Regulation Of Artemisinin Drugs On Transcription Of CYP2B6and CYP3A4in Vitro Mediated By Human Pregnane X Receptor And Human Constitutive Androstane Receptor

Objective ARTs occupy a decisive position in the treatment of malaria, nevertheless,with high recurrence rate caused by autoinduction metabolism. Previous researchshowed human pregnane X receptor and human constitutive androstane receptormediated transcription regulation of CYP2B6and CYP3A4, meanwhile CYP2B6andCYP3A4are the main isozymes in metabolism of ARTs. This study aimed at provingwhether ARTs would activate human pregnane X receptor and human constitutiveandrostane receptor to regulate the transcription of CYP2B6and CYP3A4in LS174Tcells, which could provide evidence for the study of autoinduction metabolismmechanism.Methods （1） MTT assay was used to evaluate the inhibition on proliferation of cellsby ARTs at different concentrations and time. The inhibition ratio and IC50valueswere calculated based on the results.（2） The system of dual luciferase reporter gene assay was developed and optimized.Plasmids DNA were obtained by using plasmids extracted kit. The LS174T cells weretransfected with pcDNA3-hPXR/hCAR, pGL3-CYP3A4/CYP2B6and pRL-TK usingLipofectamine2000, then treated with RIF or CITCO as positive drug, and0.1%DMSO as solvent control, respectively. The activities of firefly and renilla luciferaseswere measured by microplate reader. The fold induction was the ratio of luciferasesactivities in positive drug group to that in control group. In order to obtain high sensitivity, the dose of transfection reagent and incubation time were investigated.（3） ART、DHAand AM were choosed to be represent drugs of this kind of derivatives.Plasmids pcDNA3-hPXR, pGL3-CYP3A4/CYP2B6and pRL-TK were cotransfectedin LS174T, and treated for24h with ART of5μmol·L^-1240μmol·L^-1, DHA of3μmol·L^-160μmol·L^-1, AM of5μmol·L^-1240μmol·L^-1. The positive and solventcontrol were RIF and0.1%DMSO, respectively. Plasmids pcDNA3-hCAR,pGL3-CYP3A4/CYP2B6and pRL-TK were cotransfected in LS174T, and treatedwith ARTs in the presence of androstenol which is the inhibitor of hCAR. Thepositive and negative control were CITCO and0.1%DMSO, respectively.Statistically significant differences were analyzed by Dunnet-t test.Results （1） The inhibition rates and IC50values enlarged as dose of ARTs andexposure time increased. The IC50values for ART at24h,48h and72h were>240μmol·L^-1,>240μmol·L^-1and211.18μmol·L^-1, respectively. When treated withDHA, IC50values were91.46μmol·L^-1,80.45μmol·L^-1and45.60μmol·L^-1, as forartemether were>320μmol·L^-1,262.90μmol·L^-1and163.93μmol·L^-1, respectively.（2） Highest induction fold could be obtained by using1.8μl Lipofectamine2000tocotransfecting150ng pcDNA3-CAR/PXR,600ng pGL3-CYP2B6/3A4and50ngpRL-TK, as been incubated for5h.（3） ART、DHA and AM showed varying degrees of induction on CYP3A4and2B6:①ART could induce CYP2B6and CYP3A4expression by activation CARsignificantly at tested concentrations, but no dose-dependent induction effect wasobserved. ART induction on PXR mediated CYP2B6and CYP3A4expressionshowed dose-response effect, but5μmol·L^-120μmol·L^-1ART couldn’t induceCYP2B6significantly.②10μmol·L^-160μmol·L^-1DHA could induce CYP2B6andCYP3A4expression by activation CAR significantly, and dose-dependent inductioneffect was observed. DHA induced CYP3A4by CAR significantly at the concentration of40μmol·L^-160μmol·L^-1. And20μmol·L^-160μmol·L^-1DHA inducedCYP2B6and CYP3A4by PXR significantly.③5μmol·L^-120μmol·L^-1AM couldinduce CYP2B6by CAR significantly, and the highest fold was at20μmol·L^-1. AMcould induce CYP3A4expression by activation CAR significantly at testedconcentrations. The highest fold was also at20μmol·L^-1.20μmol·L^-1120μmol·L^-1AM could induce CYP2B6expression by activation PXR significantly, and20μmol·L^-1AM induced the most. The concentration of AM induced CYP3A4byPXR significantly was greater than10μmol·L^-1. The highest fold appeared at40μmol·L^-1.Conclusion ART, DHA and AM could activate PXR and CAR to induce CYP2B6,CYP3A4expression in LS174T cells. It was suggested that ARTs may inducedfirst-pass metabolism of CYP2B6and CYP3A4in intestinal.