Pregnane X Receptor Expression Research Articles

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.

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.

Pregnane X Receptor Activation in Liver Macrophages Protects against Endotoxin-Induced Liver Injury.

Endotoxemia-related acute liver injury has a poor prognosis and high mortality, and macrophage polarization plays a central role in the pathological process. Pregnane X receptor (PXR) serves as a nuclear receptor and xenosensor, safeguarding the liver from toxic stimuli. However, the effect and underlying mechanism of PXR activation on endotoxemic liver injury remain largely unknown. Here, the expression of PXR is reported in human and murine macrophages, and PXR activation modified immunotypes of macrophages. Moreover, PXR activation significantly attenuated endotoxemic liver injury and promoted macrophage M2 polarization. Macrophage depletion by GdCl3 confirmed the essential of macrophages in the beneficial effects observed with PXR activation. The role of PXR in macrophages is further validated using AAV8-F4/80-Pxr shRNA-treated mice; the PXR-mediated hepatoprotection is impaired, and M2 polarization enhancement is blunted. Additionally, treatment with PXR agonists inhibited lipopolysaccharide (LPS)-induced M1 polarization and favored M2 polarization in BMDM, Raw264.7, and THP-1 cells. Further analyses revealed an interaction between PXR and p-STAT6 in vivo and in vitro. Moreover, blocking Pxr or Stat6 abolished the PXR-induced polarization shift. Collectively, macrophage PXR activation attenuated endotoxin-induced liver injury and regulated macrophage polarization through the STAT6 signaling pathway, which provided a potential therapeutic target for managing endotoxemic liver injury.

A62 EXAMINING THE ACTIVATION OF XENOBIOTIC RECEPTORS PXR AND AHR IN COLONOIDS USING MICROBIAL METABOLITES AND CHEMICAL LIGANDS

Abstract Background The Aryl hydrocarbon receptor (AhR) and pregnane X receptor (PXR) are vital xenobiotic receptors activated by foreign substances, playing pivotal roles in regulating chemical metabolism. Traditionally, these receptors have been closely linked to their roles in mediating responses to toxic compounds. However, recent findings have revealed their newfound significance in maintaining gut homeostasis and regulating inflammatory processes. Aims We investigated the dual roles of AhR and PXR activation. We started with dosage response experiments to determine optimal treatment conditions, such as treatment concentration and exposure time. Subsequently, we compared transcriptomic responses induced by chemical ligands with those from microbial metabolites, aiming to understand how AhR and PXR activation can yield both adverse and beneficial effects. Methods Using mouse 3D colonoids and 2D monolayer cultures, PXR and AhR were activated using the microbial metabolites indole-3-propionic acid (IPA) and indole-3-pyruvic acid (IPyA), respectively. Concentrations were determined through dosage response experiments. Changes to PXR and AhR target gene expression were measured using qPCR. We compared the gene induction by IPA and IPyA to responses driven by the chemical ligands pregnenolone 16α-carbonitrile (PCN) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), respectively. Results In 3D cell culture, PCN induced PXR target genes Cyp2c55 and Abcb1a, while IPA had no significant effect. Cyp3a11 was absent. Both IPyA and TCDD induced AhR genes, Cyp1a1 and Cyp1b1, with varying induction, and Cyp1a2 showed no response. In 2D cell culture, PCN significantly increased Cyp2c55 gene expression, but neither PCN nor IPA significantly induced Abcb1a. In 3D culture, TCDD significantly induced Cyp1a1 and Cyp1b1, while IPyA only induced Cyp1a1, revealing differences in AhR gene induction between 2D and 3D cell culture models. Conclusions Our research reveals contrasting gene induction patterns following AhR and PXR activation through microbial metabolites and chemical ligands. These varied responses highlight the multifaceted roles these receptors play within the gut environment. This study enhances our understanding of xenobiotic receptors' contributions to maintaining gut homeostasis and regulating inflammatory processes, shedding light on the interplay between ligands and receptors. Methods First, we isolate the crypts of mice to create 3D organoids, were we have access to the basolateral side. To capture the true biological system, we convert the 3D culture to 2D. This enables us to explore both the apical and basolateral sides, retaining the crucial polarity of the cells. Funding Agencies CIHR

