Insight into the molecular recognition of human and polar bear pregnane X receptor by three organic pollutants using molecular docking and molecular dynamics simulations.

Pregnane X receptor (PXR) is a heterologous biosensor that is involved in the metabolic pathway of environmental pollutants, regulating the transcription of genes involved in biotransformation. There are significant differences in the selectivity and specificity of organic pollutants (OPs) toward polar bear PXR (pbPXR) and human PXR (hPXR), but the detailed dynamical characteristics of their interactions are unclear. Homology Modeling, molecular docking, molecular dynamics simulation, and free energy calculation were used to analyze the recognition of pbPXR and hPXR by three OPs: BPA, chlordane and toxaphene. Comparing interaction patterns along with binding free energy of pbPXR and hPXR with these three OPs revealed that although pbPXR and hPXR interact similar with these three OPs, these OPs have different effects on the internal dynamics of pbPXR and hPXR. This results in significant alterations in the interaction of key residues near Leu209, Met243, Phe288, Met323, and His407 with OPs, thereby influencing their binding energy. Non-polar interactions, especially van der Waals interactions, were found to be the dominating factors in interacting of these OPs with PXRs. The region surrounding these key residues facilitates hydrophobic contacts with PXR, which are crucial for the selective activation of PXRs in different species by these three OPs. These findings are of significant guidance in understanding the impacts of environmental endocrine disruptors on different organisms.

Pregnane X receptor inhibits the transdifferentiation of hepatic stellate cells by down-regulating periostin expression.

Pregnane X receptor (PXR) is a xenobiotic-sensing nuclear receptor that plays a key role in drug metabolism. Recently, PXR was found to attenuate the development of liver cancer by suppressing epithelial-mesenchymal transition (EMT) in liver cancer cells in a mouse model of two-stage chemical carcinogenesis. To elucidate the role of PXR in the EMT of liver cancer cells, we focused on its role in hepatic stellate cells (HSCs), which are components of the tumor microenvironment in hepatocellular carcinoma (HCC). Human HSC-derived LX-2 cells stably expressed destabilization domain (DD)-fused human PXR (hPXR-LX2 cells). Human HCC-derived HepG2 cells were transfected with the EMT marker VIM promoter-regulated reporter plasmid and co-cultured with hPXR-LX2 cells or treated with hPXR-LX2-derived conditioned medium (CM). Co-culture or CM treatment increased reporter activity in HepG2 cells. This induction was attenuated upon PXR activation in hPXR-LX2 cells by treatment with the DD-stabilizing chemical Shield-1 and the human PXR ligand rifampicin. PXR activation in hPXR-LX2 cells exhibited inhibition of TGF-ß1-induced transdifferentiation, supported by observations of morphological changes and protein or mRNA levels of the transdifferentiation markers COL1A1 and FN1. PXR activation in hPXR-LX2 cells also attenuated the mRNA levels of the key transdifferentiation factor, POSTN. Treatment of hPXR-LX2 cells with recombinant POSTN restored the PXR-mediated suppression of transdifferentiation. Reporter assays with the POSTN promoter showed that PXR inhibited the NF-κB-mediated transcription of POSTN. Consequently, PXR activation in HSCs is expected to inhibit transdifferentiation by down-regulating POSTN expression, thereby suppressing EMT of liver cancer cells.

Multigenerational effects of disperse blue 79 at environmentally relevant concentrations on zebrafish (Danio rerio) fecundity: An integrated approach.

The brominated azo dye (BAD) Disperse Blue (DB79) is a widespread environmental pollutant. The long-term toxicological effects of DB79 and the mechanisms thereof must be understood to allow assessment of the risks of DB79 pollution. A dual-omics approach employing in silico analysis, bioinformatics, and in vitro bioassays was used to investigate the transgenerational (F0-F2) toxicity of DB79 in zebrafish at environmentally relevant concentrations and identify molecular initiating events and key events associated with DB79-induced fertility disorders. Exposure to 500 µg/L DB79 decreased fecundity in the F0 and F1 generations by > 30 % and increased the condition factor of the F1 generation 1.24-fold. PPARα/RXR and PXR ligand binding activation were found to be critical molecular initiating events associated with the decrease in fecundity. Several key events (changes in fatty acid oxidation and uptake, lipoprotein metabolism, and xenobiotic metabolism and transport) involved in lipid dysregulation and xenobiotic disposition were found to be induced by DB79 through bioinformatic annotation using dual-omics data. The biomolecular underpinnings of decreased transgenerational fertility in zebrafish attributable to BAD exposure were elucidated and novel biomolecular targets in the adverse outcome pathway framework were identified. These results will inform future studies and facilitate the development of mitigation strategies.

