Pregnane X Receptor Activation Research Articles

Gut microbiota affects mPXR agonist PCN-induced hepatomegaly by regulating PXR and YAP activation.

Pregnane X receptor (PXR) is essential in the regulation of liver homeostasis and the gut microbiota is closely linked to liver physiological and pathological status. We previously found that activation of PXR significantly promotes liver enlargement through interaction with yes-associated protein (YAP). However, whether gut microbiota is contributed to PXR-induced hepatomegaly and the involved mechanisms remain unclear. In this study, C57BL/6 mice were administered the mouse-specific agonist 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 microbial. 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 experiment 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 mPXR agonist PCN-induced hepatomegaly. The treatment with an antibiotic cocktail (ABX) depletes the intestinal microbiota, leading to the impairment of liver enlargement caused by PCN. Besides, fecal microbiota transplantation (FMT) from PCN-treated mice induces liver enlargement. Further study revealed that gut microbiota is involved in hepatomegaly via regulating PXR and YAP activation.

PXR Activation Relieves Deoxynivalenol-Induced Liver Oxidative Stress Via Malat1 LncRNA m6A Demethylation.

Deoxynivalenol (DON) is a prevalent toxin causing severe liver damage through hepatocellular oxidative stress. However, the underlying mechanisms and effective therapeutic approaches remain unknown. Here, the unique role of the xenobiotic metabolism factor pregnane X receptor (PXR) in mediating DON-induced hepatocellular oxidative stress is investigated. Treatment with the PXR agonist 3-indole-propionic acid (IPA) alleviates DON-induced oxidative stress and liver injury both in vitro and in vivo. Mechanistically, it is discovered for the first time that PXR agonist IPA directly transactivates the m6A demethylase FTO expression, leading to site-specific demethylation and decreased abundance of YTHDC1-bound Malat1 lncRNA at single-nucleotide resolution. The diminished m6A modification of Malat1 lncRNA reduces its stability and augments antioxidant pathways governed by NRF2, consequently mitigating DON-induced liver injury. Furthermore, Malat1 knockout mice exhibit decreased DON-induced liver injury, emphasizing the role of Malat1 lncRNA in oxidative stress. Collectively, the findings establish that PXR-mediated m6A-dependent Malat1 lncRNA expression determines hepatocyte oxidative stress via m6A demethylase FTO, providing valuable insights into the potential mechanisms underlying DON-induced liver injury and offers potential therapeutic strategies for its treatment.

Pregnane X Receptor Signaling Pathway and Vitamin K: Molecular Mechanisms and Clinical Relevance in Human Health

This review explores the likely clinical impact of Pregnane X Receptor (PXR) activation by vitamin K on human health. PXR, initially recognized as a master regulator of xenobiotic metabolism in liver, emerges as a key regulator influencing intestinal homeostasis, inflammation, oxidative stress, and autophagy. The activation of PXR by vitamin K highlights its role as a potent endogenous and local agonist with diverse clinical implications. Recent research suggests that the vitamin K-mediated activation of PXR highlights this vitamin’s potential in addressing pathophysiological conditions by promoting hepatic detoxification, fortifying gut barrier integrity, and controlling pro-inflammatory and apoptotic pathways. PXR activation by vitamin K provides an intricate association with cancer cell survival, particularly in colorectal and liver cancers, to provide new insights into potential novel therapeutic strategies. Understanding the clinical implications of PXR activation by vitamin K bridges molecular mechanisms with health outcomes, further offering personalized therapeutic approaches for complex diseases.

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

S-nitrosylation attenuates pregnane X receptor hyperactivity and acetaminophen-induced liver injury.

Drug-induced liver injury (DILI), especially acetaminophen overdose, is the leading cause of acute liver failure. Pregnane X receptor (PXR) is a nuclear receptor and the master regulator of drug metabolism. Aberrant activation of PXR plays a pathogenic role in the acetaminophen hepatotoxicity. Here, we aimed to examine the S-nitrosylation of PXR (SNO-PXR) in response to acetaminophen. We found that PXR was S-nitrosylated in hepatocytes and the mouse livers after exposure to acetaminophen or S-nitrosoglutathione (GSNO). Mass spectrometry and site-directed mutagenesis identified the cysteine 307 as the primary residue for S-nitrosylation (SNO) modification. In hepatocytes, SNO suppressed both agonist-induced (rifampicin and SR12813) and constitutively active PXR (VP-PXR, a human PXR fused to the minimal transactivator domain of the herpes virus transcription factor VP16) activations. Furthermore, in acetaminophen-overdosed mouse livers, PXR protein was decreased at the centrilobular regions overlapping with increased SNO. In PXR-/- mice, replenishing the livers with the SNO-deficient PXR significantly aggravated hepatic necrosis, increased HMGB1 release, and exacerbated liver injury and inflammation. Particularly, we demonstrated that S-nitrosoglutathione reductase (GSNOR) inhibitor N6022 promoted hepatoprotection by increasing the levels of SNO-PXR. In conclusion, PXR is posttranslationally modified by SNO in hepatocytes in response to acetaminophen. This modification mitigated the acetaminophen-induced PXR hyperactivity. It may serve as a target for therapeutical intervention.

