甘草诱导Nrf2调控肝脏Ⅱ相代谢酶、Ⅲ相转运体抵御雷公藤化学应激的“配伍减毒”机制

Licorice is widely used in traditional and modern prescriptions owing to its unique effect on moderating the characteristics of other herbs and poison attenuation. Tripterygium wilfordii is often accompanied with licorice in clinical application to reduce the side effects. Our early studies found that the expression of P-gp can be induced by licorice, and then our research was supported by the National Natural Science Foundation of China (NSFC) Youth Fund. We further found that licorice could induce another efflux transporter MRP2 and confirmed that the effect of the main ingredients in licorice on inducing the expression of UGT1A1 and MRP2 is Nrf2-dependant by using the Nrf2 knockout mice. However, how licorice affects the terminal process of specific toxic substances via the regulation of Nrf2 and its downstream genes remains unknown. In recent years, some scholars have found that the protection of hepatotoxicity caused by tripterygium wilfordii is associated with Nrf2. Based on the previous research, we hypothesize: licorice is very likely to defense against chemical stress aroused by tripterygium wilfordii by regulating phase Ⅱ metabolizing enzymes and phase Ⅲ transporters via Nrf2, the oxidation and chemical stress defense factor, and reduce the damage of internal and external toxic substances on liver to achieve the effect of poison attenuation. Combining with cell and molecular biology, gene knockout, microdialysis and metabonomics, we aim to explore the PK-Nrf2 research model. Conducting clinical trials, we try to seek for related markers based on mass spectrometry so as to bridge signaling pathways and dynamics process. Our project aims to elucidate the essence of Compatibility and Poison Attenuation theory of Traditional Chinese Medicine (TCM) and provide scientific basis for the rational use of Chinese medicine.

甘草“和诸药，解百毒”，在古今方剂中应用普遍，雷公藤常与甘草配伍以减毒。课题组早期发现甘草能诱导P-gp表达，获得了NSFC青年基金的资助，初步证明甘草诱导另一转运体MRP2，并通过基因敲除小鼠验证了甘草主要成分对UGT1A1和MRP2等的诱导作用受Nrf2调控。然而，甘草如何通过Nrf2调控下游因子，影响毒性物质的终端过程尚不明确，亟待深入研究。近年有学者发现机体对雷公藤所致肝毒性的保护与Nrf2有关，结合前期研究基础，我们提出：甘草很可能通过防御通路Nrf2调节肝脏Ⅱ相代谢酶、Ⅲ相转运体，抵御雷公藤所致“化学应激”，减少内、外源物对肝脏的损害，从而起到“配伍减毒”的作用。项目将结合细胞分子生物学、基因敲除、微透析和代谢组学等技术，探索“PK-Nrf2”研究模式，开展临床试验，基于质谱分析寻找相关标志物，桥接前端信号通路与终端动力学过程，阐释中药配伍减毒理论精髓，为遣方用药提供科学依据。

传统中药雷公藤可引起多种毒性，因而临床应用受限。雷公藤多与甘草配伍，但中毒和减毒机制尚不明确。本项目借助基因和蛋白表达检测、基因沉默、液质联用等技术，在L02细胞和ICR小鼠上证明，雷公藤主要成分雷公藤甲素（TP）可能引起肝脏原型药物及代谢物蓄积、胆汁淤积，进而导致肝毒性，而甘草有效成分异甘草素可通过激动Nrf2通路和肝脏转运体缓解TP肝毒性；在此基础上，进一步寻找Nrf2上游因子，在体内、体外水平证明microRNA-155通过作用于Nrf2，在缓解TP肝损伤机制中发挥重要作用。考虑到TP的严重肾毒性，课题组借助细胞药动学研究方法，结合运用外排转运体抑制剂和转染技术，测定HK-2细胞内TP浓度，证明甘草提取物可降低TP细胞内浓度，对其肾毒性同样具有保护作用，且P-gp和MRPs可能参与TP细胞内转运。课题组进一步开展囊泡转运实验和细胞摄取实验，证明TP对人源MRP2和P-gp的转运活性无影响，且TP不是人源MRP2、OATP1B1和OATP1B3的底物，该结果指引课题组继续在表达水平研究雷公藤中毒机制。在探讨甘草“配伍减毒”作用的同时，建立藤茶中二氢杨梅素及其衍生物的血药浓度测定方法，通过动物实验证明，二氢杨梅素同样可作用于Nrf2通路，缓解TP肝损伤。在此基础上，借助多种耐药细胞系、荷瘤小鼠及分子对接技术，探讨了TP对耐药非小细胞肺癌的增敏作用及机制。最后，建立大鼠糖尿病肾病模型，证明了雷公藤中另一主要活性成分雷公藤红素的肾保护作用。本项目借助分子生物学和药动学方法，摸索“化学应激-PK-Nrf2”研究模式，搭建中药配伍减毒研究技术平台，并在此基础上初步探索雷公藤有效成分的其他活性，以期提高中药使用的安全性和有效性。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，为有毒中药的临床应用提供新思路。