从FXR/PXR/CYP3A4信号通路探讨雷公藤诱发胆汁淤积性肝损伤的分子机制

Tripterygium Wilfordii Hook, an effective Chinese medicine for treatment of rheumatoid arthritis, could induce cholestatic liver injury when it was used incorrectly in clinic. Our preliminary data showed that the toxic reaction of Tripterygium wilfordii was significantly different between normal and arthritic rats. The initial mechnisms was related with the bile acid metabolic pathway. However, the molecular toxicology mechanism has not been clarified yet. The TCM theory holds "You Gu Wu Yun", the logical basis or guidance for exact use of toxic herbs. The theory of "You Gu Wu Yun" not only emphasized that herbs should be used in disease state, but also pointed in the disease lasts.According to the TCM theory, it helped to getting more intuition about the mechnisms on the toxicity of toxic herbs. Regarding these, we prepare to address the collagen induce arthritic rat model (CIA) and CDCA induced HL-7702 cell injury model in the present project.And based on the models, we will explore: (1) the relationship between the hepatic injury and time-dosage-efficacy-toxicity of tripterygium wilfordii on CIA rats; (2) the relationship between the hepatic injury and the bile acid mediated FXR-PXR-CYP3A4 signal pathway; (3) what are the molecular mechanisms on regulation and modification of FXR-PXR-CYP3A4 signal pathway by tripterygium wilfordii hook...To achieve the project, some advanced experimental technology would be used, such as cocktail CYPs activity analysis, reporter gene, histological methods, cytologic methods, and molecular biological methods, and so on. The results might figure out the regulation mode of FXR-PXR-CYP3A4 signal pathway by tripterygium wilfordii, and then the studies might offer some evidences and support for clinical use of tripterygium wilfordii.

雷公藤及其制剂临床用于治疗类风湿关节炎疗效肯定，然而因使用不当可诱发胆汁淤积性肝损伤；课题申请人前期研究发现，雷公藤对正常大鼠和类风湿关节炎模型大鼠毒性反应有显著差异，其初步作用机制与胆酸代谢通路相关，但其深入的分子毒理学机制尚未阐明。中医"有故无殒"理论是有毒中药合理使用的逻辑基础，既强调中药应对"病或证"使用，也重视"中病即止"，该理论有助于理解中药临床不良反应的生物学本质。为此，本课题以胶原诱导类风湿关节炎动物模型和胆盐（CDCA）诱导的肝细胞损伤模型为研究对象，探讨雷公藤诱发肝损伤的"时-量-效-毒"关系及其与胆酸介导的FXR-PXR-CYP3A4信号通路的关系。进而采用cocktail酶活性分析、报告基因、组织学、细胞学及分子生物学等方法研究雷公藤对FXR-PXR-CYP3A4信号通路的影响及调节模式，揭示雷公藤肝毒性的分子机制，为雷公藤的临床合理使用提供实证数据和理论支撑。

雷公藤（Tripterygium wilfordii Hook. f.）是几年来最被国内外研究者及医药企业关注的中药之一，已经被开发成不同剂型的成药。国外的研究者通过大样本临床试验发现雷公藤提取物抗风湿关节炎（RA）的疗效与柳氮磺胺吡啶接近，近期有望通过FDA的审批成为治疗RA的天然产物药。国际顶级研究团队的研究表明，雷公藤红素（Celastrol）通过调控瘦素通路治疗糖尿病（包括I型糖尿病）；而且可以改善脂肪代谢起到减肥的作用。此外，雷公藤甲素、雷公藤红素还有很好的抗炎、抗肿瘤、调节免疫等药理活性。美中不足，临床观察发现长期、超量服用雷公藤制剂能诱发肝损伤，且多见胆汁淤积性肝损伤。为此我们提出如下科学假说：雷公藤活性化合物通过调控胆汁酸相关信号通路FXR-PXR-CYP3A4而实现“效-毒”的转化。. 我们选择成药（雷公藤多苷）、主要活性化合物（雷公藤甲素、雷公藤红素）为研究对象，体内与体外实验相结合，综合应用生物化学、形态学、分子生物学等多种研究手段，系统探究了雷公藤毒性反应轮廓及可能的机制。在4年的时间里，课题组取得了一些阶段性成果：. (1) 系统观测了雷公藤多苷、雷公藤甲素、雷公藤红素不同给药途径导致急性毒性的LD50值及IC50值。. (2) 梳理了与雷公藤研究相关的文献资料并采用meta分析方法挖掘出雷公藤制剂与阿司匹林联合用药增加不良反应发生的新现象。提出有毒中药对证与对症控毒的策略及研究路径。. (3) 获得了雷公藤甲素及雷公藤红素对FXR-PXR-CYP3A4信号通路影响的初步数据并借此形成了胆汁酸反馈性调节该“通路”而诱发胆汁淤积性肝炎的学术假说。该结果为胆汁代谢、肝-肠循环、核受体对胆汁代谢的调控等科学问题提供了新的研究角度。. (4) 课题组在研究过程中注意到雷公藤诱导细胞自噬的现象并开展了较深入的研究，首次发现了雷公藤甲素和雷公藤红素影响细胞命运决定的关键“开关”（即通过影响P62-PLK1蛋白相互作用而实现“正-负”双向调节）。这一发现把细胞增殖（PLK1是细胞有丝分裂调控的关键激酶）与细胞程序性死亡（自噬性凋亡）的相互调控关系彰显出来。沿着这个线索深入探究，有望揭示有毒中药抑制肿瘤细胞增殖、抗炎作用的新机制。