santonin and Inflammation

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.

Topics: Animals; Anti-Inflammatory Agents; Aryl Hydrocarbon Hydroxylases; Benzopyrans; Biological Products; Cell Line; Cells, Cultured; Constitutive Androstane Receptor; Cyclooctanes; Cytochrome P-450 CYP2B6; Cytochrome P-450 CYP3A; Cytochrome P450 Family 2; Gene Deletion; Gene Expression Regulation; Haplorhini; HEK293 Cells; Hepatocytes; Humans; Inflammation; Lipopolysaccharides; Mice; Mice, Transgenic; Pharmaceutical Preparations; Plants, Medicinal; Polycyclic Compounds; Pregnane X Receptor; Receptors, Cytoplasmic and Nuclear; Receptors, Steroid; Santonin; Steroid Hydroxylases

This protocol describes microsphere-based protease assays for use in flow cytometry and high-throughput screening. This platform measures a loss of fluorescence from the surface of a microsphere due to the cleavage of an attached fluorescent protease substrate by a suitable protease enzyme. The assay format can be adapted to any site or protein-specific protease of interest and results can be measured in both real time and as endpoint fluorescence assays on a flow cytometer. Endpoint assays are easily adapted to microplate format for flow cytometry high-throughput analysis and inhibitor screening.

Topics: Animals; Biotinylation; Flow Cytometry; Fluorescence Resonance Energy Transfer; Green Fluorescent Proteins; High-Throughput Screening Assays; Humans; Inflammation; Kinetics; Microspheres; Peptide Hydrolases; Peptides; Reproducibility of Results; Temperature