CYP1A1体外诱导活性评价模型的建立及其在人参皂苷筛选中的应用

Development of in vitro evaluation model for CYP1A1 induction and its application in screening of ginsenosides

目的建立hAhR介导的CYP1A1体外诱导活性评价模型,应用于体外快速筛选通过hAhR途径介导的对CYP1A1具有激活能力的药物。方法利用双荧光素酶报告基因系统,将CYP1A1启动子序列插入报告基因质粒上游,构建pGL4.17-CYP1A1质粒,并与含有hAhR编码区序列的pcDNA3.1-hAhR表达质粒瞬时共转染HepG2细胞, pGL4.17-control、pcDNA3.1(+)分别为pGL4.17-CYP1A1、pcDNA3.1-hAhR的空载体,建立hAhR-CYP1A1报告基因模型;应用AhR的完全激动剂TCDD(2 nmol/L)验证该报告基因模型的可靠性,应用该模型考察人参皂苷Rf、Rc、Re、Rg1、Rb、Rd、Rh2和Rg3(20μmol/L)通过hAhR途径对CYP1A1的诱导作用。结果经验证,报告基因模型构建成功;人参皂苷Rf、Re、Rg1和Rc对AhR具有激活作用,其中与对照组比较,人参皂苷Rg1、Rc对AhR激活效应显著(P<0.05、0.01),人参皂苷Rb、Rd、Rh2和Rg3对AhR无激活作用。结论本研究建立基于hAhR的CYP1A1体外诱导活性评价模型,可为具有潜在CYP1A1诱导作用的目标化合物的筛选提供有效、快速的体外筛选手段,人参皂苷Rg1、Rc对AhR-CYP1A1激活效应显著。

Objective The aim of this study is to establish and validate an in vitro model to screen potential drug inducers of CYP1A1 mediated via nuclear receptor AhR, which can be applied to evaluate the induction activity toward CYP1A1 by a large number of drugs rapidly and effectively.Methods Dual luciferase reporter gene system was performed. The promoter of CYP1A1 DNA was cloned into the firefly luciferase reporter gene vector, pGL4.17[luc2/Neo], in order to construct pGL4.17-1A1. Transiently cotransfecting the pGL4.17-1A1 reporter vector and hAhR expression plasmid pcDNA3.1-hAhR to HepG2 cells, PGL4.17-control and pcDNA3.1 (+) were empty vectors of pGL4.17-CYP1A1 and pcDNA3.1-hAhR respectively; And hAhR-mediated CYP1A1 reporter gene model was established. As the full agonist of AhR, TCDD was applied to ensure the reliability of hAhR-mediated CYP1A1 reporter gene model. The model was used to investigate the induction of CYP1A1 by hAhR pathway of ginsenoside Rf, Rc, Re, Rg1, Rb, Rd, Rh2 and Rg3. Results Nuclear receptor hAhR-mediated CYP1A1 induction model has been established successfully. Ginsenoside Rf, Re, Rg1 and Rc (20 μmol/L) could activate AhR. Compared with the control group, ginsenoside Rg1 and Rc had significant activation effect on AhR (P<0.05, 0.01). Ginsenoside Rb, Rd, Rh2 and Rg3 had no activation on AhR.Conclusion The establishment of this induction model is an efficient and rapid in vitro way of investigation the drug inducers or inhibitors of CYP1A1.