Cyclosporine-A induced cytotoxicity within HepG2 cells by inhibiting PXR mediated CYP3A4/CYP3A5/MRP2 pathway

Cyclosporine-A (CsA) is currently used to treat immune rejection after organ transplantation as a commonly used immunosuppressant. Liver injury is one of the most common adverse effects of CsA, whose precise mechanism has not been fully elucidated. Pregnane X receptor (PXR) plays a critical role in mediating drug-induced liver injury as a key regulator of drug and xenobiotic clearance. As a nuclear receptor, PXR transcriptionally upregulates the expression of drug-metabolizing enzymes and drug transporters, including cytochrome P4503A (CPY3A) and multidrug resistance-associated protein 2 (MRP2). Our study established CsA-induced cytotoxic hepatocytes in an in vitro model, demonstrating that CsA dose-dependently increased the aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) level secreted in the HepG2 cell supernatant, as well as viability and oxidative stress of HepG2 cells. CsA also dose-dependently decreased the PXR, CYP3A4, CPY3A5, and MRP2 levels of HepG2 cells. Mechanistically, altering the expression of PXR, CYP3A4, CYP3A5, and MRP2 affected the impact of CsA on AST and LDH levels. Moreover, altering the expression of PXR also changed the level of CYP3A4, CPY3A5, and MRP2 of HepG2 cells treated by CsA. Our presented findings provide experimental evidence that CsA-induced liver injury is PXR tightly related. We suggest that PXR represents an attractive target for therapy of liver injury due to its central role in the regulation of the metabolizing enzymes CYP3A and MRP2-mediated bile acid transport and detoxification.

The Role of Adopted Orphan Nuclear Receptors in the Regulation of an Organic Anion Transporting Polypeptide 1B1 (OATP1B1) under the Action of Sex Hormones.

Organic anion transporting polypeptide 1B1 (OATP1B1) is an influx transporter protein of the SLC superfamily, expressed mainly in the liver and some tumor cells. The mechanisms of its regulation are being actively studied. In the present study, the effect of sex hormones (estradiol, progesterone and testosterone) on OATP1B1 expression in HepG2 cells was examined. The role of adopted orphan receptors, farnasoid X receptor (FXR), constitutive androstane receptor (CAR), pregnane X receptor (PXR) and liver X receptor subtype alpha (LXRa), was also evaluated. Hormones were used in concentrations of 1, 10 and 100 μM, with incubation for 24 h. The protein expression of OATP1B1, FXR, CAR, PXR and LXRa was analyzed by Western blot. It was shown that estradiol (10 and 100 μM) increased the expression of OATP1B1, acting through CAR. Testosterone (1, 10 and 100 μM) increased the expression of OATP1B1, acting through FXR, PXR and LXRa. Progesterone (10 and 100 μM) decreased the expression of OATP1B1 (10 and 100 μM) and adopted orphan receptors are not involved in this process. The obtained results have important practical significance and determine ways for targeted regulation of the transporter, in particular in cancer.

Resveratrol attenuates efavirenz-induced hepatic steatosis and hypercholesterolemia in mice by inhibiting pregnane X receptor activation and decreasing inflammation

Efavirenz (EFV), a widely prescribed antiviral medication, has been implicated in dyslipidemia and can activate the pregnane X receptor (PXR), leading to hepatic steatosis and hypercholesterolemia in mice. Resveratrol (RES) can ameliorate hepatic steatosis and functions as a partial PXR agonist, capable of mitigating PXR expression induced by other PXR agonists. Therefore, we hypothesized that RES could attenuate EFV-induced hepatic steatosis and hypercholesterolemia by downregulating PXR expression and suppressing inflammatory cytokine production. Here, we conducted an in vivo study involving 6-week-old male mice, which were divided into 4 groups for a 7-day intervention: control (carrier solution), EFV (80 mg/kg), RES (50 mg/kg), and RES + EFV groups. Serum and hepatic tissue samples were collected to assess cholesterol and triglyceride concentrations. Hepatic lipid accumulation was evaluated through hematoxylin-eosin and oil red O staining. Polymerase chain reaction and western blot were performed to quantify hepatic inflammatory factors, lipogenic gene, and PXR expression. Our results indicated that hepatic lipid droplet accumulation was reduced in the RES + EFV group compared with the EFV group. Similarly, the expressions of hepatic inflammatory factors were attenuated in the RES + EFV group relative to the EFV group. Furthermore, RES counteracted the upregulation of hepatic lipid-metabolizing enzymes induced by EFV at both the transcriptional and protein levels. Importantly, PXR expression was downregulated in the RES + EFV group compared with the EFV group. Conclusively, our findings suggest that RES effectively mitigates EFV-induced hepatic steatosis and hypercholesterolemia by inhibiting PXR activation and decreasing inflammation.