Activation of PXR causes drug interactions with Paxlovid in transgenic mice.

Paxlovid is a nirmatrelvir (NMV) and ritonavir (RTV) co-packaged medication used for the treatment of coronavirus disease 2019 (COVID-19). The active component of Paxlovid is NMV and RTV is a pharmacokinetic booster. Our work aimed to investigate the drug/herb-drug interactions associated with Paxlovid and provide mechanism-based guidance for the clinical use of Paxlovid. By using recombinant human cytochrome P450s (CYPs), we confirmed that CYP3A4 and 3A5 are the major enzymes responsible for NMV metabolism. The role of CYP3A in Paxlovid metabolism were further verified in Cyp3a-null mice, which showed that the deficiency of CYP3A significantly suppressed the metabolism of NMV and RTV. Pregnane X receptor (PXR) is a ligand-dependent transcription factor that upregulates CYP3A4/5 expression. We next explored the impact of drug- and herb-mediated PXR activation on Paxlovid metabolism in a transgenic mouse model expressing human PXR and CYP3A4/5. We found that PXR activation increased CYP3A4/5 expression, accelerated NMV metabolism, and reduced the systemic exposure of NMV. In summary, our work demonstrated that PXR activation can cause drug interactions with Paxlovid, suggesting that PXR-activating drugs and herbs should be used cautiously in COVID-19 patients receiving Paxlovid.

The F-box-only protein 44 regulates pregnane X receptor protein level by ubiquitination and degradation.

Pregnane X receptor (PXR) is a ligand-activated nuclear receptor that transcriptionally upregulates drug-metabolizing enzymes [e.g., cytochrome P450 3A4 (CYP3A4)] and transporters. Although the regulation of PXR target genes is well-characterized, less is known about the regulation of PXR protein level. By screening an RNAi library, we identified the F-box-only protein 44 (FBXO44) as a novel E3 ligase for PXR. PXR abundance increases upon knockdown of FBXO44, and, inversely, decreases upon overexpression of FBXO44. Further analysis revealed that FBXO44 interacts with PXR, leading to its ubiquitination and proteasomal degradation, and we determined that the F-box associated domain of FBXO44 and the ligand binding domain of PXR are required for the functional interaction. In summary, FBXO44 regulates PXR protein abundance, which has downstream consequences for CYP3A4 levels and drug-drug interactions. The results of this study provide new insight into the molecular mechanisms that regulate PXR protein level and activity and suggest the importance of considering how modulating E3 ubiquitin ligase activities will affect PXR-mediated drug metabolism.

Ligand flexibility and binding pocket malleability cooperate to allow selective PXR activation by analogs of a promiscuous nuclear receptor ligand

The human nuclear receptor (NR) family of transcription factors contains 48 proteins that bind lipophilic molecules. Approved NR therapies have had immense success treating various diseases, but lack of selectivity has hindered efforts to therapeutically target the majority of NRs due to unpredictable off-target effects. The synthetic ligand T0901317 was originally discovered as a potent agonist of liver X receptors (LXRα/β) but subsequently found to target additional NRs, with activation of pregnane X receptor (PXR) being as potent as that of LXRs. We previously showed that directed rigidity reduces PXR binding by T0901317 derivatives through unfavorable protein remodeling. Here, we use a similar approach to achieve selectivity for PXR over other T0901317-targeted NRs. One molecule, SJPYT-318, accomplishes selectivity by favorably utilizing PXR's flexible binding pocket and surprisingly binding in a new mode distinct from the parental T0901317. Our work provides a structure-guided framework to achieve NR selectivity from promiscuous compounds.

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.

