Nrf2信号通路介导葡萄糖醛酸化转移酶和外排转运蛋白调控的黄芪个体化用药的分子机制

Astragali radix (HQ in Chinese) is a well-known traditional Chinese herbal medicine that has been widely applied for over 2000 years to reinforce vital energy and body immunity in Chinese clinics. However, the HQ doses used in Chinese clinics range from 15 g to 300 g, which far exceeds the treatment dose recorded in Chinese Pharmacopoeia. It implies that the precision personalized HQ practice is extremely important. Unfortunately, the personalized HQ practice still lacks the modern theoretical basis. In our previous studies, we found that the bioactive isoflavonoids, including calycosin, from HQ could be extensively metabolized by UDP-glucuronosyltransferases (UGTs) in vivo. On the other hand, the bioactive isoflavonoids and their glucuronides could be transported by the efflux transporters (ETs). The coupling of UGTs and ETs controlled the exposed behavior of the bioactive compounds from HQ, resulting in an extremely low bioavailability of these compounds (< 7% in mice), thereby causing striking individual differences in HQ efficacies. Moreover, we found that HQ and its main bioactive compounds could up-regulate the UGTs and ETs expression via transactivating nuclear factor erythroid 2-related factor (Nrf2) signaling pathway, further causing more striking individual differences in HQ efficacies. Taken together, we hypothesize that UGTs/ETs/Nrf2 would be the key elements for the personalized HQ practice because the diverse polymorphism of UGTs/ETs/Nrf2 could regulate the personal different exposure of the HQ bioactive isoflavonoids in vivo. .Accordingly, in the current study, we will investigate the correlation between diverse polymorphism of UGTs/ETs/Nrf2 and the HQ efficacies using diversity outbred (DO) mice and transfected cells. On the other hand, gene knock-down cells and gene knock-out animals will be employed for systematically investigating the regulatory mechanisms of HQ and its main bioactive compounds on the transcriptional activations of UGTs/ETs by Nrf2 signaling pathway. The outcomes of this study will provide the pharmacokinetic theoretical basis for the precision personalized practice of HQ in clinics.

中医临床常重用黄芪，使用剂量跨幅从15g到300g，远超出中国药典规定。这表明依据疾病的黄芪个体化用药相当重要。然而，尚未有现代理论依据。我们发现：葡萄糖醛酸化转移酶（UGTs）代谢和外排转运蛋白（ETs）外排，使黄芪的药效成分如毛蕊异黄酮等生物利用度低（小鼠<7%），产生了药效个体差异；同时，黄芪和毛蕊异黄酮等可激活Nrf2通路，上调UGTs和ETs表达，引起了更显著的个体差异。因此，我们认为UGTs/ETs/Nrf2共同决定了黄芪药效的敏感性，是个体化精准用药的重要依据。故将构建多杂交系DO小鼠模拟机体个体化特征，以肺炎和肺癌为药效评判模型，采用基因组学方法，研究UGTs/ETs/Nrf2基因多态性和黄芪药效敏感性的相关性；同时采用Nrf2基因敲除小鼠和细胞，研究黄芪通过Nrf2反调控UGTs/ETs的作用及机制。以期为黄芪个体化精准用药提供药动学基础与依据，指导黄芪临床的合理用药。

黄芪为中医临床常用中药，功效显著，但药效存在明显的个体差异，挖掘其药效差异的调控机制有助于黄芪的个体化用药。我们前期发现葡萄糖醛酸化转移酶（UGTs）代谢和外排转运蛋白（ETs）外排的共同作用，使黄芪的黄酮类药效成分生物利用度低，导致黄芪药效的个体差异。同时，黄芪及其活性成分可激活Nrf2通路上调UGTs和ETs表达，引起了更显著的个体差异。由此认为UGTs/ETs/Nrf2三元素共同决定了黄芪药效的敏感性，是黄芪个体化用药的重要靶点。为了证实这一推测，按照项目计划，开展两部分研究内容：（1）UGTs/ETs/Nrf2和黄芪药效个体差异的相关性；（2）黄芪对UGTs/ETs/Nrf2的调控作用及机制。主要结果包括：1）构建1型糖尿病模型，阐明Nrf2与黄芪药效差异的相关性及调控机制。发现小鼠对诱发型1型糖尿病的易感存在显著的个体差异，Nrf2信号通路被抑制可显著促进1型糖尿病的发生和发展，由此推测激活Nrf2是治疗T1DM的有效方法；亦发现Nrf2与黄芪改善T1DM的药效显著相关，即黄芪水提物和芒柄花黄素可激活Nrf2信号通路修复胰岛β细胞损伤，改善小鼠1型糖尿病。该研究可为黄芪临床治疗1型糖尿病个体化用药提供思路和方法；2）构建结肠癌模型，阐明UGTs/ETs与黄芪药效差异的相关性及调控机制。发现黄芪水提物和芒柄花黄素可显著抑制结肠癌，其药效差异与ETs显著相关，即ETs（MRP2和BCRP）可显著干预芒柄花黄素的药效；亦发现microRNA可靶向调控MRP2和BCRP，介导芒柄花黄素的药效，提示可基于microRNA/ETs靶标对黄芪抑制结肠癌实施个体化用药；3）阐明黄芪及其活性成分对UGTs/ETs/Nrf2的调控作用及机制，发现黄芪水提物、黄芪甲苷、毛蕊异黄酮和芒柄花黄素可显著激活Nrf2信号通路上调和增强ETs（P-gp和BCRP）的表达水平和外排功能；亦可显著调控UGT1A的蛋白表达水平。该研究结果表明黄芪可通过调控UGTs/ETs/Nrf2进一步影响其药效的差异，这亦是黄芪个体化用药的重要依据。该项目实施至今，已发表7篇相关SCI论文；已培育1名毕业硕士研究生并获得国家奖学金，正培育4名在读硕士研究生；参与获得1项广东省科学技术奖科技进步奖一等奖；参与编写1部专著；负责人获得广州中医药大学“青年英才”培养对象等。