吴茱萸碱与吴茱萸次碱经PXR、CAR核受体通路影响CYP3A4表达的研究

Effects of Evodiamine and Rutecarpine on Human CYP 3A4 through PXR and CAR Pathway

目的:研究吴茱萸碱与吴茱萸次碱能否通过孕烷X受体(PXR)、组成型雄甾烷受体(CAR)通路转录调节CYP3A4的活性。方法:PXR表达质粒与CYP3A4报告质粒、CAR表达质粒与CYP3A4报告质粒瞬时转染到LS174T细胞后,分别给予2.5、10、40μmol/L浓度的吴茱萸碱与吴茱萸次碱,采用报告基因检测CYP3A4荧光素酶活性;采用二步灌流法分离小鼠原代肝细胞,给予2.5、10、40μmol/L浓度的吴茱萸碱与吴茱萸次碱,采用实时荧光定量链式聚合酶反应(R-PCR)与液相色谱-串联质谱(LC-MS/MS)法检测小鼠原代肝细胞中CYP3A11基因表达和酶活性。结果:在10、40μmol/L浓度下,吴茱萸碱经PXR核受体通路使CYP3A4报告基因荧光素酶活性分别增加至3.41、4.15倍;10、40μmol/L浓度下吴茱萸次碱能使利福平对PXR的诱导作用减少,分别降低37.7%、45.34%,吴茱萸次碱能使CAR阳性药物CITCO对CAR的诱导作用减少29%。10、40μmol/L浓度下吴茱萸碱能使CYP3A11 mRNA表达分别增加至2.67、3.80倍;40μmol/L浓度下吴茱萸次碱能下调原代小鼠肝细胞中35%的CYP3A11 mRNA表达。10、40μmol/L浓度下吴茱萸碱能显著增加CYP3A11酶功能活性分别至3.63、3.01倍;吴茱萸次碱在40μmol/L浓度下能显著减少34.54%CYP3A11酶功能活性。结论:吴茱萸碱能通过PXR核受体通路诱导CYP3A4荧光素酶、mRNA表达及酶活性,吴茱萸次碱能通过PXR、CAR通路共同抑制CYP3A4荧光素酶、mRNA表达及酶活性。

OBJECTIVE：To study the effects of evodiamine and rutecarpine on the activity of cytochrome CYP 3A4 through pregnane X receptor（PXR）and constitutive androstane receptor（CAR）pathway.METHODS：PXR plasmid and the CYP3A4 report plasmid or CAR plasmid and the CYP3A4 report plasmid were transiently transfected into the LS174T cell,and the evodiamine and rutecarpine 2.5,10 and 40 μmol/L were incubated with the LS174T cell.The activities of luciferase were determined with CYP3A4 report gene.Primary hepatocytes were isolated from mice by two steps perfusion method and then treated with evodiamine and rutecarpine 2.5,10 and 40 μ mol/L.CYP3A11 mRNA expression and enzymatic activity of primary hepatocytes were measured using real-time PCR and LC-MS/MS.RESULTS：The evodiamine 10 and 40 μmol/L could significantly enhanced 3.41 and 4.15 folds of luciferase activity in CYP3A4 report gene through PXR pathway.Rutecarpine 10 and 40 μmol/L could reduce the effect of rifampicin on PXR,decreasing by 37.7% and 45.34%,respectively.Rutecarpine could reduce 29% induction of CAR caused by CITCO（positive activator of CAR）.After exposed to evodiamine 10,40 μmol/L,the expression of CYP3A11 mRNA was significantly 2.67 and 3.80 fold increased,respectively;the expressions of CYP3A11 mRNA in 35% primary hepatocytes were significantly down-regulated by rutecarpine 40 μmol/L.Evodiamine 10,40 μmol/L increased 3.63 and 3.01 folds of CYP3A11 enzyme activity significantly;after exposed to rutecarpine 40 μmol/L,CYP3A4 enzyme activity was decreased by 34.54% significantly.CONCLUSIONS：Evodiamine can significantly enhance the CYP3A4 luciferase,mRNA expression and enzyme activity through PXR mediated pathway.Rutecarpine can significantly inhibit the CYP3A4 luciferase,RNA expression and enzyme activity through PXR/CAR mediated pathway.