基于p53/Nrf2 crosstalk 调控肝脏转运体探讨雷公藤肝毒性及甘草配伍减毒机制

The clinical application of traditional Chinese medicine Tripterygium wilfordii Hook F (TwHF) is limited due to its hepatotoxicity. Licorice is usually used in combination with TwHF to reduce its hepatotoxicity. However, the mechanisms are not fully elucidated. We found that triptolide (TP), the main bioactive component of TwHF, could inhibit Nrf2 expressions, leading to reduced expression levels of the downstream efflux transporters, thus causing substantial injuries as well as the disorder of bile acid profiles in livers. Combined treatment with licorice could partially reverse the effects. Our preliminary experiments showed that TP also had a transient inducing effect on Nrf2 expressions, which was also called self-protection. It was reported that p53 may have bidirectional effects on Nrf2, and Nrf2 could also affect the activation and expression of p53, thus jointly influencing the expressions of downstream factors. Besides, both TP and licorice could modulate p53. Hence, we hypothesize that TP may affect p53/Nrf2 crosstalk to modulate the expressions of the downstream transporters, leading to changes in hepatic homeostasis of both endogenous bile acids and exogenous TP metabolites, thus resulting in self-protection or hepatotoxicity. Accordingly, combined treatment with licorice may show detoxification activities. By adopting the techniques of gene knockdown and silence, LC-MS/MS, the cellular thermal shift assay and so on, we aim to discuss the crosstalk between p53 and Nrf2 and its effects on the downstream transporters both in vitro and in vivo, and quantificationally analyze changes in metabolic profiles of bile acids and TP. We are also planning to confirm whether p53 is the specific molecular binding targets of TP. Our research may shed new lights on the safe application of TwHF in clinics.

传统中药雷公藤可引起肝毒性，因而临床应用受限。雷公藤多与甘草配伍，但中毒和减毒机制尚未阐明。我们发现，雷公藤主要成分雷公藤甲素（TP）通过抑制Nrf2降低下游外排转运体的表达，引起肝实质损伤及胆汁酸代谢紊乱；配伍甘草可部分缓解。预实验发现，TP也可一过性诱导Nrf2，即自保护。另有报道，p53可双相调节Nrf2，Nrf2也可反作用于p53，从而共同调控下游基因表达；且TP和甘草均可作用于p53。因此我们提出：TP可能通过影响p53/Nrf2 crosstalk，调控下游转运体表达，使胆汁酸和TP代谢产物稳态水平改变，引起肝脏自保护或中毒；配伍甘草产生相应减毒作用。本项目拟通过细胞和动物实验，运用基因敲减、液质联用、细胞热转变分析等技术，探讨p53/Nrf2 crosstalk调控转运体的机制，定量分析胆汁酸及TP代谢谱变化，确证TP与p53是否直接结合，以期为雷公藤临床安全应用提供新思路。

传统中药雷公藤具有多种药理活性，但其肝毒性严重限制其临床应用。探讨雷公藤肝毒性机制以寻求解毒策略一直是中药药理与毒理领域的研究热点。本项目基于p53通路和Nrf2通路交互对话作用（p53/Nrf2 crosstalk），从体内和体外两个水平深入探讨了雷公藤主要活性和毒性成分雷公藤甲素所致肝毒性的分子机制。我们发现，雷公藤甲素在低剂量或短时间作用时，可产生适应性自保护作用；但在高剂量或长时间作用后，可引起严重肝损伤。相应地，低剂量雷公藤甲素可显著诱导肝细胞和小鼠肝脏中转录因子Nrf2入核，并轻微诱导p53入核，进而诱导Nrf2下游III相外排转运体MRPs和BSEP的表达；高剂量雷公藤甲素则抑制Nrf2的表达和入核，进而抑制下游外排转运体的表达，并诱导p53大量表达和入核。进一步借助基因沉默和过表达等相关分子生物学技术研究发现，在低剂量雷公藤甲素所致肝细胞轻至中度应激状态下，Nrf2可增强p53的表达和转录活性，但p53对Nrf2的表达和转录活性无显著影响；在高剂量雷公藤甲素所致肝细胞重度应激状态下，Nrf2和p53相互抑制，最终导致肝细胞周期阻滞和凋亡。免疫共沉淀实验结果显示，Nrf2蛋白和p53蛋白具有直接相互作用，并随雷公藤甲素作用强度变化呈动态改变。低剂量雷公藤甲素可促进p53和Nrf2直接结合，高剂量雷公藤甲素则促进p53和Nrf2解离。随后，课题组验证了甘草与雷公藤“配伍减毒”作用，并探讨了小分子天然产物绿原酸对雷公藤甲素所致肝毒性的保护作用。最后，课题组初步探讨了雷公藤甲素体外抗肿瘤和抗耐药作用，并探讨雷公藤甲素对多种肺癌细胞株中Nrf2和p53表达水平的影响。综上，本项目搭建了中药肝毒性及配伍减毒的药理学与毒理学研究技术平台，首次从p53/Nrf2 crosstalk角度探讨了雷公藤甲素所致自保护和肝损伤的分子机制，以有助于后续对中药中毒途径中的适应性自保护和细胞损伤之间的平衡状态产生进一步的理解、调控与转换；并在此基础上初步探索雷公藤抗肿瘤与抗耐药活性，以期拓宽中药应用的安全性和有效性范围。课题组将在已取得阶段性研究成果的基础上，申请面上项目，继续探索前段信号转导通路调控与终端药物毒物代谢谱变化之间的联系与规律，为有毒中药的临床应用提供新思路。