Human Pregnane X Receptor Agonism by Ginkgo biloba Extract: Assessment of the Role of Individual Ginkgolides

Abstract

Ginkgo biloba extract activates pregnane X receptor (PXR), but how this occurs is not known. Therefore, we investigated the mechanism of PXR activation by the extract and the role of five individual terpene trilactones in the activation. In a cell-based reporter gene assay, G. biloba extract activated human PXR (hPXR), and at a concentration present in the extract, ginkgolide A, but not ginkgolide B, ginkgolide C, ginkgolide J, or bilobalide was partially responsible for the increase in hPXR activity of the extract. Likewise, in cultured human hepatocytes, only ginkgolide A contributed to the increase in hPXR target gene expression (CYP3A4 mRNA and CYP3A-mediated testosterone 6␤-hydroxylation). The extract, but none of the terpene trilactones, bound to hPXR ligand-binding domain, as analyzed by a time-resolved fluorescence resonance energy transfer competitive binding assay. Only the extract and ginkgolide A recruited steroid receptor coactivator-1, as determined by a mammalian two-hybrid assay. Compared with hPXR, rat PXR (rPXR) was activated to a lesser extent by G. biloba extract. Similar to hPXR, only ginkgolide A contributed to rPXR activation by the extract. In contrast to the effect of G. biloba extract on PXR function, it did not affect hPXR expression. Overall, the main conclusions are that G. biloba extract is an hPXR agonist, and among the five terpene trilactones investigated, only ginkgolide A contributes to the actions of the extract. Our findings provide insights into the biological and chemical mechanisms of hPXR activation by G. biloba extract.

Species-Dependent and Receptor-Selective Action of Bilobalide on the Function of Constitutive Androstane Receptor and Pregnane X Receptor

“…At the end of the treatment period, culture medium was collected and cells were lysed with 2% v/v Triton X-100 in phosphate-buffered saline (pH 7.4) containing 20 mM EDTA (0.5 ml/well for HepG2 cells and 1.25 ml/well for hepatocytes). The lactate dehydrogenase (LDH) assay was performed using the Cytotoxicity Detection Kit, as described previously (Lau et al, 2010). To determine intracellular LDH release, 5-l aliquots of culture medium or cell lysate was transferred into the wells of a 96-well microplate containing 95 l of phosphate-buffered saline (pH 7.4).…”

“…Donor demographics, isolation of human hepatocytes, and hepatocyte culture conditions were described previously (Lau et al, 2010). Human hepatocytes were treated for 72 h with bilobalide, CITCO (hCAR agonist; positive control for CYP2B6 expression assay; Faucette et al, 2006), rifampicin (hPXR agonist; positive control for CYP3A4 expression assay; Faucette et al, 2006), or DMSO (0.1% v/v;vehicle), as described in each figure legend.…”

“…Rifampicin, PCN, sodium phenobarbital, dexamethasone, TCPOBOP, 1-(2-chlorophenyl)-Nmethyl-N-(1-methylpropyl)-3-isoquinoline-carboxamide (PK11195), testosterone, bupropion hydrochloride, triprolidine hydrochloride, resorufin, 7-benzyloxyresorufin, dicumarol, dextran, and Triton X-100 (Union Carbide Corporation, Houston, TX) were purchased from Sigma-Aldrich (St. Louis, MO (Fugent, LLC, Madison, WI), Cytotoxicity Detection Kit, ␤-glucuronidase, and arylsulfatase were obtained from Roche Diagnostics (Laval, QC, Canada), and Dual-Luciferase Reporter Assay System was from Promega (Madison, WI). The suppliers of reagents for isolation and culturing of rat and human (Lau et al, 2010) hepatocytes, and those for reverse transcription and real-time polymerase chain reaction (PCR) analyses were described previously.…”

“…HepG2 human hepatocellular carcinoma cells were purchased from American Type Culture Collection (Manassas, VA) and cultured as described previously (Lau et al, 2010).…”

“…pGL3-basic-CYP2B1-luc reporter was generously provided by Dr. Thomas A. Kocarek (Wayne State University, Detroit, MI) (Kocarek and Mercer-Haines, 2002). pGL3-basic-CYP2B6-PBREM/XREM-luc reporter (Lau et al, 2011b) and pGL3-basic-CYP3A4-XREM-luc reporter (Lau et al, 2010) were constructed as described previously. All constructs were sequenced by the Nucleic Acid Protein Service Unit at the University of British Columbia (Vancouver, BC, Canada), and the identity of plasmids was confirmed by comparing their sequences with published sequences.…”