PXR Agonist Research Articles

PBPK perspective on alternative CYP3A4 inducers for rifampin.

PBPK perspective on alternative CYP3A4 inducers for rifampin.

Applanoids A—E as the First Examples of C‐15/C‐20 Michael Adducts in Ganoderma Triterpenoids and Their PXR Agonistic Activity

Comprehensive SummaryGanoderma triterpenoids (GTs), a class of major active constituents of Ganoderma fungi, possess diverse structures and remarkable activities. In the present study, nine new GTs, namely applanoids A—I (1—9), were isolated from the medicinal fungus of Ganoderma applanatum. Their structures including absolute configurations were established by comprehensive spectroscopic analyses and ECD calculation. Applanoids A—E (1—5) represent the first example of GTs with 6/6/5/6/5 pentacyclic system and the formation of the ether ring between C‐15 and C‐20 involves Michael addition reaction. Furthermore, compounds 1—8 were evaluated for their human pregnane X receptor (hPXR) agonistic activity using dual‐luciferase reporter gene assay, and the results showed that compounds 1, 2 and 4 can dose‐dependently activate hPXR. This investigation further illustrated the structural diversity of GTs and provided new insights for searching PXR agonists from GTs.

Impact of Neonatal Activation of Nuclear Receptor CAR (Nr1i3) on Cyp2 Gene Expression in Adult Mouse Liver

Abstract Perinatal exposure to environmental chemicals is proposed to reprogram development and alter disease susceptibility later in life. Supporting this, neonatal activation of the nuclear receptor constitutive androstane receptor (CAR) (Nr1i3) by TCPOBOP was previously reported to induce persistent expression of mouse hepatic Cyp2 genes into adulthood, and was attributed to long-term epigenetic memory of the early life exposure. Here, we confirm that the same high-dose neonatal TCPOBOP exposure studied previously (3 mg/kg, 15x ED50) does indeed induce prolonged (12 weeks) increases in hepatic Cyp2 expression; however, we show that the persistence of expression can be fully explained by the persistence of residual TCPOBOP in liver tissue. When the long-term presence of TCPOBOP in tissue was eliminated by decreasing the neonatal TCPOBOP dose 22-fold (0.67× ED50), strong neonatal increases in hepatic Cyp2 expression were still obtained but did not persist into adulthood. Furthermore, the neonatal ED50-range TCPOBOP exposure did not sensitize mice to a subsequent, low-dose TCPOBOP treatment. In contrast, neonatal treatment with phenobarbital, a short half-life (t1/2 = 8 h) agonist of CAR and PXR (Nr1i2), induced high-level neonatal activation of Cyp2 genes and also altered their responsiveness to low-dose phenobarbital exposure at adulthood by either increasing (Cyp2b10) or decreasing (Cyp2c55) expression. Thus, neonatal xenobiotic exposure can reprogram hepatic Cyp2 genes and alter their responsiveness to exposures later in life. These findings highlight the need to carefully consider xenobiotic dose, half-life, and persistence in tissue when evaluating the long-term effects of early life environmental chemical exposures.

Characterization of human pregnane X receptor activators identified from a screening of the Tox21 compound library

The pregnane X receptor (PXR; NR1I2) is an important nuclear receptor whose main function is to regulate enzymes within drug metabolism. The main drug metabolizing enzyme regulated by PXR, cytochrome P450 (CYP) 3A4, accounts for the metabolism of nearly 50% of all marketed drugs. Recently, PXR has also been identified as playing a role in energy homeostasis, immune response, and cancer. Due to its interaction with these important roles, alongside its drug-drug interaction function, it is imperative to identify compounds which can modulate PXR. In this study, we screened the Tox21 10,000 compound collection to identify hPXR agonists using a stable hPXR-Luc HepG2 cell line. A pharmacological study in the presence of a PXR antagonist was performed to confirm the activity of the chosen potential hPXR agonists in the same cells. Finally, metabolically competent cell lines – HepaRG and HepaRG-PXR-Knockout (KO) – were used to further confirm the potential PXR activators. We identified a group of structural clusters and singleton compounds which included potentially novel hPXR agonists. Of the 21 selected compounds, 11 potential PXR activators significantly induced CYP3A4 mRNA expression in HepaRG cells. All of these compounds lost their induction when treating HepaRG-PXR-KO cells, confirming their PXR activation. Etomidoline presented as a potentially selective agonist of PXR. In conclusion, the current study has identified 11 compounds as potentially novel or not well-characterized PXR activators. These compounds should further be studied for their potential effects on drug metabolism and drug-drug interactions due to the immense implications of being a PXR agonist.