Large-Scale Screening of Per- and Polyfluoroalkyl Substance Binding Interactions and Their Mixtures with Nuclear Receptors.

Despite their significant impact, comprehensive screenings and detailed analyses of per- and polyfluoroalkyl substance (PFAS) binding strengths at the orthosteric and allosteric sites of NRs are currently lacking. This study addresses this gap by focusing on the binding interaction analysis of both common and uncommon PFAS with the nuclear receptors (NRs) vitamin D receptor (VDR), peroxisome proliferator-activated receptor gamma (PPARγ), pregnane X receptor (PXR), and estrogen receptor alpha (ERα). Advanced docking simulations were used to screen 9507 PFAS chemicals at the orthosteric and allosteric sites of PPARγ, PXR, VDR, and ERα. All receptors exhibited strong binding interactions at the orthosteric and allosteric site with a significant number of PFAS. We verified the accuracy of the docking protocol through multiple docking controls and validations. A mixture modeling analysis indicates that PFAS can bind in various combinations with themselves and endogenous ligands simultaneously, to disrupt the endocrine system and cause carcinogenic responses. These findings reveal that PFAS can interfere with nuclear receptor activity by displacing endogenous or native ligands by binding to the orthosteric and allosteric sites. The purpose of this study is to explore the mechanisms through which PFAS exert their endocrine-disrupting effects, potentially leading to more targeted therapeutic strategies. Importantly, this study is the first to explore the binding of PFAS at allosteric sites and to model PFAS mixtures at nuclear receptors. Given the high concentration and persistence of PFAS in humans, this study further emphasizes the urgent need for further research into the carcinogenic mechanisms of PFAS and the development of therapeutic strategies that target nuclear receptors.

Metabolomics reveals dysregulated all-trans retinoic acid and polyunsaturated fatty acid metabolism contribute to PXR-induced hepatic steatosis in mice.

Activation of pregnane X receptor (PXR) by xenobiotics has been associated with metabolic diseases. This study aimed to reveal the impact of PXR activation on hepatic metabolome and explore novel mechanisms underlying PXR-mediated lipid metabolism disorder in the liver. Wild-type and PXR-deficient male C57BL/6 mice were used as in vivo models, and hepatic steatosis was induced by pregnenolone-16α-carbonitrile, a typical rodent PXR agonist. Metabolomic analysis of liver tissues showed that PXR activation led to significant changes in metabolites involved in multiple metabolic pathways previously reported, including lipid metabolism, energy homeostasis, and amino acid metabolism. Moreover, the level of hepatic all-trans retinoic acid (ATRA), the main active metabolite of vitamin A, was significantly increased by PXR activation, and genes involved in ATRA metabolism exhibited differential expression following PXR activation or deficiency. Consistent with previous research, the expression of downstream target genes of peroxisome proliferator-activated receptor α (PPARα) was decreased. Analysis of fatty acids by Gas Chromatography-Mass Spectrometer further revealed changes in polyunsaturated fatty acid metabolism upon PXR activation, suggesting inhibition of PPARα activity. Taken together, our findings reveal a novel metabolomic signature of hepatic steatosis induced by PXR activation in mice.

B3galt5 functions as a PXR target gene and regulates obesity and insulin resistance by maintaining intestinal integrity.

Pregnane X receptor (PXR) has been reported to regulate glycolipid metabolism. The dysfunction of intestinal barrier contributes to metabolic disorders. However, the role of intestinal PXR in metabolic diseases remains largely unknown. Here, we show that activation of PXR by tributyl citrate (TBC), an intestinal-selective PXR agonist, improves high fat diet (HFD)-induced obesity. The metabolic benefit of intestinal PXR activation is associated with upregulation of ß-1,3 galactosyltransferase 5 (B3galt5). Our results reveal that B3galt5 mainly expresses in the intestine and is a direct PXR transcriptional target. B3galt5 knockout exacerbates HFD-induced obesity, insulin resistance and inflammation. Mechanistically, B3galt5 is essential to maintain the integrity of intestinal mucus barrier. B3galt5 ablation impairs the O-glycosylation of mucin2, destabilizes the mucus layer, and increases intestinal permeability. Furthermore, B3galt5 deficiency abolishes the beneficial effect of intestinal PXR activation on metabolic disorders. Our results suggest the intestinal-selective PXR activation regulates B3galt5 expression and maintains metabolic homeostasis, making it a potential therapeutic strategy in obesity.

