Journal: British Journal of Pharmacology

Article Title: Activating the pregnane X receptor by imperatorin attenuates dextran sulphate sodium‐induced colitis in mice

doi: 10.1111/bph.14424

Figure Lengend Snippet: IMP activates the PXR transactivation of CYP3A4 promoter activity. HCT116 cells were transfected with pGL3‐CYP3A4‐luc (100 ng per well), as well as empty vector pCMV6‐entry (100 ng per well), (A) pCMV6‐XL4‐hPXR (100 ng per well) or (B) pCMV6‐mPXR (100 ng per well) plasmids. pGL3‐CMV Renilla luciferase control reporter vectors were used as the transfection control. After 24 h transfection, cells were treated with DMSO, rifampicin (RIF; 10 μM), ketoconazole (KET; 2.5 μM), PCN (10 μM) or IMP (6.25–25 μM) as indicated for another 24 h. CYP3A4 promoter activity is presented as relative luciferase units after being normalized against the Renilla luciferase internal control. Data are expressed as the mean ± SEM from five independent experiments. * P < 0.05 compared with the same treatment group transfected with empty vector, and the DMSO control group transfected with the receptor expression plasmid.

Article Snippet: The PXR (ab85451 for human and ab192579 for mouse), CYP3A4 (ab124921 for human and ab197053 for mouse), MDR1 (ab170904), GAPDH (ab8245) and PCNA (ab18197) primary antibodies were purchased from Abcam (Cambridge, UK).

Techniques: Activity Assay, Transfection, Plasmid Preparation, Luciferase, Expressing

Journal: British Journal of Pharmacology

Article Title: Activating the pregnane X receptor by imperatorin attenuates dextran sulphate sodium‐induced colitis in mice

doi: 10.1111/bph.14424

Figure Lengend Snippet: IMP induced PXR‐mediated gene expression and CYP3A4 enzymatic activity in LS174T cells. (A) Human PXR (NR1I2), CYP3A4 and MDR1 mRNA expression was analysed by qRT‐PCR in LS174T cells after treatment with DMSO, IMP (6.25–25 μM) or rifampicin (RIF; 10 μM) for 48 h. Results are expressed as fold changes compared with the DMSO control. Data are expressed as mean ± SEM from five independent experiments. * P < 0.05 compared with the DMSO control group. (B) Human PXR, CYP3A4 and MDR1 protein levels in the same cells were determined by Western blotting. The numbers at the bottom indicate the relative intensity of the protein bands, with the DMSO‐treated sample set as ‘1.00’. (C) IMP induced CYP3A4 enzymatic activity in LS174T cells. Cells were treated with DMSO, IMP (6.25–25 μM), ketoconazole (KET; 2.5 μM) or rifampicin (10 μM) for 48 h. CYP3A4 enzymatic activity was determined by measuring the 1′‐hydroxymidazolam concentration for specific CYP3A4‐mediated midazolam hydroxylation. The results are expressed as relative fold changes in activity compared with the vehicle control. Data are expressed as mean ± SEM from five independent experiments. * P < 0.05 compared with the DMSO control group.

Article Snippet: The PXR (ab85451 for human and ab192579 for mouse), CYP3A4 (ab124921 for human and ab197053 for mouse), MDR1 (ab170904), GAPDH (ab8245) and PCNA (ab18197) primary antibodies were purchased from Abcam (Cambridge, UK).

Techniques: Expressing, Activity Assay, Quantitative RT-PCR, Western Blot, Concentration Assay

Journal: British Journal of Pharmacology

Article Title: Activating the pregnane X receptor by imperatorin attenuates dextran sulphate sodium‐induced colitis in mice

doi: 10.1111/bph.14424

Figure Lengend Snippet: Knockdown of hPXR (NR1I2) expression decreased IMP‐induced PXR target gene expression and CYP3A4 enzymatic activity in LS174T cells. (A) LS174T cells were transiently transfected with hPXR siRNA and control non‐silencing siRNA. After transfection for 48 h, the cells were treated with DMSO, IMP (25 μM) or rifampicin (RIF; 10 μM) for another 48 h, and the mRNA expression of hPXR, CYP3A4 and MDR1 was analysed by qRT‐PCR. Results are expressed as fold changes compared with the DMSO control. Data are expressed as mean ± SEM from five independent experiments. * P < 0.05 compared with the DMSO control group. (B) Cells were transiently transfected with hPXR siRNA and control non‐silencing siRNA for 48 h and treated with DMSO, IMP (25 μM) or rifampicin (10 μM) for another 48 h. Whole‐cell lysates were collected 96 h after transfection and subjected to Western blot analysis to determine the CYP3A4, MDR1 and PXR protein levels. The numbers at the bottom indicate the relative intensity of the protein bands, with the DMSO‐treated sample set as ‘1.00’. (C) The knockdown of hPXR expression attenuated IMP‐induced CYP3A4 enzymatic activity in LS174T cells. After transient transfection with hPXR siRNA and control non‐silencing siRNA for 48 h, cells were treated with DMSO, IMP (6.25–25 μM) or rifampicin (10 μM) for another 48 h. CYP3A4 enzymatic activity was determined by measuring the 1′‐hydroxymidazolam concentration for specific CYP3A4‐mediated midazolam hydroxylation. The results are expressed as relative fold change in activity compared with the vehicle control. Data are expressed as mean ± SEM from five independent experiments. * P < 0.05 compared with the DMSO control group.

Article Snippet: The PXR (ab85451 for human and ab192579 for mouse), CYP3A4 (ab124921 for human and ab197053 for mouse), MDR1 (ab170904), GAPDH (ab8245) and PCNA (ab18197) primary antibodies were purchased from Abcam (Cambridge, UK).

Techniques: Expressing, Activity Assay, Transfection, Quantitative RT-PCR, Western Blot, Concentration Assay