PMC

HepG2 cells were transiently transfected with a mixture containing 50 ng of CYP3A4-DP-Luc, 50 ng wide type or nature human PXR variant, along with 5ng of the Null-Renilla reniformis luciferase plasmid. The transfected cells were treated with 10ng/ml IL-6 or the same volume of PBS for 24 h. Luciferase activities were determined as described in the above. To determine whether the expression is altered with certain variants, cell lysates(8µg) were analyzed by Western blotting(Anti-hPXR) for the expression level(bottom). Three independent experiments were performed, and the data were expressed as mean ± S.D.

A, a time-course effect of IL-6 on the levels of PXR and CYP3A4 in primary human hepatocytes. Human hepatocytes were treated with IL-6(10ng/ml) or the same volume of PBS for 24 h. Total RNA was isolated and subjected to the qRT-PCR analysis for the levels of PXR and CYP3A4. The qPCR Cts were 25 for PXR, 24 for CYP3A4 and 20 for GAPDH. B, repression effect of IL-6 on the PXR protein level in primary hepatocytes. Human hepatocytes were treated with IL-6(10ng/ml) or the same volume of PBS for 48 h, and cell lysates(10µg) were prepared and analyzed by Western blotting. All the experiments were repeated at least three times, and the data were expressed as mean ± S.D. p<0.05, a statistically significant decrease by IL-6 treatment.

A, effect of SiPXR on the suppression of CYP3A4. HepG2 cells were transiently transfected with the siPXR construct or the corresponding vector for 72 h, and the transfected cells were treated with IL-6(10ng/ml) or the same volume of PBS for 24 h. Total RNA was prepared and analyzed for levels of CYP3A4, PXR and GAPDH by qRT-PCR and RT-PCR. B, effect of PXR overexpression on the repression of CYP3A4 mediated by IL-6. The same procedure as the above was used for overexpression experiment except for the replacement siPXR construct with the human PXR construct or the corresponding vector for 48h. The qPCR Cts were 27 for PXR, 32 for CYP3A4 and 16 for GAPDH. All the experiments were repeated at least three times, and the data were expressed as mean ± S.D. p<0.05, a statistically significant decrease by IL-6 treatment.

A, effect of IL-6 treatment on the level of CYP3A4 mRNA in five donors’s primary hepatocytes. Human primary hepatocytes were treated with IL-6(10ng/ml) or the same volume of PBS for 24 h. Total RNA was isolated and subjected to the qRT-PCR analysis for the level of CYP3A4 mRNA probe as described under Materials and Methods. The qPCR Cts were 24 for CYP3A4 and 20 for GAPDH. B, effects of IL-6 treatment on the CYP3A4 enzymatic activity (top) and the CYP3A4 protein expression (bottom) in human primary hepatocytes. C, effects of IL-6 treatment on the CYP3A4 enzymatic activity (top) and the CYP3A4 protein expression (bottom) in HepG2 cell. Human hepatocytes or HepG2 cells were treated with IL-6(10ng/ml) or the same volume of PBS for 48 h, and cell lysates or microsomes were prepared and assayed for the activity of CYP3A4 as described in Materials and Methods. To determined the content of CYP3A4 protein, human hepatocytes lysates(8µg) or HepG2 cell microsomes(20µg) was subjected to Western analyses with an antibody against CYP3A4 or GAPDH. All the experiments were repeated at least three times, and the data were expressed as mean ± S.D. * p<0.05, a statistically significant decrease by IL-6 treatment.

A, effect of DRB on the suppression of CYP3A4 mRNA in primary human hepatocytes. Human hepatocytes were treated with 10ng/ml IL-6 for 9 h in the absence or presence of 5µM DRB. Total RNA was prepared and analyzed for the levels of CYP3A4 and GAPDH by qRT-PCR. The qPCR Cts were 24 for CYP3A4 and 20 for GAPDH. B, repression of CYP3A4-DP-Luc promoter reporter. HepG2 cells were transiently transfected by FuGENE HD with a mixture containing 50 ng of CYP3A4-DP-Luc, along with 5 ng of the Null-Renilla reniformis luciferase plasmid in the presence and absence of PXR. The transfected cells were treated with 10 ng/ml IL-6 or the same volume of PBS for 24 h. Luciferase activities were determined with a Dual-Luciferase reporter assay system, and the reporter activity was normalized based on the Null-Renilla reniformis luminescence signal. C, differential repression of CYP3A4-DP-Luc and CYP3A4-P-Luc reporters mediated by IL-6 in the presence of PXR. HepG2 cells were transfected and treated with various concentrations of IL-6(0–10ng/ml) for 24 h. The reporter activities were determined as described above. All the experiments were repeated at least three times, and the data were expressed as mean ± S.D. p<0.05, a statistically significant decrease by IL-6 treatment.