Cannabidiol promotes intestinal cholesterol uptake mediated by Pregnane X receptor.

Background: Cannabidiol (CBD), a non-psychoactive phytocannabinoid of cannabis, is therapeutically used as an analgesic, anti-convulsant, anti-inflammatory, and anti-psychotic drug. There is a growing concern about the adverse side effects posed by CBD usage. Pregnane X receptor (PXR) is a nuclear receptor activated by a variety of dietary steroids, pharmaceutical agents, and environmental chemicals. In addition to the role in xenobiotic metabolism, the atherogenic and dyslipidemic effects of PXR have been revealed in animal models. CBD has a low affinity for cannabinoid receptors, thus it is important to elucidate the molecular mechanisms by which CBD activates cellular signaling and to assess the possible adverse impacts of CBD on pro-atherosclerotic events in cardiovascular system, such as dyslipidemia. Objective: Our study aims to explore the cellular and molecular mechanisms by which exposure to CBD activates human PXR and increases the risk of dyslipidemia. Methods: Both human hepatic and intestinal cells were used to test if CBD was a PXR agonist via cell-based transfection assay. The key residues within PXR's ligand-binding pocket that CBD interacted with were investigated using computational docking study together with site-directed mutagenesis assay. The C57BL/6 wildtype mice were orally fed CBD in the presence of PXR antagonist resveratrol (RES) to determine how CBD exposure could change the plasma lipid profiles in a PXR-dependent manner. Human intestinal cells were treated with CBD and/or RES to estimate the functions of CBD in cholesterol uptake. Results: CBD was a selective agonist of PXR with higher activities on human PXR than rodents PXRs and promoted the dissociation of human PXR from nuclear co-repressors. The key amino acid residues Met246, Ser247, Phe251, Phe288, Trp299, and Tyr306 within PXR's ligand binding pocket were identified to be necessary for the agonistic effects of CBD. Exposure to CBD increased the circulating total cholesterol levels in mice which was partially caused by the induced expression levels of the key intestinal PXR-regulated lipogenic genes. Mechanistically, CBD induced the gene expression of key intestinal cholesterol transporters, which led to the increased cholesterol uptake by intestinal cells. Conclusion: CBD was identified as a selective PXR agonist. Exposure to CBD activated PXR signaling and increased the atherogenic cholesterol levels in plasma, which partially resulted from the ascended cholesterol uptake by intestinal cells. Our study provides potential evidence for the future risk assessment of CBD on cardiovascular disease, such as dyslipidemia.

HNF4α improves hepatocyte regeneration by upregulating PXR.

Hepatocyte nuclear factor 4 alpha (HNF4α) and the pregnane X receptor (PXR) are involved in hepatocyte regeneration. It is not clear whether HNF4α is involved in hepatocyte regeneration through the regulation of PXR. This study aims to explore the regulatory relationship between HNF4a and PXR, and whether it affects hepatocyte regeneration. A mouse PXR gene reporter and an HNF4α overexpression plasmid were constructed and transfected into mouse hepatoma cells (Hepa1-6). Overexpression of HNF4α, detection of the PXR gene reporter fluorescence value, PXR gene, and protein expression analysis were conducted to explore the regulatory relationship between HNF4α and PXR. Apoptosis and cell cycle data were measured to verify whether HNF4α is involved in hepatocyte regeneration through PXR. The luciferase gene reporter assay results indicated when HNF4α was overexpressed, the fluorescence value of the PXR gene reporter was higher than that in the control at 24 h. With increasing HNF4α expression, the PXR gene and protein expression increased, indicating that HNF4α binds to the PXR promoter and upregulates PXR expression. Apoptosis and cell cycle analysis results demonstrated that when the expression of HNF4α increased, the expression of PXR increased, the apoptosis rate decreased, and the proliferation rate increased. Meanwhile, when the upward trend of PXR gene expression was inhibited by ketoconazole, the proliferation rate decreased. By inhibiting HNF4α and creating a partial hepatectomy (PHx), we demonstrated that HNF4α can upregulate PXR to promote liver regeneration in vivo. Therefore, HNF4α is shown to improve hepatocyte regeneration by upregulating PXR, which provides a reference for future research on the combined application of drugs for the treatment of liver injury.