Regulation of Hepatic Long Noncoding RNAs by Pregnane X Receptor and Constitutive Androstane Receptor Agonists in Mouse Liver.

Altered expression of long noncoding RNAs (lncRNAs) by environmental chemicals modulates the expression of xenobiotic biotransformation-related genes and may serve as therapeutic targets and novel biomarkers of exposure. The pregnane X receptor (PXR/NR1I2) is a critical xenobiotic-sensing nuclear receptor that regulates the expression of many drug-processing genes, and it has similar target-gene profiles and DNA-binding motifs with another xenobiotic-sensing nuclear receptor, namely, constitutive andronstrane receptor (CAR/Nr1i3). To test our hypothesis that lncRNAs are regulated by PXR in concert with protein-coding genes (PCGs) and to compare the PXR-targeted lncRNAs with CAR-targeted lncRNAs, RNA-Seq was performed from livers of adult male C57BL/6 mice treated with corn oil, the PXR agonist PCN, or the CAR agonist 1, 4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP). Among 125,680 known lncRNAs, 3843 were expressed in liver, and 193 were differentially regulated by PXR (among which 40% were also regulated by CAR). Most PXR- or CAR-regulated lncRNAs were mapped to the introns and 3'-untranslated regions (UTRs) of PCGs, as well as intergenic regions. Combining the RNA-Seq data with a published PXR chromatin immunoprecipitation coupled with high-throughput sequencing; cytochrome P450 (P450; ChIP-Seq) data set, we identified 774 expressed lncRNAs with direct PXR-DNA binding sites, and 26.8% of differentially expressed lncRNAs had changes in PXR-DNA binding after PCN exposure. De novo motif analysis identified colocalization of PXR with liver receptor homolog (LRH-1), which regulates bile acid synthesis after PCN exposure. There was limited overlap of PXR binding with an epigenetic mark for transcriptional activation (histone-H3K4-di-methylation, H3K4me2) but no overlap with epigenetic marks for transcriptional silencing [H3 lysine 27 tri-methylation (H3K27me3) and DNA methylation]. Among differentially expressed lncRNAs, 264 were in proximity of PCGs, and the lncRNA-PCG pairs displayed a high coregulatory pattern by PXR and CAR activation. This study was among the first to demonstrate that lncRNAs are regulated by PXR and CAR activation and that they may be important regulators of PCGs involved in xenobiotic metabolism.

Exploiting Chemical Toolboxes for the Expedited Generation of Tetracyclic Quinolines as a Novel Class of PXR Agonists.

The discovery of lead compounds relies on the iterative generation of structure-activity relationship data resulting from the synthesis and biological evaluation of hit analogues. Using traditional approaches, a significant time delay may occur from compound design to results, leading to slow and expensive hit-to-lead explorations. Herein, we have exploited the use of chemical toolboxes to expedite lead discovery and optimization. In particular, the integration of flow synthesizers, automation, process analytical technologies, and computational chemistry has provided a prototype system enabling the multicomponent flow synthesis, in-line analysis, and characterization of chiral tetracyclic quinolines as a novel class of PXR agonists. Within 29 compounds, a novel template 19b (3aS,11R,11aS) was identified with an EC50 of 1.2 μM (efficacy 119%) at the PXR receptor.

Functional polarization of human hepatoma HepaRG cells in response to forskolin

HepaRG is an original human hepatoma cell line, acquiring highly differentiated hepatic features when exposed to dimethylsulfoxide (DMSO). To search alternatives to DMSO, which may exert some toxicity, we have analyzed the effects of forskolin (FSK), a cAMP-generating agent known to favor differentiation of various cell types. FSK used at 50 µM for 3 days was found to promote polarization of high density-plated HepaRG cells, i.e., it markedly enhanced the formation of functional biliary canaliculi structures. It also increased expressions of various hepatic markers, including those of cytochrome P-450 (CYP) 3A4, of drug transporters like NTCP, OATP2B1 and BSEP, and of metabolism enzymes like glucose 6-phosphatase. In addition, FSK-treated HepaRG cells displayed enhanced activities of CYP3A4, NTCP and OATPs when compared to untreated cells. These polarizing/differentiating effects of FSK were next shown to reflect not only the generation of cAMP, but also the activation of the xenobiotic sensing receptors PXR and FXR by FSK. Co-treatment of HepaRG cells by the cAMP analog Sp-5,6-DCl-cBIMPS and the reference PXR agonist rifampicin reproduced the polarizing effects of FSK. Therefore, FSK may be considered as a relevant alternative to DMSO for getting polarized and differentiated HepaRG cells, notably for pharmacological and toxicological studies.

