基于生物钟调控的雷公藤时辰代谢与毒性的分子机制研究

Tripterygium wilfordii Hook F (TWHF) is used in treatment of a variety of diseases, but it has obvious liver, kidney and other toxicity. It is of great significance to minimize the toxicity and maximize the efficacy of TWHF. In our previous studies, we found that TWHF and its active ingredient triptolide have a greater hepatic toxicity during the day than at night (chronotoxicity). However, the material basis for chronotoxicity of TWHF is unclear due to its complicated active ingredients. In addition, we found that the expression of Cyp3a11 (the major enzyme responsible for the metabolism of triptolide in mice) showed a strong circadian rhythm. Furthermore, we found that the core clock protein Bmal1 regulated the rhythm expression of Cyp3a11. Therefore, we speculated that the circadian clock may be involved in the regulation of the chronotoxicity of TWHF. Nevertheless, the mechanism by which the circadian clock control chronotoxicity of TWHF through regulation of metabolic enzymes has not been clarified. This project intends to clarify the chronotoxicity of TWHF and the chemical constituents causing the chronotoxicity of TWHF. Based on cell models and gene knockout mice, we aim to clarify the regulation of the main metabolic enzymes and the chronotoxicity of TWHF by the circadian clock. Also, the molecular mechanism by which the circadian clock regulates the main metabolic enzymes and the chronotoxicity of TWHF will be clarified by means of a variety of molecular biological methods. The study will provide scientific basis for the rational use and further development of TWHF as well as other similar toxic Chinese medicines.

中药雷公藤可用于治疗多种疾病，但其具有明显的肝、肾等毒性。规避雷公藤毒性而充分发挥其疗效具有重要的科学意义。在前期工作中，我们发现雷公藤及其活性成分雷公藤甲素的肝毒性具有明显时辰依赖性。然而，雷公藤的化学成分复杂而多样，造成其时辰毒性的物质基础尚不明确。此外我们发现，雷公藤甲素主要代谢酶Cyp3a11的表达具有明显昼夜节律性。再者，生物钟核心时钟蛋白Bmal1可调控Cyp3a11的节律表达。由此我们推测生物钟可能参与雷公藤时辰毒性的调控。然而，生物钟通过调控代谢酶而控制雷公藤时辰毒性的机制尚未明确。本项目拟对雷公藤的时辰毒性及造成雷公藤时辰毒性的化学成分进行阐明。基于细胞和基因敲除小鼠，明确生物钟对雷公藤主要代谢酶及其时辰毒性的调控作用，并通过各种分子生物学手段，阐明生物钟调控雷公藤主要代谢酶和雷公藤时辰毒性的分子机制，为雷公藤及类似有毒中药的临床合理用药和进一步开发提供科学依据。

本研究在前期工作的基础上，发现了雷公藤肝毒性具有昼夜节律性，并探究了造成雷公藤肝毒性昼夜节律的机制，并取得了以下进展：1）发现了雷公藤在0.45~1.35 mg/kg剂量范围内产生了明显的肝毒性，且ZT2毒性均高于ZT14，1.35 mg/kg剂量下差异最大。在该剂量下测定6个时间点（ZT2、ZT6、ZT10、ZT14、ZT18和ZT22）的肝毒性，发现雷公藤的肝毒性有明显的时辰依赖性，即雷公藤的肝毒性具有昼夜节律。2）探究了雷公藤在ZT2给药时，其主要活性/毒性成分雷公藤甲素在野生型小鼠血浆中的浓度以及AUC值比ZT14高；而其他四种主要成分（雷公藤乙素、雷公藤红素、雷公藤定碱、雷公藤春碱）的AUC值在两个给药时点（ZT2和ZT14）之间没有差异。雷公藤甲素的药代动力学时辰差异（暴露量：ZT2＞ZT14）可能是导致雷公藤肝毒性（ZT2＞ZT14）具有时辰依赖性的原因。3）发现了Clock基因是调控雷公藤时辰毒性和雷公藤甲素的时辰药代动力学的主要基因，缺失Clock基因可以消除雷公藤药代动力学的时辰依赖性。4）发现了Clock可以调节雷公藤甲素的肝微粒体代谢，在野生型小鼠的肝微粒体中，雷公藤甲素在ZT14的代谢速率及生成的雷公藤甲素代谢物更多。而缺失Clock基因显著降低雷公藤甲素在肝微粒体中的代谢水平，且代谢的时辰差异消失，生成的雷公藤甲素代谢物无时辰差异。5）发现了Cyp3a11的mRNA表达水平具有昼夜节律，Clock基因缺失会扰乱Cyp3a11的表达节律，多种Cyp2c基因的表达没有节律，Clock基因缺失也不影响它们的表达水平。通过本项目的研究，目前已在Journal of Pharmacy and Pharmacology期刊上发表SCI论文1篇，依托本项目已培养硕士研究生1名。