Molecular targets of PXR-dependent ethanol-induced hepatotoxicity in female mice.

The pregnane X receptor (PXR, NR1I2), a xenobiotic-sensing nuclear receptor signaling potentiates ethanol (EtOH)-induced hepatotoxicity in male mice, however, how PXR signaling modulates EtOH-induced hepatotoxicity in female mice is unknown. Wild type (WT) and Pxr-null mice received 5 % EtOH-containing diets or paired-fed control diets for 8 weeks followed by assessment of liver injury, EtOH elimination rates, histology, and changes in gene and protein expression; microarray and bioinformatic analyses were also employed to identify PXR targets in chronic EtOH-induced hepatotoxicity. In WT females, EtOH ingestion significantly increased serum ethanol and alanine aminotransferase (ALT) levels, hepatic Pxr mRNA, constitutive androstane receptor activation, Cyp2b10 mRNA and protein, oxidative stress, endoplasmic stress (phospho-elF2α) and pro-apoptotic (Bax) protein expression. Unexpectedly, EtOH-fed female Pxr-null mice displayed increased EtOH elimination and elevated levels of hepatic acetaldehyde detoxifying aldehyde dehydrogenase 1a1 (Aldh1a1) mRNA and protein, EtOH-metabolizing alcohol dehydrogenase 1 (ADH1), and lipid suppressing microsomal triglyceride transport protein (MTP) protein, aldo-keto reductase 1b7 (Akr1b7) and Cyp2a5 mRNA, but suppressed CYP2B10 protein levels, with evidence of protection against chronic EtOH-induced oxidative stress and hepatotoxicity. While liver injury was not different between the two WT sexes, female sex may suppress EtOH-induced macrovesicular steatosis in the liver. Several genes and pathways important in retinol and steroid hormone biosynthesis, chemical carcinogenesis, and arachidonic acid metabolism were upregulated by EtOH in a PXR-dependent manner in both sexes. Together, these data establish that female Pxr-null mice are resistant to chronic EtOH-induced hepatotoxicity and unravel the PXR-dependent and -independent mechanisms that contribute to EtOH-induced hepatotoxicity.

Characterization of NR1J1 Paralog Responses of Marine Mussels: Insights from Toxins and Natural Activators.

The pregnane X receptor (PXR) is a nuclear hormone receptor that plays a pivotal role in regulating gene expression in response to various ligands, particularly xenobiotics. In this context, the aim of this study was to shed light on the ligand affinity and functions of four NR1J1 paralogs identified in the marine mussel Mytilus galloprovincialis, employing a dual-luciferase reporter assay. To achieve this, the activation patterns of these paralogs in response to various toxins, including freshwater cyanotoxins (Anatoxin-a, Cylindrospermopsin, and Microcystin-LR, -RR, and -YR) and marine algal toxins (Nodularin, Saxitoxin, and Tetrodotoxin), alongside natural compounds (Saint John's Wort, Ursolic Acid, and 8-Methoxypsoralene) and microalgal extracts (Tetraselmis, Isochrysis, LEGE 95046, and LEGE 91351 extracts), were studied. The investigation revealed nuanced differences in paralog response patterns, highlighting the remarkable sensitivity of MgaNR1J1γ and MgaNR1J1δ paralogs to several toxins. In conclusion, this study sheds light on the intricate mechanisms of xenobiotic metabolism and detoxification, particularly focusing on the role of marine mussel NR1J1 in responding to a diverse array of compounds. Furthermore, comparative analysis with human PXR revealed potential species-specific adaptations in detoxification mechanisms, suggesting evolutionary implications. These findings deepen our understanding of PXR-mediated metabolism mechanisms, offering insights into environmental monitoring and evolutionary biology research.

Pregnane X receptor activation induces liver enlargement and regeneration and simultaneously promotes the metabolic activity of CYP3A1/2 and CYP2C6/11 in rats.