Sequence Variations in pxr (nr1i2) From Zebrafish (Danio rerio) Strains Affect Nuclear Receptor Function.

Regulators of biotransformation are of particular interest in pharmacology and toxicology, determining in part the metabolism, disposition, and toxicity of chemicals. The nuclear receptor NR1I2 (pregnane X receptor, PXR) is a prominent xenosensor that regulates the expression of biotransformation enzymes governing elimination of many exogenous as well as endogenous compounds. Zebrafish (Danio rerio) has only one gene locus for pxr, but different genetic variants have been identified in zebrafish. However, the prevalence and significance of these variants are unknown. We hypothesize that sequence variation occurring in the Pxr gene of zebrafish may affect the action and fate of many chemicals in this species, a key model organism in various fields of research, including environmental toxicology. Here, we examine variation in Pxr sequences from four different strains of zebrafish and assess the responses of each Pxr to clotrimazole and butyl-4-aminobenzoate. The Pxr variants differed in both their ability to bind these structurally different ligands and to regulate reporter gene expression in vitro. We infer that the observed sequence variations in zebrafish Pxrs likely affect the response to putative Pxr agonists in vivo and potentially cause strain-specific biotransformation of xenobiotics in zebrafish. Thus, the choice of zebrafish strain could affect the outcome of downstream toxicological studies.

Activating the pregnane X receptor by imperatorin attenuates dextran sulphate sodium-induced colitis in mice.

Activation of the human pregnane X receptor (PXR; NR1I2) has potential therapeutic uses for inflammatory bowel disease (IBD). Imperatorin (IMP), a naturally occurring coumarin, is the main bioactive ingredient of Angelica dahurica Radix, which is regularly used to treat the common cold and intestinal disorders. However, there are no data on the protective effects of IMP against IBD. The effects of IMP on PXR-modulated cytochrome P450 3A4 (CYP3A4) expression were assessed using a PXR transactivation assay, a mammalian two-hybrid assay, a competitive ligand-binding assay, analysis of CYP3A4 mRNA and protein expression levels and measurement of CYP3A4 activity using a cell-based reporter gene assay and in vitro model. The inhibitory effects of IMP on NF-κB activity were evaluated by a reporter assay and NF-κB p65 nuclear translocation. The anti-IBD effects of IMP were investigated in a dextran sulphate sodium (DSS)-induced colitis mouse model. Colon inflammatory cytokines were assessed by elisa. IMP activated CYP3A4 promoter activity, recruited steroid receptor coactivator 1 to the ligand-binding domain of PXR and increased the expression and activity of CYP3A4. PXR knockdown substantially reduced IMP-induced increase in CYP3A4 expression. Furthermore, IMP-mediated PXR activation suppressed the nuclear translocation of NF-κB and down-regulated LPS-induced expression of pro-inflammatory genes. Nevertheless, PXR knockdown partially reduced the IMP-mediated inhibition of NF-κB. IMP ameliorated DSS-induced colitis by PXR/NF-κB signalling. IMP acts as a PXR agonist to attenuate DSS-induced colitis by suppression of the NF-κB-mediated pro-inflammatory response in a PXR/NF-κB-dependent manner.

