The Regulatory Mechanism Of PXT On CYP3A4 By Nuclear Receptor CAR In HepG2 Cells

Background:Panaxytriol(PXT)is one of the important active ingredients in Ginseng,Red Ginseng extract and Shenmai injection,which inhibits the activity of many tumor cells,enhances the effect of anti-tumor and helps to alleviate the adverse reactions of certain cancer therapies,it is a clinical option to assist cancer treatment.Thus PXT has a good application prospect in anti-tumor.Our team previous studies showed that PXT can up-regulate the expression of CYP3A4 mRNA and protein level by mediating the pregnane X receptor(PXR;NR1I2)pathway in HepG2 cells.The constitutive androstane receptor(CAR;NR1I3)is a sister of PXR in orphan nuclear receptor family.Whether PXT can regulate expression of CYP3A4 through mediating CAR,and PXT may be able to regulate expression of CYP3A4 through cross-talk between CAR and PXR.Objectives:To study the effect of PXT on CAR-CYP3A4 regulatory pathway in HepG2 cells and the effect of CAR expression on PXR transcriptional regulate expression of CYP3A4,this study aim to provide experimental and theoretical basis for the further exploration of PXT in clinical rational drug use and combinational therapy.Methods:1.In HepG2 cells,draw the growth curve of HepG2 cells,the toxic effect of serial concentrations of PXT on HepG2 cells were examined using MTS,and investigate the effect of PXT on the expression of CAR and CYP3A4 mRNA and protein2.To construct hCAR over-expressing HepG2 cells,and use RT-qPCR,Western blot and other experimental techniques to investigate the effect of PXT on the expression of CAR,PXR,and CYP3A4 mRNA and protein.3.To construct HepG2 cells transiently co-transfected with hCAR.The dual-luciferase reporter gene assay was used to investigate the effect of PXT on theregulatory pathway of CAR-CYP3A4 in HepG2 cells.4.To construct hCAR-silencing HepG2 cells,and use RT-qPCR,Western blot and other experimental techniques to investigate the effect of PXT on expression of PXR and CYP3A4 mRNA and protein.Results:1.In HepG2 cells,PXT up-regulate the expression of CYP3A4 mRNA and protein in serial concentrations for 24 h,CYP3A4 mRNA were up-regulated 93.06%,131.18%,237.30% and 376.33% respectively,CYP3A4 protein were up-regulated47.04%,64.99%,115.81% and 214.58% respectively.However,PXT up-regulate the expression of CAR m RNA and protein only at high concentration of 80 ?M,93.28%and 59.19%,respectively.2.In hCAR over-expressing HepG2 cells,PXT up-regulate the expression of CAR mRNA and protein only at high concentration of 80?M for 24 h,132.18% and106.57% respectively.However,the effect of PXT on the expression of PXR and CYP3A4 mRNA and protein was only up-regulated at 80 ?M,PXR mRNA and protein were up-regulated 125.69% and 57.25%,CYP3A4 mRNA and protein were up-regulated 108.79% and 67.02%.There was no significant difference on the expression of PXR and CYP3A4 mRNA and protein at 10?M,20?M,40?M.3.In transient co-tranfected hCAR HepG2 cells,PXT only up-regulate the activity of CYP3A4 reporter gene at 80?M were examined by the dual-luciferase reporter gene assay,the fold induction is 1.54.4.In hCAR-silencing HepG2 cells,PXT also up-regulate the expression of PXR and CYP3A4 mRNA and protein in serial concentrations for 24 h.PXR m RNA were up-regulated 183.55%,212.63%,255.65% and 308.66% respectively,CYP3A4 mRNA were up-regulated 185.83%,235.88%,337.16% and 493.55% respectively,PXR protein were up-regulated 108.55%,138.44%,166.98% and 238.66%respectively,CYP3A4 protein were up-regulated 131.83%,146.62%,211.58% and288.55% respectively.Compared with HepG2 cells,PXT significantly increases the up-regulation of PXR and CYP3A4 mRNA and protein expression.The mRNA expression of PXR were increased 45.39%,39.49%,34.69% and 24.98% respectively,the mRNA expression of CYP3A4 were increased 48.57%,45.29%,29.61% and24.61% respectively,the protein expression of PXR were increased 63.37%,32.99%,39.09% and 32.00% respectively,the protein expression of CYP3A4 were increased57.66%,49.47%,44.38% and 23.51%.Conclusion:1.In HepG2 cells,PXT mediate the expression of CYP3A4 by CAR regulatory pathway only at high concentrations,but not at low concentrations.2.PXT mediate the expression of CYP3A4 by cross-talk between CAR and PXR,and CAR inhibits the regulation of PXR-CYP3A4 pathway.