Human pregnane X receptor (PXR) is critical for regulating the expression of key drug-metabolizing enzymes such as CYP3A and CYP2C. Our recent study revealed that treatment with rodent-specific PXR agonist pregnenolone-16α-carbonitrile (PCN) significantly induced hepatomegaly and promoted liver regeneration after two-thirds partial hepatectomy (PHx) in mice. However, it remains unclear whether PXR activation induces hepatomegaly and liver regeneration and simultaneously promotes metabolic function of the liver. Here, we investigated the metabolism activity of CYP1A2, CYP3A1/2 and CYP2C6/11 during PXR activation-induced liver enlargement and regeneration in rats after cocktail dosing of CYP probe drugs. For PCN-induced hepatomegaly, a notable increase in the metabolic activity of CYP3A1/2 and CYP2C6/11, as evidenced by the plasma exposure of probe substrates and the AUC ratios of the characteristic metabolites to its corresponding probe substrates. The metabolic activity of CYP1A2, CYP3A1/2 and CYP2C6/11 decreased significantly after PHx. However, PCN treatment obviously enhanced the metabolic activity of CYP2C6/11 and CYP3A1/2 in PHx rats. Furthermore, the protein expression levels of CYP3A1/2 and CYP2C6/11 in liver were up-regulated. Taken together, this study demonstrates that PXR activation not only induces hepatomegaly and liver regeneration in rats, but also promotes the protein expression and metabolic activity of the PXR downstream metabolizing enzymes such as CYP3A1/2 and CYP2C6/11 in the body.

Persistent organic pollutants disrupt the oxidant/antioxidant balance of INS-1E pancreatic ß-cells causing their physiological dysfunctions.

BACKGROUND: Persistent organic pollutants (POPs) have emerged as potent diabetogenic agents, but their mechanisms of action remain poorly identified. OBJECTIVES: In this study, we aim to determine the mechanisms regulating the damaging effects of POPs in pancreatic ß-cells, which have a central role in the development of diabetes. METHODS: We treated INS-1E pancreatic ß-cells with PCB-153, p,p'-DDE, PCB-126, or TCDD at doses ranging from 1 × 10-15to 5 × 10-6M. We measured insulin content and secretion, cell viability and assessed the mRNA expression of the xenobiotic nuclear receptors Nr1i2 and Nr1i3, and the aryl hydrocarbon receptor (Ahr). In addition, we evaluated the antioxidant defense and production of reactive oxygen species (ROS). Finally, we studied the ability of the antioxidant N-acetyl-L-cysteine (NAC) to counteract the effects of POPs in INS-1E cells. RESULTS: When exposed to environmental POP levels, INS-1E cells had impaired production and secretion of insulin. These defects were observed for all tested POPs and were paralleled by reduced Ins1 and Ins2 mRNA expression. While POP treatment for 3 days did not affect INS-1E cell viability, longer treatment progressively killed the cells. Furthermore, we found that the xenobiotic detoxification machinery is poorly expressed in the INS-1E cells, as characterized by the absence of Nr1i2 and Nr1i3 and their respective downstream targets Cyp3a1/Cyp3a2 and Cyp2b1/Cyp2b3, and the weak functionality of the Ahr/Cyp1a1 signaling. Interestingly, POPs dysregulated key antioxidant enzymes such as glutathione peroxidases, peroxiredoxins, thioredoxins, and catalases. In parallel, the production of intracellular ROS, including superoxide anion (O2•-) and hydrogen peroxide (H2O2), was increased by POP exposure. Improving the oxidant scavenging capacity of INS-1E cells by NAC treatment restored the production and secretion of insulin. CONCLUSION: By promoting oxidative stress and impairing the ability of INS-1E cells to produce and secrete insulin, this study reveals how POPs can mechanistically act as diabetogenic agents, and provides new scientific evidence supporting the concept that POPs are fueling the diabetes epidemics.

The effect of new atypical antipsychotic drugs on the expression of transcription factors regulating cytochrome P450 enzymes in rat liver.

