The Chinese herbal medicine Sophora flavescens activates pregnane X receptor

Abstract

Sophora flavescens (SF) is an herbal medicine widely used for the treatment of viral hepatitis, cancer, viral myocarditis, gastrointestinal hemorrhage, and skin diseases. It was recently reported that SF up-regulates CYP3A expression. The mechanism of SF-induced CYP3A expression is unknown. In the current study, we tested the hypothesis that SF-induced CYP3A expression is mediated by the activation of pregnane X receptor (PXR). We used two cell lines, DPX2 and HepaRG, to investigate the role of PXR in SF-induced CYP3A expression. The DPX2 cell line is derived from HepG2 cells with the stable transfection of human PXR and a luciferase reporter gene linked with a human PXR response element identified in the CYP3A4 gene promoter. In DPX2 cells, SF activated PXR in a concentration-dependent manner. We used a metabolomic approach to identify the chemical constituents in SF, which were further analyzed for their effect on PXR activation and CYP3A regulation. One chemical in SF, N-methylcytisine, was identified as an individual chemical that activated PXR. HepaRG is a highly differentiated hepatoma cell line that mimics human hepatocytes. In HepaRG cells, N-methylcytisine significantly induced CYP3A4 expression, and this induction was suppressed by the PXR antagonist sulforaphane. These results suggest that SF induces CYP3A expression via the activation of PXR.

Fig. 1.

A cell-based PXR gene reporter assay of SF aqueous extract. DPX2 cells were incubated with SF aqueous extract for 24 h. Results are shown as the fold induction of luciferase activity over the vehicle control. A, the effect of SF aqueous extract (480 mg/l) on PXR activation in DPX2 cells. SCB and GUF aqueous extracts (480 mg/l) served as positive controls for herb-mediated PXR activation. The data are presented as means ± S.D. (n = 3; *, p < 0.05 versus control). B, concentration-dependent PXR activation in DPX2 cells by SF aqueous extract. The data are presented as means (n = 3 at each concentration).

Fig. 2.

Global profiling of the chemical constituents of SF aqueous extract using a liquid chromatography-mass spectrometry-based metabolomic approach. UPLC-TOFMS was used to analyze SF constituents. Centroid and integrated mass chromatographic data were processed by MarkerLynx software to generate a multivariate data matrix. PCA and OPLS-DA were conducted on Pareto-scaled data to analyze the chemical constituents of SF. A, separation of SF aqueous extract group and vehicle group in a PCA score plot. The t[1] and t[2] values represent the scores of each sample in principal component 1 and 2, respectively. B, loading S-plot generated by OPLS-DA. The y-axis represents the correlation of each ion to the model, and the x-axis represents the relative abundance of ions. The ions from SF aqueous extract are presented in the upper-right window, and several top-ranking ions were identified as oxymatrine (I), oxysophocarpine (II), matrine (III), sophocarpine (IV), and N-methylcytisine (V).

Fig. 3.

Structural elucidation of N-methylcytisine in SF extract. The analysis of N-methylcytisine and SF extract was performed under the same conditions using UPLC-TOFMS. A, a chromatogram of authentic N-methylcytisine, retention time at 0.97 min. B, a chromatogram of N-methylcytisine detected in SF extract, retention time at 0.97 min. C, tandem mass spectrometry fragmentation of N-methylcytisine. Tandem mass spectrometry fragmentation was conducted with collision energy ramping from 10 to 30 eV. Major daughter ions from fragmentation are interpreted in the inlaid structural diagram.

Fig. 4.

Identification of PXR activator(s) in SF aqueous extract. DPX2 cells were incubated for 24 h with individual chemicals identified from SF aqueous extract. Results are shown as the fold induction of luciferase activity over the vehicle control. A, effect of individual chemicals (10 μM) on PXR activation. These chemicals were identified in SF aqueous extract. Rifampicin served as a positive control for PXR activation. The data are presented as mean ± S.D. (n = 3; *, p < 0.05 versus control). B, concentration-dependent PXR activation in DPX2 cells by N-methylcytisine. The data are presented as means (n = 3 at each concentration).

Fig. 5.

Induction of CYP3A4 by SF aqueous extract and N-methylcytisine. DPX2 cells and HepaRG cells were incubated with SF aqueous extract and N-methylcytisine (10 μM) for 48 h. CYP3A4 mRNA expression was analyzed by qPCR. Values were quantified using the comparative cycle threshold method, and samples were normalized to glyceraldehyde-3-phosphate dehydrogenase. Rifampicin (10 μM) served as a positive control of CYP3A4 inducer. A, effect of SF aqueous extract and N-methylcytisine on CYP3A4 expression in DPX2 cells. The data are shown as the fold induction versus control (n = 3; *, p < 0.05 versus control). B, effect of SF aqueous extract and N-methylcytisine on CYP3A4 expression in HepaRG cells. The data are shown as the fold induction versus control (n = 3; *, p < 0.05 versus control). C, effect of PXR antagonist sulforaphane (20 μM) on N-methylcytisine-mediated CYP3A4 induction in HepaRG cells. CYP3A4 expression in the N-methylcytisine-treated group was set as 100% (n = 3; *, p < 0.05 versus N-methylcytisine-treated group).

Fig. 6.

The effect of the pretreatment of SF aqueous extract and its constituents on CYP3A activity in DPX2 cells. DPX2 cells were exposed to SF aqueous extract and its constituents (10 μM each) for 48 h. After the treatment, the culture medium containing SF aqueous extract or its constituents was withdrawn and changed to the medium containing 50 μM midazolam. Midazolam was used as a probe for CYP3A activity analysis. 1′-Hydroxymidazolam was analyzed by UPLC-TOFMS. CYP3A activity in the control group was set as 1. Rifampicin (10 μM) served as a positive control. All data are presented as means ± S.D. (n = 3; *, p < 0.05 versus control).