灯盏花素对细胞色素P450酶和蛋白转运体活性的影响及机制探讨

The ethnic medicine Breviscapine is used mainly for supportive treatment of heart cerebrovascular disease subsidiarily and has extensive effects on other aspects. Consequently, there are also an increasing number of combination therapies between Breviscapine and other clinical drugs. Up to date, a lot of studies on Breviscapine pharmacological actions have been done. However, there is little reports on pharmacokinetics and combination therapy from the ethnic medicine Breviscapine. Under the guidance of pharmacogenetic theory to discuss interaction between Breviscapine andrelated clinical drugs through researching the effect of Breviscapine on drug metablics enzymes and transporters, which is used modern new science technologics and methods based on previous correlative studies. In addition, it will make futher discussion of effect on nuclear receptor and microRNA from the ethnic medicine Breviscapine according to have been reported researches that expression of downstream gene CYP3A4 was influenced with genetic polymorphism of PXR and the expression of PXR was regulated by microRNA. On the basic of above mentioned, it will be studied influence of Breviscapine on drug metablic enzymes and transport proteins in national minority population of Yunnan province to decide the correlation between efficacy and CYP3A5, transport MDR1 gene type. The methods that are involed have not been reported at home and abroad, which will provide trains of thought for combination therapy between Breviscapine and other clinical drugs, as well as for rational use of Brebiscapine.

地方药灯盏花素一直以来被用于心脑血管疾病的辅助治疗，同时还具有其他广泛的作用，故与其他临床药物联合应用的机会也越来越多。过去的研究主要集中在该药的药效学方面，而对它的药动学和药物相互作用方面研究较少。本项目拟以遗传药理学理论为指导，在前期相关研究的基础上应用现代科学新技术，通过研究灯盏花素对药物代谢酶（CYP）和转运体活性影响来探讨灯盏花素与临床相关药物相互作用，近年来核受体PXR等在CYP及转运体蛋白的表达调控中的重要作用逐渐被认识，PXR的相关研究发现其活性能影响下游靶基因CYP3A4的表达，而且研究还发现microRNA能调控PXR的表达，故还将进一步探讨灯盏花素对核受体及microRNA表达影响，然后通过人体内实验判断其效应与CYP3A5及编码转运体基因型之间的相关性。目前国内外尚未见相关报道，故通过此研究将为灯盏花素与相关药物联合应用提供理论依据，并为合理应用灯盏花素提供新思路。

灯盏花素(Breviscapine) 是菊科飞蓬属植物短亭飞蓬 Erigeron breviscapus（Vant.）Hand.- Mazz.〔Aster breuiscapus Vant.〕灯盏花中的一类总黄酮，临床常作为长期治疗药物使用，广泛用于治疗心脑血管系统疾病，故与不可避免地会与一些临床上长期服用的常用药物发生合用现象，但对该药的药物相互作用研究较少。本项目首先建立并验证CYP3A4、CYP3A5、CYP2C9 mRNA和蛋白检测方法，在Chang Liver细胞系中发现灯盏花素可抑制CYP3A4、CYP3A5、CYP2C9 mRNA和蛋白的表达，提示灯盏花素可能通过影响CYP3A、CYP2C9酶活性而发生临床药物之间相互作用。在人肝肿瘤细胞株HepG2细胞和人结肠癌细胞株Caco2细胞中，用瞬时转染报告基因试验检测灯盏花素对核受体PXR或 CAR介导的CYP3A4、CYP2C19的转录调节作用，结果表明灯盏花素可能通过PXR途径影响CYP3A4的表达；通过CAR途径促进CYP2C19的表达。建立血清中microRNA提取方法以及实时荧光定量PCR检测miR-27a，27b，148a，298，451a表达水平的方法，然后在Chang Liver细胞系中观察灯盏花素对microRNA表达的影响，发现血清hsa-miR-27a，27b，148a，298，451a表达量，与安慰剂组相比没有明显的变化（p＞0.05），提示这几种microRNAs可能与灯盏花素影响CYP3A酶活性无关。建立并验证了CYP3A5、CYP2C9、MDR1基因多态性检测方法。分别以咪达唑仑、洛沙坦为探药在不同CYP3A5*3、CYP2C9基因型个体中研究灯盏花素对人体CYP代谢活性的影响，结果表明灯盏花素对CYP3A酶活性有抑制作用但与CYP3A5*3基因多态性无明显关联性，灯盏花素对CYP2C9无影响、和CYP2C9基因型无相关性，通过体内实验明确灯盏花素对P-糖蛋白底物非索非那定人体药物代谢动力学无影响，对人体内P-gp的影响与MDR1 C3435T基因多态性无关。