Establishment Of In Vitro Evaluation Model For CYP286Induction And Its Application On Screeing Active Constituents In TCMs

CYP2B6, an important member in CYP450superfamily, is mainly expressed in the liver, accounting for2%～10%of the total microsomal CYPs. CYP2B6participatesin the biotransformation of a large number of endogeneous and exogenous compound, especially plays key roles in the metabolism of clinically used drugs, drug abuse and enviomental compounds. CYP2B6is highly inducible, previous studies have demonstrated that CYP2B6gene can be induced by phenobarbital-type inducers, CITCO, rifampicin etc. Recent reports have revealed that nuclear receptors (NRs), PXR (Pregnane X Receptor, NR1I2) and CAR (Constitutive Androstane Receptor, NR1I3) co-regulate the transcription of CYP2B6. Induction of CYP2B6can easily cause the drug toxicity or reduced efficacy; even lead to the drug-drug interaction (DDI) and clinical adverse drug reaction. It’s necessary to develop the in vitro model for screening CYP2B6inducers. This study constructed the hPXR/CAR3mediated reporter gene model and applied it to screen CYP2B6inducers in TCMs in vitro. Cell lysates treated with potential CYP2B6inducers were used to measure analysed for CYP2B6gene expression, protein expression and enzyme catalytic activity, respectively. The research results can provide theoretical basis for prediction and study of induction of CYP2B6based drug-drug interaction.Part1Establishment and validation of the human CYP2B6reporter gene assay systemThe proximal and diatal promoters, amplified from human genomic DNA, were inserted into the upstream of reporter gene in pGL3-promoter to construct the reporter gene plasmid pGL3-CYP2B6-Luc.The human CAR3code sequence, amplified from human liver cDNA, was inserted into pcDNA3.1(+) empty vector to construct hCAR3expression plasmid. The hPXR expression plasmid was stored in our lab. The CYP2B6reporter gene plasmids and hPXR/CAR3expression plasmids were transciently co-transfected into HepG2cells. The hPXR/CAR3positive activators rifampicin and CITCO were applied to optimize the proportion of the plasmids. The empty vectors were applied to investigate the interference of transcripitional factors in the blank cells. The hPXR/CAR mediated CYP2B6reporter gene assay was successfully established and could be utilized to screen hPXR/CAR ligands.Part2Screening CYP2B6inducers from active constituents in TCM by reporter gene assayTwenty-four hours prior to transfection, HepG2cells were seeded in24-well plates. After4-6h, the media containing inducers or vehicle were added and the cells were incubated for48hours. Following treatment, the relative luciferase activities were then determined. The TCM extracts and active constituents, resolved in DMSO, were diluted with medium to100μg/ml and10μmol/L respectively to treat with the cells. The lower concentration was used when toxicity happened. The5kinds of TCM extracts and84kinds of active constituents were investigated through hPXR/CAR3mediated CYP2B6induction. The extracts, Atractylodes and Ginkgo Biloba P.E, and the active constituents apigenin, tanshinone IIA, cryptotanshinone, schisandrin A, schisandrin B, schisandrin C, schisantherin A, schizandrol A, ginkgolide B, ginkgolide B, piperine, rhynchophylline, vindoline, artemether, columbianadin, ursolic acid, curcumol, praeruptorin A, atractylenolide I and atractylenolide II, were found to activate hPXR to induce CYP2B6, active constituents artemether, and praeruptorin A, can activate hCAR3to induce CYP2B6. The activation of hPXR or hCAR3was dose-dependent.Part3Confirmation of the TCM constituents mediated CYP2B6inductionThe LS174T cells were treated with active constituents for72hours. Total RNA was extracted from the LS174T cells and the primers for human CYP2B6and GAPDH were designed. The CYP2B6gene expression was detected with real-time PCR. The lysates were extracted from LS174T cells and western blot assays were presented to detect CYP2B6protein expression. The probe substrate bupropion can be biotransformed to hydroxy bupropion. A UPLC-MS/MS method was developed for the determination of the hydroxybupropion in cell lysates. The amount of the hydroxybupropion metabolized by the cell lysate was analysed. The results revealed that apigenin, curcumol, prearuptorin A, artemether, tanshinone IIA, cryptotanshinone and rhynchophylline could induce the RNA, proteinexpression and catalytic activity of CYP2B6.