Switching on/off aryl hydrocarbon receptor and pregnane X receptor activities by chemically modified tryptamines

Microbial indoles have been demonstrated as selective or dual agonists and ligands of the pregnane X receptor (PXR) and aryl hydrocarbon receptor (AhR). However, structural determinants of microbial indoles selectivity towards both receptors remain elusive. Here, we studied the effects of existing and newly synthesized derivatives of indole microbial metabolite tryptamine on the activity of AhR and PXR receptors. We show that the elongation of indolyl-3-alkaneamine chain, indole N-methylation and conversion of indolyl-3-alkaneamines to oleamides resulted in a major increase of PXR activity and in parallel loss of AhR activity. Using reporter gene assays, RT-PCR and TR-FRET techniques, we have characterized in detail the activation of PXR by novel indolyl-3-alkanyl-oleamides, 1-methyltryptamine and 1-methyltryptamine-acetamide. As a proof of concept, we demonstrated anti-inflammatory and epithelial barrier-protective activity of lead derivatives in intestinal Caco-2 cells, employing the measurement of expression of pro-inflammatory chemokines, tight junction genes, trans-epithelial electric resistance TEER, and dextran-FITC permeability assay. In conclusion, we show that a subtle chemical modifications of simple microbial indole metabolite tryptamine, leads to substantial changes in AhR and PXR agonist activities.

NcBAF enhances PXR-mediated transcriptional activation in the human and mouse liver

Pregnane X receptor (PXR) is one of the key regulators of drug metabolism, gluconeogenesis, and lipid synthesis in the human liver. Activation of PXR by drugs such as rifampicin, simvastatin, and efavirenz causes adverse reactions such as drug-drug interaction, hyperglycemia, and dyslipidemia. The inhibition of PXR activation has merit in preventing such adverse events. Here, we demonstrated that bromodomain containing protein 9 (BRD9), a component of non-canonical brahma-related gene 1-associated factor (ncBAF), one of the chromatin remodelers, interacts with PXR. Rifampicin-mediated induction of CYP3A4 expression was attenuated by iBRD9, an inhibitor of BRD9, in human primary hepatocytes and CYP3A/PXR-humanized mice, indicating that BRD9 enhances the transcriptional activation of PXR in vitro and in vivo. Chromatin immunoprecipitation assay reveled that iBRD9 treatment resulted in attenuation of the rifampicin-mediated binding of PXR to the CYP3A4 promoter region, suggesting that ncBAF functions to facilitate the binding of PXR to its response elements. Efavirenz-induced hepatic lipid accumulation was attenuated by iBRD9 in C57BL/6J mice, suggesting that the inhibition of BRD9 would be useful to reduce the risk of efavirenz-induced hepatic steatosis. Collectively, we found that inhibitors of BRD9, a component of ncBAF that plays a role in assisting transactivation by PXR, would be useful to reduce the risk of PXR-mediated adverse reactions.

Pregnane X receptor activation remodels glucose metabolism to promote NAFLD development in obese mice

ObjectiveBoth obesity and exposure to chemicals may induce non-alcoholic fatty liver disease (NAFLD). Pregnane X Receptor (PXR) is a central target of metabolism disrupting chemicals and disturbs hepatic glucose and lipid metabolism. We hypothesized that the metabolic consequences of PXR activation may be modified by existing obesity and associated metabolic dysfunction. MethodsWildtype and PXR knockout male mice were fed high-fat diet to induce obesity and metabolic dysfunction. PXR was activated with pregnenolone-16α-carbonitrile. Glucose metabolism, hepatosteatosis, insulin signaling, glucose uptake, liver glycogen, plasma and liver metabolomics, and liver, white adipose tissue, and muscle transcriptomics were investigated. ResultsPXR activation aggravated obesity-induced liver steatosis by promoting lipogenesis and inhibiting fatty acid disposal. Accordingly, hepatic insulin sensitivity was impaired and circulating alanine aminotransferase level increased. Lipid synthesis was facilitated by increased liver glucose uptake and utilization of glycogen reserves resulting in dissociation of hepatosteatosis and hepatic insulin resistance from the systemic glucose tolerance and insulin sensitivity. Furthermore, glucagon-induced hepatic glucose production was impaired. PXR deficiency did not protect from the metabolic manifestations of obesity, but the liver transcriptomics and metabolomics profiling suggest diminished activation of inflammation and less prominent changes in the overall metabolite profile. ConclusionsObesity and PXR activation by chemical exposure have a synergistic effect on NAFLD development. To support liver fat accumulation the PXR activation reorganizes glucose metabolism that seemingly improves systemic glucose metabolism. This implies that obese individuals, already predisposed to metabolic diseases, may be more susceptible to harmful metabolic effects of PXR-activating drugs and environmental chemicals.

NEAT1_2 and DAZAP1, Paraspeckle Components, Interact with PXR to Negatively Regulate CYP3A4 Induction.