PXR: Structure-specific activation by hepatotoxic pyrrolizidine alkaloids

Pyrrolizidine alkaloids (PAs) comprise a large group of more than 660 secondary metabolites found in more than 6000 plant species worldwide. Acute PA intoxication induces severe liver damage. Chronic exposure to sub-lethal doses may cause cumulative damage or cancer. Nuclear receptor activation often constitutes a molecular event for xenobiotic-induced toxicity. However, so far nothing is known about potential interactions of PAs with nuclear receptors as a toxicological mode of action. Thus, in the present study PA-dependent activation of a comprehensive panel of nuclear receptors (PPARs, LXRα, RARα, RXRα, FXR, CAR, PXR, ERα/β) was investigated using GAL4/UAS-based transactivation reporter gene assays. To cover the most frequently occurring PA structure types (retronecine, heliotridine and otonecine type; as well as monoester, open-chain diester and cyclic diester) different PAs were analyzed for interaction with nuclear receptors.Most of the nuclear receptors investigated were not affected by the tested PAs. However, significant activation was found for PXR, which was exclusively activated by the open-chain diesters, echimidine and lasiocarpine. Induction of the model PXR target gene CYP3A4 by PAs was verified at the mRNA, protein and enzyme activity level.In conclusion, PXR activation and PXR-mediated induction of CYP3A4 expression by PAs seem to be structure-dependent. Data suggest that only open-chain diesters act as PXR agonists. This might imply that a PXR-mediated mode of action may contribute to the hepatotoxicity of PAs that is dependent on PA structure.

Resveratrol modifies biliary secretion of cholephilic compounds in sham-operated and cholestatic rats.

AIMTo investigate the effect of resveratrol on biliary secretion of cholephilic compounds in healthy and bile duct-obstructed rats.METHODSResveratrol (RSV) or saline were administered to rats by daily oral gavage for 28 d after sham operation or reversible bile duct obstruction (BDO). Bile was collected 24 h after the last gavage during an intravenous bolus dose of the Mdr1/Mrp2 substrate azithromycin. Bile acids, glutathione and azithromycin were measured in bile to quantify their level of biliary secretion. Liver expression of enzymes and transporters relevant for bile production and biliary secretion of major bile constituents and drugs were analyzed at the mRNA and protein levels using qRT-PCR and Western blot analysis, respectively. The TR-FRET PXR Competitive Binding Assay kit was used to determine the agonism of RSV at the pregnane X receptor.RESULTSRSV increased bile flow in sham-operated rats due to increased biliary secretion of bile acids (BA) and glutathione. This effect was accompanied by the induction of the hepatic rate-limiting transporters for bile acids and glutathione, Bsep and Mrp2, respectively. RSV also induced Cyp7a1, an enzyme that is crucial for bile acid synthesis; Mrp4, a transporter important for BA secretion from hepatocytes to blood; and Mdr1, the major apical transporter for xenobiotics. The findings were supported by increased biliary secretion of azithromycin. The TR-FRET PXR competitive binding assay confirmed RSV as a weak agonist of the human nuclear receptor PXR, which is a transcriptional regulator of Mdr1/Mrp2. RSV demonstrated significant hepatoprotective properties against BDO-induced cirrhosis. RSV also reduced bile flow in BDO rats without any corresponding change in the levels of the transporters and enzymes involved in RSV-mediated hepatoprotection.CONCLUSIONResveratrol administration for 28 d has a distinct effect on bile flow and biliary secretion of cholephilic compounds in healthy and bile duct-obstructed rats.

Metabolism of the Marine Phycotoxin PTX-2 and Its Effects on Hepatic Xenobiotic Metabolism: Activation of Nuclear Receptors and Modulation of the Phase I Cytochrome P450.

PTX-2 is a marine biotoxin frequently found in shellfish that can lead to food intoxication in humans. Information regarding PTX-2 metabolism is scarce, and little is known of its effect on xenobiotic-metabolizing enzymes (XME) or its molecular pathways. The aim of this study was consequently to examine PTX-2 Phase I metabolism using rat and human liver S9 fractions, and also to assess the capability of PTX-2: (i) to modulate the gene expression of a panel of Phase I (CYP450) and II (UGT, SULT, NAT, and GST) enzymes, as well as the Phase III or 0 (ABC and SLCO) transporters in the human hepatic HepaRG cell line using qPCR; (ii) to induce specific CYP450 in HepaRG cells measured by immunolabeling detection and the measurement of the cells’ activities; and (iii) to activate nuclear receptors and induce CYP promoter activities in HEK-T and HepG2 transfected cell lines using transactivation and reporter gene assay, respectively. Our results indicate that PTX-2 hydroxylation occurred with both rat and human S9 fractions. Whereas PTX-2 mostly upregulated the gene expression of CYP1A1 and 1A2, no induction of these two CYP activities was observed. Lastly, PTX-2 did not act as an agonist of CAR or PXR. Due to its effects on some key XME, more attention should be paid to possible drug–drug interactions with phycotoxins, especially as shellfish can accumulate several phycotoxins as well as other kinds of contaminants.