Identifying hepatoprotective mechanism and effective components of Yinchenzhufu decoction in chronic cholestatic liver injury using a comprehensive strategy based on metabolomics, molecular biology, pharmacokinetics, and cytology

Ethnopharmacological relevanceIn Traditional Chinese Medicine (TCM), cholestasis liver disease belongs to jaundice. Yinchenzhufu decoction (YCZFD) is a classic formula used for treating jaundice. Aim of the studyThis study was aimed to investigate the potential mechanism and effective components of YCZFD in chronic cholestatic liver injury (CCLI). Materials and methodsA chronic cholestatic mouse model induced by 3, 5-diethoxycarbonyl-1, 4-dihydroxychollidine was used to investigate the effect of YCZFD. Then, metabolomics was used to investigate the metabolites influenced by YCZFD. Serum and liver bile acid (BA) levels were measured using liquid chromatography coupled with triple quadruple mass spectrometry (LC-MS/MS), and the gene and protein expressions of BA transporters and metabolic enzymes were detected. Additionally, the pharmacokinetics of multiple components of YCZFD was explored to clarify the potential effective components. The effects of absorbed components of YCZFD on BA metabolism and transporter function, inflammation, and farnesoid X receptor (FXR) and pregnane X receptor (PXR) activation were analyzed using sandwich cultured rat hepatocytes, AML12 cells, and dual-luciferase receptor systems, respectively. ResultsYCZFD decreased the liver damage in chronic cholestatic mice. Serum metabolomics results indicated that the main pathways influenced by YCZFD involved primary BA biosynthesis and arachidonic acid metabolism. YCZFD upregulated the expression of FXR, PXR, and BA efflux transporters and the metabolic enzymes of liver tissues, promoting BA excretion and metabolism in cholestatic mice. Additionally, YCZFD downregulated the expression of genes and proteins of the toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-κB) pathway and decreased liver inflammation. The pharmacokinetic study indicated that multiple components showed different pharmacokinetic properties. Among the absorbed components of YCZFD, multiple components activated the transcription of FXR and PXR, regulated BA transporters and metabolic enzyme function, and reduced the gene expression of TLR4 and NF-κB1. ConclusionYCZFD can ameliorate CCLI by promoting the excretion and metabolism of BAs and inhibiting inflammation via the TLR4/NF-κB signaling pathway. The multiple components of YCZFD could act on BA homeostasis regulation and anti-inflammation, exhibiting a combined effect against CCLI.

Pregnane X receptor (PXR) deficiency protects against spinal cord injury by activating NRF2/HO-1 pathway.

