Panaxytriol up-regulates CYP2B6 expression through PXR and CAR and their interactions with RXRα and SRC-1

Cytochrome P450 2B6 (CYP2B6) is a critical drug-metabolizing enzyme, and its expression is regulated by the nuclear receptors. Panaxytriol (PXT), a prominent bioactive constituent extracted from the roots of Panax ginseng C.A. Meyer, was found to induce cytochrome P450 3A4 (CYP3A4) via pregnane X receptor (PXR) and constitutive androstane receptor (CAR) in our previous work. Nevertheless, it remains unclear whether PXT exerts a similar regulatory effect on CYP2B6 through PXR and CAR pathways.

This study aims to elucidate the concentration-dependent molecular mechanisms by which PXT regulates CYP2B6 expression through PXR and CAR, and their interactions with retinoid X receptor α (RXRα) and steroid receptor coactivator-1 (SRC-1).

HepG2 and Huh7 cells, along with those transfected with short-hairpin RNAs (shRNAs) targeting PXR (shPXR) and CAR (shCAR), and their respective non-targeting short-hairpin RNA negative control (shNC), were exposed to PXT at concentrations ranging from 10 to 80 μM. The expression levels of CYP2B6, PXR, and CAR were determined by reverse transcription-quantitative PCR (RT-qPCR) and western blotting. Furthermore, co-immunoprecipitation (Co-IP) and immunofluorescence (IF) assays were conducted to evaluate the associations and subcellular colocalization of PXR and CAR with RXRα and SRC-1.

PXT concentration-dependently upregulated CYP2B6 expression in both cell lines. At low-to-moderate concentrations (10-40 μM) of PXT, CYP2B6 induction was primarily mediated by PXR, whereas high concentration (80 μM) activated both PXR and CAR pathways. Both 20 μM and 80 μM concentrations of PXT can promote the association of PXR with RXRα and SRC-1; however, only high concentrations of PXT (80 μM) can enhance the association of CAR with RXRα and SRC-1. Silencing PXR enhanced the nuclear translocation of CAR, increased the association between CAR-RXRα and CAR-SRC-1, and consequently elevated the basal expression level of CYP2B6. Conversely, downregulation of CAR increased PXR nuclear translocation and its association with RXRα and SRC-1, but significantly reduced the baseline expression of CYP2B6.

PXT upregulates CYP2B6 through a concentration-dependent dual-pathway mechanism involving competitive PXR-CAR interactions for shared co-factors RXRα and SRC-1.