钩吻-玉叶金花调控核受体PXR/CAR表达“相畏”减毒的分子机制研究

The experimental data from the National Natural Science Foundation of China have already shown that the detoxification and analgesic efficacy of Gelsemium elegans combined with Mussaenda pubescens were closely related to efflux of indole alkaloids by P-gp and metabolism of indole alkaloids by CYP2E1. It is generally acknowledged that nuclear receptor PXR/CAR is in charge of detoxification and excretion process of exogenous toxins or endogenous lipid. They can effectively adjust CYP450 metabolic enzymes and ABC transporter. Therefore this research hypothesis is put forward: the detoxification mechanism of mutual restraint between Gelsemium elegans and Mussaenda pubescens works by regulating of CYP450 enzyme-ABC transporter through PXR/CAR nuclear receptor. The research is carried out to check that whether the detoxification mechanism is connected with CYP450 enzyme-ABC transporter regulated by PXR/CAR nuclear receptor. The therapeutic targets and detoxification mechanism will be elucidated from different body state between normal cells and PXR/CAR transfected cells, toxication mice and detoxification mice by determinating gene and protein expression levels of upstream nuclear receptors (PXR, CAR), downstream CYP450 enzymes (CYP2E1, CYP1A2, CYP2B6, CYP2C9, CYP3A4) and ABC transporter (P-gp, MRP2). Thus, the study will clarify the characteristics and advantages of detoxification research by mutual restraint, and provide a new method for the detoxification research of mutual restraint in traditional Chinese medicine.

课题组前期完成的国家自然科学基金表明：钩吻配玉叶金花能减毒存效，与转运体P-gp外排、代谢酶CYP2E1代谢吲哚类生物碱有关。现已知核受体PXR/CAR负责外源性毒物或内源毒性脂类的解毒和排泌过程，能有效调节ABC转运体及CYP450代谢酶。因而研究提出“钩吻畏玉叶金花是通过调控核受体PXR/CAR来上调药物代谢酶及转运体的活性，而达到有效减缓钩吻毒性的目的”假说，对相畏是否通过核受体PXR/CAR介导的CYP450酶-ABC转运体途径展开探讨，通过正常与PXR/CAR转染细胞、中毒与解毒C57BL/6小鼠，检测其上游核受体（PXR、CAR）、下游CYP450酶（CYP2E1、CYP1A2、CYP2B6、CYP2C9、CYP3A4）与ABC转运体（P-gp、MRP2）基因与蛋白表达，从不同机能状态层面诠释相畏的作用靶点及分子机制。不仅阐明了相畏减毒的特色和优势，又为相畏减毒研究提供新思路。

“七情”配伍减毒是中医药对毒性中药应用的特色和优势之一，本项目在前期的文献调研和实验基础上，进一步深入探索毒性中药钩吻畏玉叶金花减毒作用机制，为钩吻的安全合理应用提供依据。.项目通过正常与转染LS174T/Caco-2细胞、中毒与解毒C57BL/6小鼠，检测其上游核受体（PXR、CAR、VDR、PPARγ等）、下游CYP450酶（CYP2B6、CYP3A4等）与ABC转运体（P-gp、MRP2、BCRP）基因与蛋白表达，并给予抑制剂验证，开展钩吻配伍前后对体内外核受体/CYP450-ABC转运体表达影响的研究。.研究结果发现：钩吻配伍能减轻对LS174T细胞的毒性，减少对细胞的损害；与空白组相比，钩吻组CAR、CYP3A4、CYP2B6 mRNA和蛋白表达量显著下降（P<0.01或P<0.05）；与钩吻组相比，配伍组则显著升高（P<0.01或P<0.05）。钩吻总碱及钩吻素甲、钩吻素子对Caco-2细胞存活率，随着浓度的增大而减少；与空白组相比，钩吻总碱及单体对P-gp、MRP2、BCRP mRNA和蛋白表达量均显著下降（P<0.01或P<0.05）；与空白组相比，钩吻总碱及单体对核受体CAR、PXR、VDR、PPARγ mRNA和蛋白表达量均显著下降（P<0.01或P<0.05），而对核受体GR、AhR、Nrf2无显著影响；PXR核受体抑制剂干预后，钩吻更显著下调了Caco-2细胞中PXR、P-gp、MRP2、BCRP的表达；将PXR过表达质粒及BCRP报告基因质粒转入细胞内，钩吻可显著下调转染后细胞中BCRP的表达。钩吻配伍后小鼠存活率明显提升，肝靶器官病变减轻；钩吻组Bcl-2/Bax mRNA和蛋白比值显著降低（P<0.01或P<0.05）；配伍后比值显著升高（P<0.05）；与空白组相比，钩吻组CAR、Cyp3a11、BCRP mRNA和蛋白表达显著下降（P<0.01或P<0.05）；与钩吻组相比，钩吻配伍组CAR、Cyp3a11、BCRP mRNA和蛋白表达显著升高（P<0.01或P<0.05），且CAR蛋白主要分布在细胞核周围。.钩吻配伍玉叶金花具有减毒作用，该机制可能与调控核受体PXR/CAR介导的CYP450酶-ABC转运体的表达相关。该研究不仅阐明了相畏减毒的特色和优势，又为相畏减毒研究提供了思路。