As a devastating neurological disease, spinal cord injury (SCI) results in severe tissue loss and neurological dysfunction. Pregnane X receptor (PXR) is a ligand-activated nuclear receptor with a major regulatory role in xenobiotic and endobiotic metabolism and recently has been implicated in the central nervous system. In the present study, we aimed to investigate the role and mechanism of PXR in SCI. The clip-compressive SCI model was performed in male wild-type C57BL/6 (PXR+/+ ) and PXR-knockout (PXR-/- ) mice. The N2a H2 O2 -induced injury model mimicked the pathological process of SCI in vitro. Pregnenolone 16α-carbonitrile (PCN), a mouse-specific PXR agonist, was used to activate PXR in vivo and in vitro. The siRNA was applied to knock down the PXR expression in vitro. Transcriptome sequencing analysis was performed to discover the relevant mechanism, and the NRF2 inhibitor ML385 was used to validate the involvement of PXR in influencing the NRF2/HO-1 pathway in the SCI process. The expression of PXR decreased after SCI and reached a minimum on the third day. In vivo, PXR knockout significantly improved the motor function of mice after SCI, meanwhile, inhibited apoptosis, inflammation, and oxidative stress induced by SCI. On the contrary, activation of PXR by PCN negatively influenced the recovery of SCI. Mechanistically, transcriptome sequencing analysis revealed that PXR activation downregulated the mRNA level of heme oxygenase-1 (HO-1) after SCI. We further verified that PXR deficiency activated the NRF2/HO-1 pathway and PXR activation inhibited this pathway in vitro. PXR is involved in the recovery of motor function after SCI by regulating NRF2/HO-1 pathway.

In vitro and in silico assessment of endocrine disrupting effects of food contaminants through pregnane X receptor.

As a promiscuous xenobiotic receptor, pregnane X receptor (PXR) has been confirmed to participate in numerous physiological process. In addition to the conventional estrogen/androgen receptor, PXR also serves as an alternative target for environmental chemical contaminants. In this work, the PXR-mediated endocrine disrupting effects of typical food contaminants were explored. Firstly, the time-resolved fluorescence resonance energy transfer assays confirmed the PXR binding affinities of 2,2',4,4',5,5'-hexachlorobiphenyl, bis(2-ethylhexyl) phthalate, dibutyl phthalate, chlorpyrifos, bisphenol A, and zearalenone, with IC50 values ranging from 1.88 to 4284.00nM. Then their PXR agonist activities were assessed by PXR-mediated CYP3A4 reporter gene assays. Subsequently, the regulation of gene expressions of PXR and its targets CYP3A4, UGT1A1, and MDR1 by these compounds was further investigated. Intriguingly, all the tested compounds interfered with these gene expressions, confirming their endocrine disrupting effects via PXR-mediated signaling. The compound-PXR-LBD binding interactions were explored by molecular docking and molecular dynamics simulations to unravel the structural basis of their PXR binding capacities. The weak intermolecular interactions are key players in stabilizing these compound-PXR-LBD complexes. During the simulation process, 2,2',4,4',5,5'-hexachlorobiphenyl remained stable while the other 5 compounds underwent relatively severe disturbances. In conclusion, these food contaminants might exhibit endocrine disrupting effects via PXR.

Effects of atorvastatin on the Sirtuin/PXR signaling pathway in Mugilogobius chulae.

Atorvastatin (ATV) is a hypolipidemic drug widely detected in the aquatic environment. Nevertheless, limited information is provided about the toxic effects of ATV on estuary or coastal species and the underlying mechanisms. In the present study, the responses of genes expression in pregnane X receptor (PXR) signaling pathway and enzymatic activities in the liver of the estuarine benthic fish (Mugilogobius chulae) were investigated under acute and sub-chronic ATV exposure. Results showed that PXR was significantly inhibited in the highest exposure concentration of ATV for a shorter time (24h, 500μg L-1) but induced in a lower concentration (72h, 5μg L-1). The downstream genes in PXR signaling pathway such as CYP3A, SULT, UGT, and GST showed similar trends to PXR. P-gp and MRP1 were repressed in most treatments. GCLC associated with GSH synthesis was mostly induced under ATV exposure for a long time (168h), suggesting that reactive oxygen species (ROS) were generated under ATV exposure. Similarly, GST and SOD enzymatic activities significantly increased in most exposure treatments. Under ATV exposure, SIRT1 and SIRT2 displayed induction to some extent in most treatments, suggesting that SIRTs may affect PXR expression by regulating the acetylation levels of PXR. The investigation demonstrated that ATV exposure affected the expression of the Sirtuin/PXR signaling pathway, thus further interfered adaption of M. chulae to the environment.

