雷公藤甲素通过孕烷X受体抑制CYP3A4表达协同促进底物药物肝毒性

Triptolide (TP) is a widely used immunosuppressive agents and anticancer agent with clearly defined efficacy. It is originated from traditional Chinese herb Tripterygium wilfordii and mainly used in China. TP-induced liver injury has greatly limited it clinical application and further development. However, the mechanism of TP-induced hepatotoxicity has not been thoroughly elucidated. Available basic studies showed that TP acts specifically on RNA polymerase II, blocks the synthesis of new mRNA and inhibits the expression of inducible functional proteins. It is well known that expression induction of CYP3A4 caused by exposure of exogenous drugs leads to activated enzyme activity and increased metabolic clearance of drugs. We speculate that, besides nonspecific transcriptional inhibition mediated toxicity in hepatocytes, TP may increase the exposure level of drugs that are metabolically detoxified in liver, by inhibiting the expression and activity of CYP3A4, and synergistically promote hepatotoxicity. Since the incidence of CYP3A4 inhibition mediated drug interactions is high in clinic, and the drug interaction induced toxicity may cause severe consequences, this study is designed to investigate the regulatory role of TP on the downstream CYP3A4 target gene of progesterone X receptor in sandwich culture primary hepatocytes or hepatic cell lines in vitro or experimental animals in vivo. A number of advanced techniques or methods will be used including luciferase EGFR reporting system, High-Content Analysis, LC-MS/MS based metabolite identification. The synergistic effect of TP with CYP3A4 substrate drugs, such as statins, on their CYP3A mediated detoxification or activation will also be evaluated to reveal the mechanism of TP-induced hepatotoxicity. The outcomes of this study will provide a scientific basis for guiding the safe use of drug in clinic.

雷公藤甲素（TP）是我国特有、临床疗效明确的中药单体，其肝毒性限制了临床应用。然而，TP特异性肝毒性机制不明。鉴于基础研究表明，TP特异性抑制RNA聚合酶，阻断全新mRNA合成；而肝脏药酶受核受体诱导调控。我们推测，TP可能通过抑制代谢酶的表达和活性增加底物药物的暴露水平，协同诱发固有毒性药物的肝脏毒性，增加临床肝毒性的风险。基于CYP3A4代谢药物相互作用在临床的发生率较高且后果最为严重，本课题拟以原代、传代肝细胞及大、小鼠为研究对象，综合应用现代分子毒理学与药物代谢的相关技术方法，包括体外细胞三明治培养，荧光素酶EGFR报告系统，高内涵分析、代谢酶活性及代谢产物HPLC/MS鉴定等，考察TP对孕烷X受体及其下游CYP3A4靶基因的调控作用，并验证TP与CYP3A4解毒药物他汀协同产生肝毒性作用，系统阐明TP肝毒性作用机制，为指导TP的安全合理用药提供理论与实验依据。

雷公藤甲素（Triptolide, TP）作为免疫抑制剂在我国已广泛应用40多年。近年来，TP所致的肝损伤引起了广泛关注，但其真正的肝毒性机制，特别是毒性事件发生的起始原因尚不清楚。前期研究结果表明，TP对细胞整体转录的抑制作用发生在细胞死亡早期和较低浓度时。因此，全转录组抑制及其驱动的细胞功能障碍在TP诱导的肝毒性中起着核心作用。鉴于以上基础研究结果，TP特异性抑制RNA聚合酶，阻断全新mRNA合成。因此，我们推测，TP可能通过抑制肝组织药物暴露条件下药物消除相关代谢酶的表达和活性增加底物药物的暴露水平，协同诱发固有毒性药物的肝脏毒性，增加临床肝毒性的风险。基于CYP3A4代谢药物相互作用在临床的发生率较高且后果最为严重，本课题以原代、传代肝细胞及大鼠为研究对象，综合应用现代分子毒理学与药物代谢的相关技术方法，包括体外细胞三明治培养，荧光素酶EGFR报告系统，代谢酶活性及代谢产物HPLC/MS鉴定等，考察TP对孕烷X受体及其下游CYP3A4靶基因的调控作用，并验证TP通过抑制CYP3A4表达与底物药物他汀钙（Atorvastatin calcium，ATR）协同产生肝毒性作用，为指导TP的安全合理用药提供理论与实验依据。主要结果如下：.1. TP处理pcDNA3.1-hPXR，pGL3-Cyp3A4和pRL-TK瞬时转染HepG2细胞36 h后，PXR介导的荧光素酶活性随着浓度的增大显著降低，TP抑制hPXR，从而抑制CYP3A4基因的转录活性。.2. 夹心培养的大鼠原代肝细胞（SCRH）中PXR靶基因mRNA水平随TP处理时间和浓度增加依赖性降低。同时，大鼠体内实验结果表明TP对CYP3A的抑制作用随着给药次数的增加而加强，连续7天口服TP （0.5 mg/kg/day）后CYP3A探针底物咪达唑仑暴露水平与阴性对照组相比增加最为显著。此外，雌性SD大鼠中CYP3A4，CYP2C8和P-gp蛋白表达水平显著低于对照组。TP抑制广谱mRNA转录和肝组织中相关肝药酶和转运蛋白的表达。.3. CYP3A4底物药物ATR在72 h剂量依赖性降低HepG2，HepRG及SCRH细胞活力，产生细胞毒性。TP在三种肝细胞系中均显示出与ATR有效协同相互作用。.4. 与对照给药组大鼠的肝组织相比，ATR组肝切片病理学检查显示肝细胞水肿变性和空泡化，联合组最为严重。