BACKGROUND: Our recent studies showed that prolonged administration of novel atypical antipsychotics affected the expression and activity of cytochrome P450 (CYP), as demonstrated in vitro on human hepatocytes and in vivo on the rat liver. The aim of the present work was to study the effect of repeated treatment with asenapine, iloperidone, and lurasidone on the expression of transcription factors regulating CYP drug-metabolizing enzymes in rat liver. METHODS: The hepatic mRNA (qRT-PCR) and protein levels (Western blotting) of aryl hydrocarbon receptor (AhR), pregnane X receptor (PXR), constitutive androstane receptor (CAR) and peroxisome proliferator-activated receptor (PPARγ) were measured in male Wistar rats after 2 week-treatment with asenapine, iloperidone or lurasidone. RESULTS: The 2-week treatment with asenapine significantly diminished the AhR and PXR expression (mRNA, protein level), and CAR mRNA level in rat liver. Iloperidone lowered the AhR and CAR expression and PXR protein level. Lurasidone did not affect the expression of AhR and CAR, but increased PXR expression. The antipsychotics did not affect PPARγ. CONCLUSIONS: Prolonged treatment with asenapine, iloperidone, or lurasidone affects the expression of transcription factors regulating the CYP drug-metabolizing enzymes. The changes in the expression of AhR, CAR, and PXR mostly correlate with alterations in the expression and activity of respective CYP enzymes found in our previous studies. Since these transcription factors are also engaged in the expression of phase II drug metabolism and drug transporters, changes in their expression may affect the metabolism of endogenous substrates and pharmacokinetics of concomitantly used drugs.

Gut Microbiota Affects Mouse Pregnane X Receptor Agonist Pregnenolone 16α-Carbonitrile-Induced Hepatomegaly by Regulating Pregnane X Receptor and Yes-Associated Protein Activation.

Pregnane X receptor (PXR) is essential in the regulation of liver homeostasis, and the gut microbiota is closely linked to liver physiologic and pathologic status. We previously found that activation of PXR significantly promotes liver enlargement through interaction with yes-associated protein (YAP). However, whether gut microbiota contributes to PXR-induced hepatomegaly and the involved mechanisms remain unclear. In this study, C57BL/6 mice were administered the mouse-specific agonist pregnenolone 16α-carbonitrile (PCN) for 5 days. Depletion of gut microbiota was achieved using broad-spectrum antibiotics (ABX) and fecal microbiota transplantation (FMT) was performed to restore the gut microbia. The composition of gut microbiota was analyzed by 16S rRNA sequencing, while the expression of PXR, YAP, and their downstream target genes and proteins were assessed. The results indicated that PCN treatment altered the composition and abundance of specific bacterial taxa. Furthermore, depletion of gut microbiota using ABX significantly attenuated PCN-induced hepatomegaly. FMT experiments further demonstrated that the fecal microbiota from PCN-treated mice could induce liver enlargement. Mechanistic studies revealed that ABX treatment impeded the PXR and YAP activation induced by PCN, as evidenced by decreased expression of PXR, YAP, and their downstream targets. Moreover, alterations in PXR and YAP activation were likely contributing to hepatomegaly in recipient mice following FMT from PCN-treated mice. Collectively, the current study demonstrated that gut microbiota is involved in PCN-induced hepatomegaly via regulating PXR and YAP activation, providing potential novel insights into the involvement of gut microbiota in PXR-mediated hepatomegaly. SIGNIFICANCE STATEMENT: This work describes that the composition of gut microbiota is altered in mouse pregnane X receptor (PXR) agonist pregnenolone 16α-carbonitrile (PCN)-induced hepatomegaly. Treatment with an antibiotic cocktail depletes the intestinal microbiota, leading to the impairment of liver enlargement caused by PCN. Additionally, fecal microbiota transplantation from PCN-treated mice induces liver enlargement. Further study revealed that gut microbiota is involved in hepatomegaly via regulating PXR and yes-associated protein activation.

Chemical manipulation of an activation/inhibition switch in the nuclear receptor PXR.

Nuclear receptors are ligand-activated transcription factors that can often be useful drug targets. Unfortunately, ligand promiscuity leads to two-thirds of receptors remaining clinically untargeted. PXR is a nuclear receptor that can be activated by diverse compounds to elevate metabolism, negatively impacting drug efficacy and safety. This presents a barrier to drug development because compounds designed to target other proteins must avoid PXR activation while retaining potency for the desired target. This problem could be avoided by using PXR antagonists, but these compounds are rare, and their molecular mechanisms remain unknown. Here, we report structurally related PXR-selective agonists and antagonists and their corresponding co-crystal structures to describe mechanisms of antagonism and selectivity. Structural and computational approaches show that antagonists induce PXR conformational changes incompatible with transcriptional coactivator recruitment. These results guide the design of compounds with predictable agonist/antagonist activities and bolster efforts to generate antagonists to prevent PXR activation interfering with other drugs.