Regulation of hepatic abcb4 and cyp3a65 gene expression and multidrug/multixenobiotic resistance (MDR/MXR) functional activity in the model teleost, Danio rerio (zebrafish)

Multidrug/multixenobiotic resistance (MDR/MXR) confers resistance to a diverse range of potentially toxic pharmaceuticals and environmental contaminants through a cellular response that involves the coordinated induction and activity of the ATP-binding cassette (ABC) transporter P-glycoprotein (P-gp) and the Phase I metabolizing enzyme cytochrome P450 3A (CYP3A). In mammals, ligand-mediated pregnane X receptor (PXR) transcriptional activity regulates the induction of P-gp and CYP3A; however, this mechanism has not been well-characterized in piscine species. Zebrafish (Danio rerio) treated with the Pxr agonist pregnenolone 16α-carbonitrile (PCN) showed decreased P-gp (zebrafish Abcb4) and CYP3A (zebrafish Cyp3a65) mRNA levels after 48h exposure; however, treatment with PCN also resulted in increased hepatic MDR/MXR functional activity (i.e. increased Rhodamine 123 efflux) in vivo. Consistent with mammalian-like MDR/MXR regulated by PXR, the PCN-mediated modulation of hepatic Abcb4 and Cyp3a65 mRNA levels and MDR/MXR functional activity was attenuated by co-treatment with PCN and the mammalian PXR antagonist, ketoconazole (KTC). These results provide evidence that zebrafish Pxr may play a role in MDR/MXR through transcriptional regulation of abcb4 and cyp3a65 gene expression.

MiR-148a-mediated estrogen-induced cholestasis in intrahepatic cholestasis of pregnancy: Role of PXR/MRP3.

Intrahepatic cholestasis of pregnancy (ICP) is an idiopathic liver disease while the biochemical characteristic is the elevated level of total bile acid (TBA). The present study investigated whether miR-148a mediates the induced effect of estrogen on the development of ICP and the proper mechanism: PXR/MRP3 signal pathway. mRNA expression was detected by qPCR, protein expression was detected by western blotting, the concentration of estrogen and TBA were detected by reagent kit respectively. In the cinical research, it was found that miR-148a expression was positive related with the concentration of TBA in the serum of ICP patients. In in vitro research, estradiol (500 nmol/L, 12 h) significantly upregulated miR-148a expression and LV-148a-siRNA inhibited the function of estradiol (500 nmol/L, 48 h) on TBA secretion. In addition, gene silence of miR-148a upregulated PXR expression which was inhibited by estradiol in LO2 cells. Pretreatment of rifampin (10 μmol/L), the agonist of PXR alleviated the TBA secretion induced by estradiol (500 nmol/L, 48 h). miR-148a-siRNA and PXR had a synergistic action on TBA secretion of LO2. Both of miR-148a-siRNA and rifampin (10 μmol/L) inhibited the upregulated effect of estradiol on MRP3 expression. This research has demonstrated that miR-148a may be involved in the induction of estrogen on ICP via PXR signal pathway, and MRP3 may be involved.

Mo1371 - Murine Primary Hepatocytes Exposed to AhR, PXR, CAR or LXR Agonists Produce Differential Transcriptomes which Partially Overlap with One Another and the Transcriptome Induced by PCB Exposure

Mo1371 - Murine Primary Hepatocytes Exposed to AhR, PXR, CAR or LXR Agonists Produce Differential Transcriptomes which Partially Overlap with One Another and the Transcriptome Induced by PCB Exposure

Crypt Organoids Culture as an In Vitro Model in Studying Regulation of Xenobiotic Metabolism and drug‐induced Cytotoxicity