TPX2 enhances the transcription factor activation of PXR and enhances the resistance of hepatocellular carcinoma cells to antitumor drugs

The pregnane X receptor (PXR) is an important regulator of hepatocellular carcinoma cellular resistance to antitumor drugs. Activation of PXR was modulated by the co-regulators. The target protein for the Xenopus plus end-directed kinesin-like protein (Xklp2) known as TPX2 that was previously considered as a tubulin regulator, also functions as the regulator of some transcription factors and pro-oncogenes in human malignances. However, the actions of TPX2 on PXR and HCC cells are still unclear. In the present study, our results demonstrate that the high expression of endogenous mRNA level of TPX2 not only correlated with the poor prognosis of advanced HCC patients who received sorafenib treatment but also with expression of PXR’s downstream genes, cyp3a4 and/or mdr-1. Results from luciferase and real-time polymerase chain reaction (qPCR) showed that TPX2 leads to enhancement of the transcription factor activation of PXR. Protein–protein interactions between PXR and TPX2 were identified using co-immunoprecipitation. Mechanically, overexpression of TPX2 led to enhancement of PXR recruitment to its downstream gene cyp3a4’s promoter region (the PXRE region) or enhancer region (the XREM region). Treatment of HCC cells with paclitaxel, a microtubule promoter, led to enhancement of the effects of TPX2, whereas vincristine, a microtubule depolymerizing agent caused a decrease in TPX2-associated effects. TPX2 was found to cause acceleration of the metabolism or clearance of sorafenib, a typical tyrosine kinase inhibitor (TKI) in HCC cells and in turn led to the resistance to sorafenib by HCC cells. By establishing novel actions of TXP2 on PXR in HCC cells, the results indicate that TPX2 could be considered a promising therapeutic target to enhance HCC cells sensitivity to antitumor drugs.

Pregnane X receptor gene variant rs7643645 and total mortality in subjects with nonalcoholic fatty liver disease.

Pregnane X receptor (PXR) gene variants rs7643645 and rs2461823 are reported to associate with clinically and histologically more severe liver injury in nonalcoholic fatty liver disease (NAFLD). It is known that the more progressive the NAFLD, the higher the hepatic and extra-hepatic mortality and morbidity. Thus, we investigated the total mortality in Finnish middle-aged ultrasonographically verified NAFLD patients with PXR rs7643645 AA/AG ( n = 217) or GG ( n = 27) variants and rs2461823 CC/CT ( n = 215) or TT ( n = 27) variants. In up to 30 years of follow-up, PXR rs7643645 GG subjects were at an increased risk of total mortality compared with AA/AG subjects, 1.676 (1.014-2.772), P = 0.044. The statistically significant difference prevailed after multiple adjustments for potentially confounding factors, RR, 2.024 (1.191-3.440), P = 0.009. In the subjects without NAFLD ( n = 731), the mortality risk was not associated with rs7643645 variants, 1.051 (0.708-1.560; P = 0.804). There was no difference in the total mortality between the PXR rs2461823 variant subgroups, 1.141 (0.663-1.962; P = 0.634). As the rs7643645 G variant disrupts a putative hepatocyte nuclear factor 4α binding site located in the PXR gene promoter and is associated with lower hepatic expression of PXR and its target genes, our result suggests that genetic disruption of xenobiotic metabolism increases mortality in subjects with NAFLD. Further studies are needed to confirm the results of the present study.

Screening Method for the Identification of Compounds That Activate Pregnane X Receptor.

The pregnane X receptor (PXR) is a nuclear receptor found mainly in the liver and intestine, whose main function is to regulate the expression of drug-metabolizing enzymes and transporters. Recently, it has been noted that PXR plays critical roles in energy homeostasis, immune response, and cancer. Therefore, identifying chemicals or compounds that can modulate PXR is of great interest, as these can result in downstream toxicity or, alternatively, may have therapeutic potential. Testing one compound at a time for PXR activity would be inefficient and take thousands of hours for large compound libraries. Here, we describe a high-throughput screening method that encompasses plating and treating HepG2-CYP3A4-hPXR cells in a 1536-well plate, as well as reading and interpreting assay (e.g., luciferase reporter gene activity) endpoints. These cells are stably transfected with a human PXR expression vector and CYP3A4-promoter-driven luciferase reporter vector, allowing the identification of compounds that activate PXR through cytochrome 450 3A4. We also describe how to analyze the data from each assay and explain follow-up steps, namely pharmacological characterization and quantitative polymerase chain reaction (qPCR) assays, which can be performed to confirm results from the original screen. These methods can be used to identify and confirm hPXR activators after completion of a compound screening. Published 2022. This article is a U.S. Government work and is in the public domain in the USA. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Establishment of a high-throughput assay to identify hPXR activators Basic Protocol 2: Quantitative high-throughput screening a compound library to classify hPXR activators Basic Protocol 3: Performing pharmacological characterization and qPCR assays to confirm hPXR activators.

