基于Nrf2/ARE信号通路调控MRP2探讨甘草与有毒中药配伍减毒的机制

Licorice can reconcile various drugs and have detoxification effect. Some toxic herbs often accompany licorice in clinical application. Modern studies suggest that its mechanism including precipitation effects, antagonistic action, inducting the expression of P450 enzyme, etc. With the development of the research, new mechanisms have been put forward and these mechanisms coordinate with each other. The pharmacokinetics of poisonous components is a new entry point. P-gp and MRP attract the most attention in drug transporters. They can influence the absorption, distribution and excretion of their substrates. We found that licorice can induce the outward transport of P-gp in our earlier study. We consider that licorice maybe intervene Nrf2/ARE signal pathway to induce MRP2, then licorice can accelerate the elimination of poisonous components and reduce the exposure of poisonous components. Maybe this is a new mechanism of decreasing toxicity of licorice. This study will be carried out from the angle of Ⅲ phase drug transports. We will combine molecular biology, gene knockout and metabolite analysis techniques to build a research platform of mechanism of decreasing toxicity by compatibility. Tripterygium wilfordii and semen strychni will be selected as models of compatibility. We will discuss the mechanism of decreasing toxic herbs toxicity by compatibility of licorice based on Nrf2/ARE signal transduction pathway regulating MRP2. We will explore the relation and discipline between the regulation and control of signal transduction pathway and the change of metabolic profiling. Our study will supply a new approach for improving the safety of compound prescription of Traditional Chinese Medicine.

甘草具有调和诸药和解毒的作用，一些有毒中药常与甘草配伍以减少毒性。现代研究认为其机制主要有沉淀毒物、药效学拮抗作用、提高P450酶表达增强毒物代谢等。随着研究的深入，新的配伍减毒机制不断被提出，且各种机制相互协同，毒性成分体内药动学是目前新的切入点。P-gp和MRP是被关注最多的药物转运体，能影响其底物的吸收分布和排泄，本课题组在前期发现甘草能够诱导P-gp外向转运的基础上，认为甘草可能通过Nrf2/ARE通路诱导MRP2表达，加速毒物从机体消除，减少毒物暴露，起到与有毒中药配伍减毒的作用。本研究从Ⅲ相药物转运体的角度，结合分子生物学和基因敲除技术，与代谢产物定性定量分析相对接，选择雷公藤、马钱子为配伍对象，搭建中药配伍减毒研究技术平台，探讨甘草通过Nrf2/ARE 通路调控MRP2的配伍减毒机制，探索信号转导通路调控与药物毒物代谢谱变化之间的联系与规律，为提高中药复方安全性提供新的思路。

甘草“和诸药，解百毒”，在古今方剂中应用普遍，一些有毒中药常与甘草配伍以减少毒性。课题组结合文献和前期研究，提出假设：甘草可能通过Nrf2/ARE信号通路诱导MRP2 表达，加速毒物从机体消除，减少毒物暴露，起到与有毒中药配伍减毒的作用。本项目围绕重点问题进行了探索，选用HepG2细胞和ICR小鼠进行体内外实验，证实甘草提取物显著诱导了Nrf2及其下游基因MRP2、UGT1A1等，摸索了甘草的有效诱导剂量，并通过ARE-荧光酶报告基因法发现异甘草素对ARE诱导效果最为明显；进一步通过基因敲除小鼠验证了异甘草素对MRP2、UGT1A1等基因表达的诱导作用依赖于Nrf2/ARE的调控；通过GC-MS分析异甘草素对Nrf2基因敲除/野生型小鼠肝脏小分子代谢物谱的影响，发现了Nrf2通路与糖代谢的联系，提示了异甘草素可能通过Nrf2通路调节II相解毒酶，影响甘氨酸、谷胱甘肽等的浓度，从而起到保护肝脏的作用；通过LC-MS/MS测定Nrf2野生型和基因敲除型小鼠肝脏16种胆汁酸并进行代谢轮廓分析，异甘草素能够诱导Nrf2的表达，影响小鼠部分内源性胆汁酸的显著性变化，表征了这些胆汁酸可能为异甘草素通过Nrf2通路对小鼠肝脏起保护作用的生物标识物；在此基础上，以甘草雷公藤配伍减毒为实例，通过测定大鼠血浆中胆汁酸及应用于甘草雷公藤配伍减毒研究，从胆汁酸浓度变化可初步推断，甘草可缓减雷公藤所致肝毒性；在细胞水平考察了甘草及其有效成分对雷公藤甲素所致氧化应激的改善作用，又从动物水平进行了考察，雷公藤甲素可显著升高血清中的ALT、AST等，异甘草素可一定程度降低其含量；雷公藤甲素还可降低GSH含量，减弱GPx、SOD等活性同时升高MDA含量，异甘草素可改善这一现象。雷公藤甲素可下调细胞Nrf2表达，可能会损伤细胞或机体的抗氧化能力，异甘草素可一定程度上调Nrf2的表达，起到增强抗氧化防御的作用。本研究从Ⅲ相药物转运体的角度，通过一系列研究，结合分子生物学和基因敲除技术，与代谢组学技术相对接，选择雷公藤为配伍研究对象，搭建了中药配伍减毒研究技术平台，从信号通路的角度提出了甘草新的解毒机制，取得了阶段性的成果，为中药配伍减毒应用提供了新的理论依据。课题组将在新的面上项目资助下，继续探索信号转导通路调控与药物毒物代谢谱变化之间的联系与规律，为提高中药复方安全性提供新的思路。