The gastrointestinal (GI) tract is the largest absorption organ in the body and is also enriched with many of the Xenobiotic Processing Genes (XPGs), which play an important role in xenobiotic bioactivation, metabolism, and detoxification. The application of genetically modified mouse models to study drug metabolism and toxicity has proven beneficial over the past decade, and with the ability to culture primary cells from these animal experiments can now be developed in vitro to examine mechanistic properties. Intestinal stem cells (ISCs), located in the crypt zone at the bottom of the villi, produce and differentiate into intestinal epithelial cells (IECs) as a supply for continuous intestinal renewal. Methods have been established in long‐term culture conditions in which one single Lgr5+ stem cell embedded in matrix gel can independently generate villus‐like epithelial domains with the presence of all differentiated IECs. Here, we report the application of 3D crypt organoid cultures as a tool for the study of intestinal drug metabolism and toxicity evaluation. Crypt cells were isolated from mouse intestinal tissue, suspended into matrigels, and cultured in the advanced DMEM/F12 medium supplemented with growth factors. Following continuous budding, crypts were further expanded to create organoids and the function of genes and enzymes involved in drug metabolism were evaluated. Crypt cultures were treated with vehicle (control) or different prototypical agonists of the xenobiotic nuclear receptors (XNR), including the AhR agonist TCDD, PXR agonist PCN, CAR agonist TCPOBOP, PPARa agonist Wy14643, and LXRa agonist T0901317. RT‐QPCR results demonstrated that in response to the different XNR agonists, the representative downstream target genes, including Cyp1a1 (by TCDD), Cyp3a11 (by PCN), Cyp2b10 (by TCPOBOP), Cyp4a10 (by Wy14643), and Abca1 (by T0901317) were largely induced. To further validate receptor‐specific gene induction by their corresponding ligands, we validated these findings using XNR‐null mice. In crypts isolated from Pxr−/−, PCN failed to induce Cyp3a11 gene expression; similarly, TCPOBOP and Wy14643 failed to induce Cyp2b10 or Cyp4a10 in the crypt organoids isolated from Car−/− and Ppara−/− mice, respectively. Camptothecin (CPT)‐11 (Irinotecan), an anticancer drug that generates substantial intestinal toxicity, is bioactivated by carboxylesterases to form the active topoisomerase 1 inhibitor SN‐38. Previous studies in our laboratory have shown that the deletion of intestinal UGT1A1 greatly sensitizes mice toward irinotecan induced intestinal toxicity induced by SN‐38. Bioactivation of irinotecan and cytotoxicity of SN‐38 can also be evaluated in novel crypt organoid cultures. This study suggests that crypt organoid cultures can be exploited as an effective in vitro model for studying the impact of xenobiotics on intestinal XPGs, drug metabolism, and toxicity.Support or Funding InformationSupported by ES010337 and ES024818.

Alisol B 23-acetate from the rhizomes of Alisma orientale is a natural agonist of the human pregnane X receptor

BackgroundPregnane X receptor (PXR) is a key regulator of the induction of drug metabolizing enzymes. PXR has been studied for its importance in drug-drug or herb-drug interactions, and it is also a molecular target for the treatment of inflammatory and metabolic diseases. PurposeThis study aims to determine new natural PXR-ligands from traditional plant medicines. MethodsThe PXR activation activity was measured by a mammalian one hybrid assay of PXR. Identification of the active compound from Alisma rhizome (the rhizomes of Alisma orientale) was carried out by bioassay-guided fractionation method. The transcriptional activity of the liver-enriched nuclear receptors was measured by the luciferase reporter assay. The interaction between the SRC-1 and PXR was measured by a mammalian 2-hybrid assay. The expression of endogenous CYP3A4 mRNA in both cultured hPXR-overexpressing hepatoma cells and human primary hepatocytes were measured by quantitative RT-PCR method. ResultsThe extract of Alisma rhizome showed the most potent activation activity by screening of a library of medicinal plant extracts. Alisol B 23-acetate (ABA) was identified to be the active compound of Alisma rhizome. ABA caused a concentration-dependent increase on the PXR-dependent transactivation of a luciferase reporter gene, but did not affect the ligand binding activity of the liver-enriched nuclear receptors, such as CAR, LXR, FXR, PPARα, PPARδ and PPARγ, emphasizing that ABA is a potent and specific agonist of PXR. With ABA treatment, the direct interaction between the ligand-binding domain of PXR and the receptor interaction domain of SRC1 was observed. ABA also induced the expression of endogenous CYP3A4 mRNA in both cultured hPXR-overexpressing hepatoma cells and human primary hepatocytes. ConclusionSince the rhizomes of Alisma orientale are used for a wide range of ailments in traditional Chinese medicine and Japanese Kampo medicine, this study could possibly extend into the clinical usage of these medicines via the mechanism of PXR activation.