Comparison of Sweated and Non-Sweated Ethanol Extracts of Salvia miltiorrhiza Bge. (Danshen) Effects on Human and Rat Hepatic UDP-Glucuronosyltransferase and Preclinic Herb-Drug Interaction Potential Evaluation.

The ethanol of Danshen (DEE) preparation has been widely used to treat cardiac-cerebral disease and cancer. Sweating is one of the primary processing methods of Danshen, which greatly influences its quality and pharmacological properties. Sweated and non-sweated DEE preparation combined with various synthetic drugs, add up the possibility of herbal-drug interactions. This study explored the effects of sweated and non-sweated DEE on human and rat hepatic UGT enzyme expression and activity and proposed a potential mechanism. The expression of two processed DEE on rat UGT1A, UGT2B, and nuclear receptors, including pregnane X receptor (PXR), constitutive androstane receptor (CAR), and peroxisome proliferator-activated receptor α (PPARα), were investigated after intragastric administration in rats by Western blot. Enzyme activity of DEE and its active ingredients (Tanshinone I, Cryptotanshinone, and Tanshinone I) on UGT isoenzymes was evaluated by quantifying probe substrate metabolism and metabolite formation in vitro using Ultra Performance Liquid Chromatography. The two processed DEE (5.40 g/kg) improved UGT1A (P<0.01) and UGT2B (P<0.05) protein expression, and the non-sweated DEE (2.70 g/kg) upregulated UGT2B expression protein (P<0.05), compared with the CMCNa group. On day 28, UGT1A protein expression was increased (P<0.05) both in two processed DEE groups meanwhile, the non-sweated DEE significantly enhanced UGT2B protein expression (P<0.05) on day 21, compared with the CMCNa group. The process underlying this mechanism involved the activation of nuclear receptors CAR, PXR, and PPARα. In vitro, sweated DEE (0-80 μg/mL) significantly inhibited the activity of human UGT1A7 (P<0.05) and rat UGT1A1, 1A8, and 1A9 (P<0.05). Non-sweated DEE (0-80 μg/mL) dramatically suppressed the activity of human UGT1A1, 1A3, 1A6, 1A7, 2B4, and 2B15, and rat UGT1A1, 1A3, 1A7, and 1A9 (P<0.05). Tanshinone I (0-1 μM) inhibited the activity of human UGT1A3, 1A6, and 1A7 (P<0.01) and rat UGT1A3, 1A6, 1A7, and 1A8 (P<0.05). Cryptotanshinone (0-1 μM) remarkably inhibited the activity of human UGT1A3 and 1A7 (P<0.05) and rat UGT1A7, 1A8, and 1A9 (P<0.05). Nonetheless, Tanshinone IIA (0-2 μM) is not a potent UGT inhibitor both in humans and rats. Additionally, there existed significant differences between two processed DEE in the expression of PXR, and the activity of human UGT1A1, 1A3, 1A6, and 2B15 and rat UGT1A3, and 2B15 (P<0.05). The effects of two processed DEE on hepatic UGT enzyme expression and activity differed. Accordingly, the combined usage of related UGTs substrates with DEE and its monomer components preparations may call for caution, depending on the drug's exposure-response relationship and dose adjustment. Besides, it is vital to pay attention to the distinction between sweated and non-sweated Danshen in clinic, which influences its pharmacological activity.