Induction effect of antiretroviral bictegravir on the expression of Abcb1, Abcg2 and Abcc1 genes associated with P-gp, BCRP and MRP1 transporters present in rat peripheral blood mononuclear cells.

BACKGROUND: Antiretrovirals have the potential to cause drug interactions leading to inefficacy or toxicity via induction of efflux transporters through nuclear receptors, altering drug concentrations at their target sites. RESEARCH DESIGN AND METHODS: This study used molecular dynamic simulations and qRT-PCR to investigate bictegravir's interactions with nuclear receptors PXR and CAR, and its effects on efflux transporters (P-gp, BCRP, MRP1) in rat PBMCs. PBMC/plasma drug concentrations were measured using LC-MS/MS to assess the functional impact of transporter expression. RESULTS: Bictegravir significantly increased the expression of ABC transporters, with Car identified as a key mediator. This suggests that bictegravir's influence on nuclear receptors could affect drug transport and efficacy at the cellular level. CONCLUSIONS: Bictegravir activates nuclear receptors enhancing efflux transporter expression. Understanding these interactions is crucial for preventing drug-drug interactions and reducing toxicity in clinical use. Combining CAR antagonists with bictegravir may prevent drug resistance and toxicity. However, these findings are based on preclinical data and necessitate further clinical trials to confirm their applicability in clinical settings.

The reversal of PXR or PPARα activation-induced hepatomegaly.

The activation of pregnane X receptor (PXR) or peroxisome proliferator-activated receptor α (PPARα) can induce liver enlargement. Recently, we reported that PXR or PPARα activation-induced hepatomegaly depends on yes-associated protein (YAP) signaling and is characterized by hepatocyte hypertrophy around the central vein area and hepatocyte proliferation around the portal vein area. However, it remains unclear whether PXR or PPARα activation-induced hepatomegaly can be reversed after the withdrawal of their agonists. In this study, we investigated the regression of enlarged liver to normal size following the withdrawal of PCN or WY-14643 (typical agonists of mouse PXR or PPARα) in C57BL/6 mice. The immunohistochemistry analysis of CTNNB1 and KI67 showed a reversal of hepatocyte size and a decrease in hepatocyte proliferation after the withdrawal of agonists. In details, the expression of PXR or PPARα downstream proteins (CYP3A11, CYP2B10, ACOX1, and CYP4A) and the expression of proliferation-related proteins (CCNA1, CCND1, and PCNA) returned to the normal levels. Furthermore, YAP and its downstream proteins (CTGF, CYR61, and ANKRD1) also restored to the normal states, which was consistent with the change in liver size. These findings demonstrate the reversibility of PXR or PPARα activation-induced hepatomegaly and provide new data for the safety of PXR and PPARα as drug targets.

Pregnane X receptor (PXR) deficiency promotes hepatocarcinogenesis via induction of Akr1c18 expression and prostaglandin F2α (PGF2α) levels.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Pregnane X receptor (PXR), a xenobiotic-sensing nuclear receptor, plays a critical role in the metabolism of endogenous and exogenous substances in the liver. Here, we investigate whether PXR plays a role in pathogenesis of HCC. We show that liver tumors were developed in diethylnitrosamine (DEN)-treated in PXR knockout (KO) mice. Hepatic levels of prostaglandin F2α (PGF2α) and aldo-keto reductase family 1 member C18 (Akr1c18), a prostaglandin synthase of catalyzing reduction of PGH2 to PGF2α, were significantly elevated in DEN-treated PXR KO mice. Hepatic mRNA levels of alpha fetoprotein (AFP), cyclin D1 (Ccnd1), fibroblast growth factor 21 (FGF21), and inflammatory cytokine interleukin 6 (IL-6) were significantly increased in DEN-treated PXR KO mice. Other members of Akr1c family, liver metabolizing enzymes including Cyp1a2, Cyp2b10 and Cyp3a11, and bile acid synthesis enzyme Cyp7a1 mRNA levels were significantly decreased in DEN-treated PXR KO mice. Our findings revealed that PXR deficiency promoted DEN-induced HCC in mice via induction of Akr1c18 expression and PGF2α levels and the increased PGF2α levels synthetized by Akr1c18 enhanced hepatocytes proliferation and induced inflammatory cytokine production, which accelerated liver tumor development after DEN treatment, suggesting that PXR deficiency may create a microenvironment that is more prone to DEN-induced liver tumors and targeting PXR and Akr1c18 to reduce PGF2α biosynthesis may be a potential and novel therapeutic strategy for HCC.

