Fig. 6.

Functional characterization of the metabolites. (A) Differential induction of CES1 and BSEP Huh7 cells were treated with Z-guggulsterone (2 μM) or a metabolite (2 μM) for 24 h. The mRNA levels of CES1 and BSEP were determined. An asterisk indicates statistical significance of a metabolite over Z-guggulsterone, and an alpha indicates statistical significance between two columns linked by a line (P < 0.05). (B) Differential activation of CES1 and BSEP element reporters. Huh7 cells were seeded in 48-well plates. After overnight incubation, the cells were transfected with a reporter (50 ng) and 5 ng of the null-Renilla luciferase plasmid. The transfected cells were treated with Z-guggulsterone (2 μM), a metabolite (2 μM), or the same volume of DMSO for 24 h. The reporter luciferase activities (firefly) were normalized according to the null-Renilla luciferase activities. An asterisk indicates statistical significance of a metabolite over Z-guggulsterone, and an alpha indicates statistical significance between two columns linked by a line (P < 0.05). (C) Effect of CYP3A4 on the induction of CES1 and BSEP. Huh7 cells were seeded in 24-well plates at a density of 1.5 × 10⁵ and cultured overnight. The cells were transfected with a CYP3A4 expression construct or the corresponding vector (0.5 µg). The transfected cells were cultured for 24 h and then treated with Z-guggulsterone at 5 μM for 24 h. Total RNA was isolated and analyzed for the levels of CES1 and BSEP mRNA. The data were collected from three separate experiments. *P < 0.05.