Long Noncoding RNAs Hepatocyte Nuclear Factor 4A Antisense RNA 1 and Hepatocyte Nuclear Factor 1A Antisense RNA 1 are Involved in Ritonavir‐induced Cytotoxicity in Hepatoma Cells

Ritonavir (RTV), a pharmacoenhancer used in anti‐HIV regimens, can induce liver damage. RTV is mainly metabolized by cytochrome P450 3A4 (CYP3A4) in the liver. HNF4A antisense RNA 1 (HNF4A‐AS1) and HNF1A antisense RNA 1 (HNF1A‐AS1) are long noncoding RNAs (lncRNAs) that regulate the expression of pregnane X receptor (PXR) and CYP3A4. This study investigated the role and molecular mechanisms of HNF4A‐AS1 and HNF1A‐AS1 in RTV‐induced hepatotoxicity. Small hairpin RNAs were used to knock down HNF4A‐AS1 and HNF1A‐AS1 in Huh7 and HepG2 cells. Lactate dehydrogenase and reactive oxygen species assays were performed to assess RTV‐induced hepatotoxicity. Chromatin immunoprecipitation qPCR was used to detect PXR enrichment and histone modifications in the CYP3A4 promoter. HNF4A‐AS1 knockdown increased PXR and CYP3A4 expression and exacerbated RTV‐induced cytotoxicity, whereas HNF1A‐AS1 knockdown generated the opposite phenotype. Mechanistically, enrichment of PXR and trimethylation of histone 3 lysine 4 (H3K4me3) in the CYP3A4 promoter was increased, and trimethylation of histone 3 lysine 27 (H3K27me3) was decreased after HNF4A‐AS1 knockdown. However, PXR and H3K4me3 enrichment decreased after HNF1A‐AS1 knockdown. Alterations in RTV‐induced hepatotoxicity caused by decreasing HNF4A‐AS1 or HNF1A‐AS1 were reversed by knockdown or overexpression of PXR. Increased susceptibility to RTV‐induced liver injury caused by the PXR activator rifampicin was attenuated by HNF4A‐AS1 overexpression or HNF1A‐AS1 knockdown. Taken together, these results revealed that HNF4A‐AS1 and HNF1A‐AS1 modulated RTV‐induced hepatotoxicity by regulating CYP3A4 expression, primarily by affecting the binding of PXR and histone modification status in the CYP3A4 promoter.

Gardenia jasminoides Ellis polysaccharide ameliorates cholestatic liver injury by alleviating gut microbiota dysbiosis and inhibiting the TLR4/NF-κB signaling pathway.

Gardenia jasminoides Ellis is a well-known herbal medicine. In this study, the effect of G. jasminoides Ellis polysaccharide (GPS) on liver injury in an alpha-naphthylisothiocyanate (ANIT)-induced cholestatic mouse model and the associated molecular mechanisms were investigated. GPS administration dose-dependently ameliorated impaired hepatic function, including a 2-7-fold decrease in aminotransferase levels, ameliorating tissue damage, upregulating the expression of farnesoid X receptor (FXR) and pregnane X receptor (PXR) and their downstream efflux transporters, and decreasing the levels of 12 bile acids (BAs), in cholestatic mice. Furthermore, GPS ameliorated gut microbiota dysbiosis, improved intestinal barrier function, and reduced serum and hepatic lipopolysaccharide levels 1.5-fold. GPS also inhibited the Toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-κB) signaling, decreased the expression of inflammatory factor genes, and ameliorated hepatic inflammation. Notably, fecal microbiota transplantation from GPS-fed mice also increased the hepatic expression of FXR, PXR, and efflux transporters; decreased the levels of 12 BAs; restored intestinal barrier function; and decreased hepatic inflammation mediated by the TLR4/NF-κB pathway. In conclusion, GPS has a protective effect against cholestatic liver injury through modulation of gut microbiota and inhibition of the TLR4/NF-κB pathway. Regulating gut microbiota using herbal medicine polysaccharides may hold unique therapeutic promise for cholestatic liver diseases.

Niclosamide induces miR-148a to inhibit PXR and sensitize colon cancer stem cells to chemotherapy.