Modulation of CYP3A4 activity alters the cytotoxicity of lipophilic phycotoxins in human hepatic HepaRG cells

The aim of this study was to investigate (i) the cytotoxic effects of lipophilic phycotoxins, including okadaic acid (OA) and dinophysistoxin-1 and -2 (DTX-1 and DTX-2), pectenotoxin-2 (PTX-2), yessotoxin (YTX), spirolide (SPX), and azaspiracids-1, -2 and -3 (AZA-1, AZA-2 and AZA-3), in human HepaRG cells using a multiparametric high content analysis approach, (ii) the ability of nine lipophilic phycotoxins to act as PXR agonists in a HepG2-PXR cell line, (iii) their potential to induce CYP450 activity, and (iv) the role of CYP3A4 in cytotoxicity induced by lipophilic phycotoxins. Our results indicate that while OA, DTX-1 and DTX-2 activated PXR-dependent transcriptional activity in HepG2 cells, no increase of CYP450 (1A2, 3A4, 2C9, 2C19) activities were observed in HepaRG cell following a 72h treatment with these toxins. Multiparametric analysis showed that OA, DTX-1, DTX-2, and PTX-2 were highly cytotoxic in HepaRG cells; inducing cell loss, activation of caspase-3 and γ-H2AX formation. However, no toxicity was observed for YTX, SPX, and AZAs. Moreover, we found that inhibition of CYP3A4 activity by ketoconazole enhances the toxic effects of OA, DTX-1, DTX-2, and PTX-2 in HepaRG cells. Taken together, these results suggest that CYP3A4-mediated metabolism of some lipophilic phycotoxins decreases their in vitro toxicity.

Cholestatic Itch Management.

Cholestatic itch is a feature of numerous hepatobiliary disorders such as primary biliary cirrhosis, primary sclerosing cholangitis, the inherited form of cholestasis, and intrahepatic cholestasis of pregnancy. Although undervalued by physicians, cholestatic itch can be a source of great discomfort to the patient and significantly affects quality of life. Many pruritogens such as bile salts, opioids, serotonin, and histamine have been implicated in the pathogenesis of cholestatic itch, but no causative link has ever been established. Recent findings indicate that the potent neuronal activator lysophosphatidic acid and autotaxin, the enzyme forming lysophosphatidic acid, may be key elements in its pathogenesis. Treatment options for patients with cholestatic itch include the anion exchange resin cholestyramine, bile acid ursodeoxycholic acid, PXR agonist rifampicin, opioid antagonist naltrexone, and the serotonin inhibitor sertraline. These drugs can be used as a stepwise therapeutic approach. The body of evidence for many of these options, however, is not very robust. Patients who do not respond to medical therapy can be candidates for interventional measures, such as albumin dialysis, plasmapheresis, or nasobiliary drainage, or certain experimental approaches such as UVB phototherapy. Research over the past decade has elucidated many of the receptors and neuropeptides involved in itch sensation and transmission; it is hoped that in the future this will lead to the development of novel antipruritic medication for cholestatic itch.

Tanshinone IIA exerts protective effects in a LCA-induced cholestatic liver model associated with participation of pregnane X receptor

Tanshinone IIA (Tan IIA) is one of the main natural active ingredients purified from Salvia miltiorrhiza radix, which has long been used in clinical practice in China to treat diseases including liver fibrosis, Alzheimer׳s disease, and cardiovascular diseases. Tan IIA has hepatoprotective properties, and is an efficacious PXR agonist. Our study was designed to observe the function and mechanism of the hepatoprotective properties of Tan IIA. HepG2 cells were used to investigate the vitrol effects of Tan IIA on PXR and CYP3A4. Gut-formed LCA is hepatotoxic, and has been implicated in the pathogenesis of cholestatic diseases. To further investigate the hepatoprotective mechanisms of Tan IIA against LCA-induced cholestasis in vivo, we choose the normal mice and siRNA-treated mice. The in vitro study demonstrated that the effect of Tan IIA on CYP3A4 was mediated by transactivation of PXR in a dose- and time-dependent manner. The in vivo experiments using PXR siRNA revealed that Tan IIA could protect against LCA-induced hepatotoxicity and cholestasis in a dose-dependent manner. These effects were partially caused by the upregulation of PXR, as well as Cyp3a11, Cyp3a13, and Mdr1, which are the enzymes responsible for LCA metabolism. This is the first report showing that the hepatoprotective effects of Tan IIA are partly mediated by PXR.