核受体REV-ERB调控药物处置及其作用机制研究

Nuclear receptors REV-ERBα/β, as important components of circadian clock, are involved in regulation of biological rhythms and metabolic homeostasis, and are closely related to various metabolic diseases. However, the regulations of metabolic enzyme and transporter expressions as well as drug disposition by REV-ERBs remain largely unknown. In preliminary work, we found that Cyp2b10 and Ugt2b36 expressions were inhibited by REV-ERBα via transcriptional regulation or interaction with nuclear receptor CAR (which activates Cyp2b10 and Ugt2b36 expressions). Furthermore, REV-ERBα knockdown significantly increased the cell metabolism of cyclophosphamide and morphine, which are substrates of Cyp2b10 and Ugt2b36, respectively. Therefore, it is reasonable to speculate that REV-ERBs have regulatory functions on drug metabolizing enzymes and drug disposition. In this study, we will screen other drug metabolizing enzymes and transporters regulated by REV-ERBs through RNA-seq and ChIP-seq. Systematical regulations of drug disposition by REV-ERBs will be performed using mouse and human hepatoma cells, primary hepatocytes and gene knockout mice. We will uncover the underlying mechanisms by integrating various molecule biology techniques. Our study will help to clarify the regulatory effects of REV-ERBs on drug disposition, and provide scientific foundations for drug development, rational drug uses, and revealing the molecular mechanisms of drug-drug interactions.

核受体REV-ERBα/β，作为生物钟重要组成部分，参与调节生物节律及代谢稳态，与多种代谢性疾病密切相关。然而，REV-ERBs对代谢酶和转运体及药物处置的调控作用尚不明确。在前期工作中，我们发现Rev-erbα在体内外均可抑制代谢酶Cyp2b10和Ugt2b36的表达。此外，还发现Rev-erbα通过转录或与CAR等核受体相互作用来调控这两个酶的表达。再者，敲低Rev-erbα显著增加环磷酰胺（Cyp2b10底物）和吗啡（Ugt2b36底物）在细胞中的代谢。因此，有理由推测REV-ERBs对代谢酶及药物处置具有调控作用。本项目拟在前期基础上，通过组学进一步筛查受REV-ERBs调控的药物代谢酶和转运体；结合肝癌细胞、原代肝细胞和基因敲除鼠，系统探究REV-ERBs及其配体对药物处置的调控作用，并联合各分子生物学技术解析调控机制，为新药开发、合理用药及药物相互作用分子机制解析提供科学依据。

核受体REV-ERBα是生物钟系统的重要组成部分，是连接生物钟与代谢稳态的关键桥梁。然而，REV-ERBα对药物代谢的影响尚不明确。本课题主要阐明了REV-ERBα对代谢酶、转运体和药物处置的节律调控作用及机制。重要结果：（1）基于REV-ERBα敲除鼠和野生型鼠，通过转录组技术，筛查发现多个药物代谢酶和转运体的表达受REV-ERBα调控；（2）通过qPCR、WB和活性验证，发现代谢酶（CYP2B10、FMO5、UGT1A9和UGT2B）和转运体（SLC6A1和SLC6A11）的表达具有明显昼夜节律，且其节律受到REV-ERBα调控；（3）基于体内外实验，进一步确证REV-ERBα正调控FMO5、UGT1A9、SLC6A1和SLC6A11，而负调控CYP2B10和UGT2B；（4）肝微粒体孵育和药动学实验表明，REV-ERBα敲除降低UGT1A9底物药物丙泊酚的代谢，而增加CYP2B10和UGT2B底物药物环磷酰胺和吗啡的代谢，同时REV-ERBα敲除削弱了这三个药物代谢的时辰依赖性；（5）基于双荧光报告基因、凝胶迁移和染色质免疫沉淀分析表明，REV-ERBα通过靶基因启动子区的REV-RE元件直接抑制CYP2B10和UGT2B表达，通过抑制E4bp4上调FMO5、SLC6A1和SLC6A11的表达，以及通过DEC2促进UGT1A9的表达。以上结果表明，核受体REV-ERBα在调节药物代谢酶和转运体的节律表达，以及药物处置中发挥着重要作用。该结果对解析REV-ERBα的生理功能和临床合理用药提供了重要理论依据。