG水解速率明显快于BG和BG’。结果表明WG为MRP2的底物是两类成分体内暴露差异的原因，WG水解速率快和WG与MRPs亲和力高是肠和肝中存在形式差异的原因；BG和WG由OATP2B1和1B3摄取进入肝脏，再经beta-GUS水解成B和W，使肝脏B和W浓度高于血浓度。③生物碱：小檗碱(Ber)等在肝肾切片和rOct2-MDCK主动摄取被Oct抑制剂抑制,在酸化的rMate1-MDCK摄取被乙胺嘧啶抑制；伊曲康唑抑制Ber在MDR1-MDCK中B->A的跨膜转运；Ber被rOct2-MDCK摄取>rOct2-rMate1-MDCK,经rOct2-rMate1-MDCK跨细胞转运>rOct2-MDCK；乙胺嘧啶和伊曲康唑使肝肾Ber浓度增加而尿及胆汁排泄降低。分子对接显示Ber可与MATE1和P-gp结合,乙胺嘧啶与MATE1、伊曲康唑与P-gp的结合能力高于Ber。结果表明OCT摄取导致肝肾中Ber浓度高于血浓度，P-gp和MATE1共同介导Ber肝肾排泄。这些研究结果将促进泻心汤抗肝纤维作用机理及配伍规律的研究和大黄、黄芩及黄连与其他药物合理联用的相关研究，对经典方剂的传承及创新有重要意义，并有助于对方剂与转运体相互作用研究方法进行探索。