葡萄糖醛酸化通路及其调控在大黄蒽醌类肝肾代谢及解毒作用中的基础研究

Polygonum multiflorum and Rheum officinale can lead to severe liver or kidney injury, and the Rheum Athraquinoines are the main toxic components. Clarifying the metabolic mechanism of these components is important to their detoxification. We have proven that glucuronidation is the main metabolic and detoxification pathway for Rheum Athraquinoines; UGT1A9 and UGT2B7 are the major metabolic enzymes; HNF4A may regulate the transcription of both UGT1A9 and UGT2B7 by binding their promoter region. In order to prove our hypothesis that the hepatic/renal detoxification of Rheum Athraquinoines is regulated by HNF4A-UGT1A9/UGT2B7-mediated metabolic pathway, we plan to: 1) Explore the individual glucuronidation of Rheum Athraquinoines in human liver and kidney microsomes, as well as the relationship between the UGT1A9/UGT2B7-mediated metabolism and the HNF4A expression based on the Genome-wide association study and RNA-seq association study; 2) Explore the detoxification of Rheum Athraquinoines through HNF4A-UGT1A9/UGT2B7-mediated pathway in vitro using HNF4A-overexpressing HepG2 cell, HNF4A-RNAi HepG2 cell, site-directed mutation cells, UGT1A9-overexpressing HEK293 and UGT2B7-overexpressing HEK293; 3) Explore the pharmacokinetics/toxicokinetics of Rheum Athraquinoines using UGT-inhibited mice, UGT-induced mice and HNF4A-RNAi FVB mice to further demonstrate our hypothesis. The study will clarify a new HNF4A-UGT1A9/UGT2B7-mediated regulatory metabolic pathway for the detoxification of Rheum Athraquinoines in liver/kidney and provide theoretical and experimental basis for the clinical security applications of traditional Chinese medicine such as Polygonum multiflorum and Rheum officinale.

大黄蒽醌类是何首乌、大黄等中药引起肝肾损伤的主要致毒物。从蒽醌类自身代谢角度寻找解毒途径已成为关键科学问题，但目前其代谢调控机制与毒性关系尚不清楚。我们前期发现葡萄糖醛酸化是大黄蒽醌重要解毒途径，葡萄糖醛酸转移酶UGT1A9与UGT2B7为代谢关键酶，其表达极可能同时受转录因子HNF4A调控。为证实此代谢解毒调控通路，本项目拟：①采用人肝肾微粒体代谢模型，结合转录组、基因型数据及生物信息学分析阐明HNF4A-UGT1A9/2B7-大黄蒽醌代谢调控机制；②沉默/过表达细胞中HNF4A及UGT1A9/2B7，进行细胞毒性实验和代谢研究，阐明代谢调控与毒性关系；③以HNF4A-RNAi敲低小鼠及UGT酶抑制/诱导小鼠，结合肝肾毒性评价研究大黄蒽醌药代/毒代动力学，确证上述通路作用。研究结果可为发现UGT代谢酶诱导解毒剂促进大黄蒽醌类代谢降低其毒性提供科学理论依据，并为此类中药安全应用开辟新思路。

大黄素是主要存在于大黄和何首乌等药材中的天然蒽醌类物质，具泻下、抗菌、抗炎、抗肿瘤和保护肝肾等作用。然而大黄素对肝脏具双重作用，小剂量起保护作用，大剂量产生毒性。目前关于大黄素的致肝毒性机理及代谢与毒性的关系尚不明确，本研究主要考察大黄素的致毒机理，药物代谢酶和代谢调控介导的大黄素毒性。为大黄素的临床合理使用提供理论基础和科学依据。.研究发现：①高浓度大黄素通过诱发氧化应激和耗竭还原型谷胱甘肽致肝损伤，外源性补充还原型谷胱甘肽和N-乙酰半胱氨酸可缓解大黄素对细胞的毒性。②UGT2B7是大黄素的体内主要II相代谢酶，4个新的基因突变位点rs12186237、 rs56724509、rs135297和 rs11726899显著影响大黄素II相代谢。大黄素原药毒性大于其葡萄糖醛酸化代谢物的毒性，因此UGT2B7介导的大黄素II相代谢起解毒作用。高浓度大黄素可同时抑制HNF4A和UGT2B7，而敲低HNF4A可显著下调UGT2B7，由此证实大黄素可通过抑制HNF4A下调UGT2B7使II相代谢减弱，毒性增加。③长期给予大剂量大黄素可致大鼠肝损伤，且大黄素的肝毒性和毒代动力学具性别差异，UGT2B7和多药耐药相关蛋白2（Multidrug resistance-associated protein 2, MRP2）是导致大黄素的肝毒性具性别差异的潜在机制之一。④长期合用丙磺舒和大黄素可导致大鼠严重肝损伤（33.3%大鼠死亡）。丙磺舒通过抑制大鼠和细胞内UGT1A9、UGT2B7的酶活性/表达及MRP2的表达，增加大黄素及其代谢物的血药浓度，进而加重大黄素诱导的肝损伤和细胞毒性。