Identification of Three Novel Natural Product Compounds that Activate PXR and CAR and Inhibit Inflammation
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To investigate the effects of three natural product compounds, carapin, santonin and isokobusone, on the activity of pregnane X receptor (PXR) and constitutive androstane receptor (CAR) in induction of drug-metabolizing enzymes and inhibition of inflammation.
The monkey kidney-derived fibroblast (CV-1) cells and human embryonic kidney HEK293 cells were used for transient transfection and luciferase reporter gene assays. Human primary hepatocytes and primary hepatocytes from wild type, PXR−/−, and hPXR transgenic mice were used to study the induction of drug-metabolizing enzymes and the implication of these compounds in inflammation.
Carapin, santonin and isokobusone activated both PXR and CAR in transient transfection and luciferase reporter gene assays. Mutagenesis studies showed that two amino acid residues, Phe305 of the rodent PXR and Leu308 of the human PXR, are critical for the recognition of these compounds by PXR. Importantly, the activation of PXR and CAR by these compounds induced the expression of drug-metabolizing enzymes in primary human and mouse hepatocytes. Furthermore, activation of PXR by these compounds inhibited the expression of inflammatory mediators in response to lipopolysaccharide (LPS). The effects of these natural compounds on drug metabolism and inflammation were abolished in PXR−/− hepatocytes.
Our results show that carapin, santonin and isokobusone activate PXR and CAR and induce drug-metabolizing enzymes. In addition, these compounds inhibited the expression of inflammatory mediators in response to LPS through the activation of PXR.
KEY WORDSdrug metabolism gene regulation natural products nuclear receptor
Constitutive androstane receptor
Inflammatory bowel disease
Pregnane X receptor
1,4-bis [2-(3,5-Dichloropyridyloxy)] benzene
ACKNOWLEDGMENTS AND DISCLOSURES
We thank Yanhong Shi for her assistance in the initial library screen. We also thank Lushan Yu for his assistance in the P450 enzymatic assay. Wen Xie is the Joseph Koslow Endowed Chair in Pharmaceutical Sciences at the University of Pittsburgh School of Pharmacy.
This work was supported in part by an NIH grants ES019629, DK083952 and HD073070 (to W.X.), ES010337 and CA014195 (to R.M.E.), the Leona M. and Harry B. Helmsley Charitable Trust, the Glenn Foundation for Medical Research, and the Thailand Research Fund through the Royal Golden Jubilee Ph.D. Program (Grant PHD/0204/2549 to S.K.). R.M.E. is an investigator of the Howard Hughes Medical Institute and March of Dimes Chair in Molecular and Developmental Biology at the Salk Institute. Normal human hepatocytes were obtained through the Liver Tissue Procurement and Distribution System, Pittsburgh, Pennsylvania, which was funded by NIH Contract [N01-DK-7-0004/HHSN267200700004C].
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