Regulation of hepatic xenosensor function by HNF4alpha.

Nuclear receptors such as constitutive androstane receptor (CAR), pregnane X receptor (PXR), and peroxisome proliferator-activated receptor-alpha (PPARα), and transcription factors with nuclear receptor type activity such as aryl hydrocarbon receptor (AhR) function as xenobiotic sensors. Hepatocyte nuclear factor 4alpha (HNF4α) is a highly conserved orphan nuclear receptor essential for liver function. We tested the hypothesis that HNF4α is essential for the function of these 4 major xenosensors. Wild-type (WT) and hepatocyte-specific Hnf4a null (HNF4α-KO) mice were treated with the mouse-specific activators of AhR (TCDD, 30 µg/kg), CAR (TCPOBOP, 2.5 µg/g), PXR, (PCN, 100 µg/g), and PPARα (WY-14643, 1 mg/kg). Blood and liver tissue samples were collected to study receptor activation. TCDD (AhR agonist) treatment did not affect the liver-to-body weight ratio (LW/BW) in either WT or HNF4α-KO mice. Further, TCDD activated AhR in both WT and HNF4α-KO mice, confirmed by increase in expression of AhR target genes. TCPOBOP (CAR agonist) significantly increased the LW/BW ratio and CAR target gene expression in WT mice, but not in HNF4α-KO mice. PCN (a mouse PXR agonist) significantly increased LW/BW ratio in both WT and HNF4α-KO mice however, failed to induce PXR target genes in HNF4α-KO mice. The treatment of WY-14643 (PPARα agonist) increased LW/BW ratio and PPARα target gene expression in WT mice but not in HNF4α-KO mice. Together, these data indicate that the function of CAR, PXR, and PPARα but not of AhR was disrupted in HNF4α-KO mice. These results demonstrate that HNF4α function is critical for the activation of hepatic xenosensors, which are critical for toxicological responses.

NR1I2 as a core biological target in chronic venous ulcer tissues treated with ultrasound therapy.

Ultrasound therapy is a method of applying ultrasonic energy to the stimulation produced by human body to change the function and tissue state of the body in order to achieve the purpose of treating diseases. Chronic venous ulcer is a common chronic skin ulcer. GSE222503 for ultrasound therapy of chronic venous ulcers was downloaded from gene expression omnibus database, which were used to identify differentially expressed genes. Weighted gene co-expression network analysis, functional enrichment analysis, gene set enrichment analysis, immune infiltration analysis and construction and analysis of protein-protein interaction network were performed. Draw gene expression heatmaps. Comparative toxicogenomics database analysis was performed. Two hundred thirty-five differentially expressed genes were obtained. According to gene ontology analysis, in biological process analysis, they were mainly enriched in positive regulation of cellular biosynthetic process, reproductive cell development, vasculogenesis, vascular morphogenesis, and inflammatory response. In cellular component analysis, they were mainly enriched in leading edge of growing cell, extracellular matrix binding organelle, F-actin capping protein complex. In molecular function analysis, they were mainly concentrated in receptor ligand activity, cytokine receptor binding. In Kyoto encyclopedia of genes and genomes analysis, they were mainly enriched in cytokine-cytokine receptor interaction, PI3K-Akt signaling pathway, HIF-1 signaling pathway, heme biosynthesis. In weighted gene co-expression network analysis, the soft threshold power was set to 9. Thirty modules were generated. PF4, NR1I2, TTC16, H3C12, KLRB1, CYP21A2 identified by 4 algorithms (MCC, EPC, closeness, stress). Heatmap of core gene expression showed that H3C12, KLRB1, PF4, NR1I2 were all underexpressed in samples of ultrasound-treated chronic venous ulcers and overexpressed in samples of untreated chronic venous ulcers. Comparative toxicogenomics database analysis showed that H3C12, KLRB1, PF4, NR1I2 are associated with thrombophlebitis, phlebitis, vascular malformations, metabolic syndrome, ulcers, and inflammation. In samples of chronic venous ulcer tissue treated with ultrasound, NR1I2 shows low expression, while in samples of chronic venous ulcer tissue without ultrasound treatment, it shows high expression. This finding suggests a potential role of NR1I2 in the process of ultrasound therapy for chronic venous ulcers, which may be related to the therapeutic effect of ultrasound therapy on chronic venous ulcers.