SummaryTumor recurrence is often attributed to cancer stem cells (CSCs). We previously demonstrated that down-regulation of Pregnane X Receptor (PXR) decreases the chemoresistance of CSCs and prevents colorectal cancer recurrence. Currently, no PXR inhibitor is usable in clinic. Here, we identify miR-148a as a targetable element upstream of PXR signaling in CSCs, which when over-expressed decreases PXR expression and impairs tumor relapse after chemotherapy in mouse tumor xenografts. We then develop a fluorescent reporter screen for miR-148a activators and identify the anti-helminthic drug niclosamide as an inducer of miR-148a expression. Consequently, niclosamide decreased PXR expression and CSC numbers in colorectal cancer patient-derived cell lines and synergized with chemotherapeutic agents to prevent CSC chemoresistance and tumor recurrence in vivo. Our study suggests that endogenous miRNA inducers is a viable strategy to down-regulate PXR and illuminates niclosamide as a neoadjuvant repurposing strategy to prevent tumor relapse in colon cancer.

Resveratrol ameliorates nutritional steatohepatitis through the mmu‑miR‑599/PXR pathway.

The aim of the present study was to elucidate the effect of resveratrol on non-alcoholic steatohepatitis (NASH), and the molecular basis in mice and Hepa1-6 cells, in order to verify its therapeutic effect. C57BL/6J mice were fed a methionine-choline-deficient (MCD) diet to induce steatohepatitis and were treated with resveratrol. Mouse sera were collected for biochemical analysis and enzyme-linked immunosorbent assay, and livers were obtained for histological observation, and mmu-microRNA (miR)-599 and inflammation-related gene expression analysis. Hepa1-6 cells were treated with palmitic acid to establish a NASH cell model, and were then treated with resveratrol, or transfected with mmu-miR-599 mimic, mmu-miR-599 inhibitor or recombinant pregnane X receptor (PXR) plasmid. Subsequently, the cells were collected for mmu-miR-599 and inflammation-related gene expression analysis. Reverse transcription-quantitative polymerase chain reaction and western blotting were used to assess mmu-miR-599 expression levels, and the mRNA and protein expression levels of PXR and inflammation-related genes. The binding site of mmu-miR-599 in the PXR mRNA was verified by the luciferase activity assay. Mice fed an MCD diet for 4 weeks exhibited steatosis, focal necrosis and inflammatory infiltration in the liver. Resveratrol significantly reduced serum aminotransferase and malondialdehyde levels, and ameliorated hepatic injury. These effects were associated with reduced mmu-miR-599 expression, enhanced PXR expression, and downregulated levels of nuclear factor-κB, tumour necrosis factor-α, interleukin (IL)-1β, IL-6, NOD-like receptor family pyrin domain-containing protein 3 and signal transducer and activator of transcription 3. Administration of the mmu-miR-599 mimic inhibited PXR expression in Hepa1-6 cells, whereas the mmu-miR-599 inhibitor exerted the opposite effect. A binding site for mmu-miR-599 was identified in the PXR mRNA sequence. Furthermore, overexpression of PXR inhibited the expression of inflammatory factors in Hepa1-6 cells. The present study provided evidence for the protective role of resveratrol in ameliorating steatohepatitis through regulating the mmu-miR-599/PXR pathway and the consequent suppression of related inflammatory factors. Resveratrol may serve as a potential candidate for steatohepatitis management.

The Nuclear Receptor PXR in Chronic Liver Disease.

Pregnane X receptor (PXR), a nuclear receptor known for modulating the transcription of drug metabolizing enzymes and transporters (DMETs), such as cytochrome P450 3A4 and P-glycoprotein, is functionally involved in chronic liver diseases of different etiologies. Furthermore, PXR activity relates to that of other NRs, such as constitutive androstane receptor (CAR), through a crosstalk that in turn orchestrates a complex network of responses. Thus, besides regulating DMETs, PXR signaling is involved in both liver damage progression and repair and in the neoplastic transition to hepatocellular carcinoma. We here summarize the present knowledge about PXR expression and function in chronic liver diseases characterized by different etiologies and clinical outcome, focusing on the molecular pathways involved in PXR activity. Although many molecular details of these finely tuned networks still need to be fully understood, we conclude that PXR and its modulation could represent a promising pharmacological target for the identification of novel therapeutical approaches to chronic liver diseases.