乌头碱、新乌头碱和次乌头碱对CYP3A4抑制作用研究

摘要

Objective:To investigate inhibitory effects of aconitum alkaloids,the effective components of Radix Aconiti Lateralis Praeparata,on cytochrome P450 3A4 (CYP3A4).Methods:Dual luciferase reporter gene system was applied.Six kinds of aconitum alkaloids (aconitine,mesaconitine,hypaconitine,acetylaconitine,aconine and benzoylaconine) were screened for Pregnane X receptor activation by reporter gene assays,and ketoconazole was used as the positive control.Furthermore,LS174T cells were treated with various concentrations of aconitin,mesaconitine and hypaconitine.Then,their RNA and total protein were extracted to evaluate the regulating effect of alkaloids on CYP3A4 mRNA and protein expression by real time PCR and Western blot methods,respectively.Results:Reporter gene screening showed that aconitum alkaloids did not activate PXR,and had potential inhibitory effects on CYP3A4 enzyme.Meanwhile,dose-dependent inhibiton of the gene expression were observed in response to aconitine,mesaconitine and hypaconitine,and the levels of CYP3A4 protein expression were decreased by aconitine,mesaconitine and hypaconitine in varying degrees.Conclusion:Aconitine,mesaconitine and hypaconitine can down-regulate the gene and protein expression of CYP3A4 which could induce significant drug-drug interactions.%目的:研究附子主要药效成分乌头类生物碱对C YP3 A4活性的抑制作用.方法:利用前期实验室构建的pGI4.17-CYP3A4报告基因质粒、pcDNA3.1-hPXR表达质粒与pRL-TK内参质粒共转染HepG2细胞,检测乌头碱、新乌头碱、次乌头碱、乙酰乌头碱、乌头原碱和苯甲酰乌头碱对PXR的转录激活效应;并利用real time PCR与Western blot技术对乌头碱、新乌头碱和次乌头碱的不同时间及不同浓度处理组进行mRNA和蛋白的检测.结果:报告基因模型检测结果显示,乌头碱、新乌头碱、次乌头碱、乙酰乌头碱、乌头原碱和苯甲酰乌头原碱对PXR无转录激活作用,且能下调CYP3A4报告基因荧光值的比活性,其中乌头碱、新乌头碱、次乌头碱下调作用明显;同时不同浓度乌头碱、新乌头碱、次乌头碱均能下调LS174T细胞中的CYP3A4 mRNA与蛋白表达水平.结论:乌头碱、新乌头碱、次乌头碱对CYP3A4 mRNA与蛋白质水平的表达具有抑制作用.在临床用药过程中,可能会导致药物-药物相互作用.