The Effect Of Organotin Compound DBDCT On Cytochrome P4503A(CYP3A) Mediated By Pregnane X Receptor(PXR)

Objective:The major aims of this study were to further clarify the mechanism of cytochrome P450 3A(CYP3A) inhibition induced by diorganotin compound di-n-butyl-di-(4-chlorobenzohydroxamato)tin(IV)(DBDCT). To explore the effect of DBDCT on pregnane X receptor(PXR)-mediated CYP3 A expression and analyzes the role of PXR in the inhibition of CYP3 A effected by DBDCT. By invesgating the influences of DBDCT on the expression of nuclear factor NF-κB and the effect of NF-κB on the PXR-mediated CYP3 A pathway, the research intends to discuss and confirm the possible inhibition of PXR-mediated CYP3 A expression by DBDCT via NF-κB activation.Methods:(1) The effect of DBDCT on PXR-mediated CYP3 A expression was analyzed using the dual-luciferase reporter assay system.(2) The changes of NF-κB contents in total protein, cytosolic protein, and nuclear protein of Hep G2 cells following DBDCT treatment were determined and analyzed by western blot; in addition, the nuclear translocation of NF-κB following DBDCT treatment was examined by immunofluorescence staining, to investigate the possible activation of NF-κB by DBDCT.(3) The effect of NF-κB activation induced by DBDCT on PXR-mediated transcriptional regulation of CYP3 A was investigated by fluorescent quantitation PCR(FQ-PCR) and dual-luciferase reporter assay after treated with DBDCT.(4) To further clarify the inhibition mechanisms, the expressions of PXR and NF-κB were subjected to RNA interference using RNA interference. The effect of DBDCT on CYP3 A m RNA expression in Hep G2 cells and the correlation between DBDCT mediated CY3 PA inhibition and PXR or NF-κB were elucidated byRT-PCR.Results:(1) Both the expression of human or rat PXR-mediated CYP3 A transcription could be induced by DBDCT in dose-effect relationship. Compared with the blank group, the induced expression CYP3A1 by r PXR were 1.37, 2.06 and 4.34 times higher(p<0.05,p<0.001, p<0.001) after using 0.5μM、1μM and 2μM DBDCT. Under the same condition,the induced expression CYP3A4 by h PXR were 2.16, 3.72 and 4.86 times higher(p<0.05,p<0.001, p<0.001). Meanwhile, the inductive effect also relate to the effect time. Cells were treated with 0.5μM DBDCT for 24 h, 48 h, 72 h. Longer duration brings stronger induction.(2) Results of the western blot analysis revealed that the overall NF-κB content(in total protein) showed no significant changes(p>0.05) after DBDCT treatment.However, compared with the control group, 1μM and 2μM DBDCT could reduce the expression of NF-κB in the cytoplasm to 0.70 and 0.61 times(p<0.01, p<0.01) and enhance the expression of NF-κB in the nucleus to 1.31 and 2.00 times(p<0.01, p<0.01).Immunofluorescence staining also confirmed NF-κB transported from the cytoplasm to the nucleus following DBDCT treatment. That is to say NF-κB could be activated by DBDCT.(3) The NF-κB activator LPS effected a significant decrease in CYP3A4 m RNA expression;DBDCT also caused a reduction in CYP3A4 m RNA expression, which was reversed using the NF-κB inhibitor PDTC. In addition, in the test of dual-luciferase reporter assay system,DBDCT was used to increase CYP3A4 expression. But the increased expression could be reduced by the transfection of NF-κB plasmid, the declination depends on the addition of NF-κB plasmid. This down regulation effect of NF-κB could be weakened by the transfection of its inhibitor IκBα. The results indicated that NF-κB downregulated PXR-mediated CYP3A4 expression induced by DBDCT.(4) All of 0.5μM, 1μM and 2μM DBDCT could cause a downregulation in CYP3A4 m RNA expression. Compared with the control group, the expression were reduced to 0.75, 0.64 and 0.44 times(p<0.05, p<0.05,p<0.01). DBDCT has stronger inhibit effect on CYP3A4 m RNA expression when the expression of PXR was subjected to RNA interference. The inhibition of DBDCT wasweakened in sh RNA interference of NF-κB p65 expression.Conclusions:(1) DBDCT induced CYP3 A expression by PXR regulation.(2) DBDCT was observed to activate NF-κB, which in turn affected PXR-mediated CYP3A4 transcription,leading to the downregulation of CYP3A4 expression.