Human pregnane X receptor (PXR) is a major nuclear receptor that upregulates the expression of drug-metabolizing enzymes such as CYP3A4. In our recent study, it was revealed that PXR interacts with DAZ-associated protein 1 (DAZAP1), which is an essential component of the paraspeckle, a membraneless nuclear body, and the interaction was disassociated by rifampicin, a ligand of PXR. The purpose of this study was to clarify the roles of paraspeckles in PXR-mediated transcriptional regulation. Immunoprecipitation assays using PXR-overexpressing HepG2 (ShP51) cells revealed that PXR interacts with not only DAZAP1 but also NEAT1_2, a long noncoding RNA included in the paraspeckle, and that the interaction between PXR and NEAT1_2 was disassociated by rifampicin. These results suggest that PXR is trapped in paraspeckles and that the activation of PXR by its ligands facilitates its disassociation from paraspeckles. Induction of CYP3A4 by rifampicin was significantly enhanced by the knockdown of NEAT1_2 or DAZAP1 in ShP51 cells and their parental HepG2 cells. A luciferase assay using a plasmid containing the PXR response elements of CYP3A4 revealed that the increased CYP3A4 induction by siNEAT1_2 or siDAZAP1 was due to the increased transactivation by PXR. These results suggest that paraspeckles play a role in trapping nuclear PXR in the absence of the ligand to negatively regulate transactivation of its downstream gene. Collectively, this is the first study to demonstrate that the paraspeckle components NEAT1_2 and DAZAP1 negatively regulate CYP3A4 induction by PXR. SIGNIFICANCE STATEMENT: This study revealed that PXR interacts with paraspeckle components NEAT1_2 and DAZAP1 to suppress CYP3A4 induction by PXR, and the interaction is dissociated by PXR ligands. This finding provides a novel concept that paraspeckles formed by liquid-liquid phase separation potentially affect drug metabolism via negative regulation of PXR function.

Pregnane X receptor activation alleviates renal fibrosis in mice via interacting with p53 and inhibiting the Wnt7a/β-catenin signaling.

Renal fibrosis is a common pathological feature of chronic kidney disease (CKD) with various etiologies, which seriously affects the structure and function of the kidney. Pregnane X receptor (PXR) is a member of the nuclear receptor superfamily and plays a critical role in regulating the genes related to xenobiotic and endobiotic metabolism in mammals. Previous studies show that PXR is expressed in the kidney and has protective effect against acute kidney injury (AKI). In this study, we investigated the role of PXR in CKD. Adenine diet-induced CKD (AD) model was established in wild-type and PXR humanized (hPXR) mice, respectively, which were treated with pregnenolone-16α-carbonitrile (PCN, 50 mg/kg, twice a week for 4 weeks) or rifampicin (RIF, 10 mg·kg-1·d-1, for 4 weeks). We showed that both PCN and RIF, which activated mouse and human PXR, respectively, improved renal function and attenuated renal fibrosis in the two types of AD mice. In addition, PCN treatment also alleviated renal fibrosis in unilateral ureter obstruction (UUO) mice. On the contrary, PXR gene deficiency exacerbated renal dysfunction and fibrosis in both adenine- and UUO-induced CKD mice. We found that PCN treatment suppressed the expression of the profibrotic Wnt7a and β-catenin in AD mice and in cultured mouse renal tubular epithelial cells treated with TGFβ1 in vitro. We demonstrated that PXR was colocalized and interacted with p53 in the nuclei of tubular epithelial cells. Overexpression of p53 increased the expression of Wnt7a, β-catenin and its downstream gene fibronectin. We further revealed that p53 bound to the promoter of Wnt7a gene to increase its transcription and β-catenin activation, leading to increased expression of the downstream profibrotic genes, which was inhibited by PXR. Taken together, PXR activation alleviates renal fibrosis in mice via interacting with p53 and inhibiting the Wnt7a/β-catenin signalingpathway.

Effects of perfluorooctanoic acid (PFOA) on gene expression profiles via nuclear receptors in HepaRG cells: Comparative study with in vitro transactivation assays

Perfluorooctanoic acid (PFOA), a synthetic perfluorinated eight-carbon organic chemical, induces hepatotoxicity in rodents, indicated increased liver weight, hepatocellular hypertrophy, necrosis, and peroxisome proliferation. Epidemiological studies have demonstrated the association between serum PFOA levels and various adverse effects. In this study, we investigated the gene expression profiles of human HepaRG cells exposed to 10 and 100 μM PFOA for 24 h. Treatment with 10 and 100 μM PFOA significantly modulated the expression of 190 and 996 genes, respectively. Genes upregulated or downregulated by 100 µM PFOA included peroxisome proliferator-activated receptor (PPAR) signaling genes related to lipid metabolism, adipocyte differentiation, and gluconeogenesis. Moreover, we identified the “Nuclear receptors-meta pathways” following the activation of other nuclear receptors: constitutive androstane receptor (CAR), pregnane X receptor (PXR) and farnesoid X receptor (FXR), as well as the transcription factor nuclear factor E2-related factor 2 (Nrf2). The expression levels of some target genes (CYP4A11, CYP2B6, CYP3A4, CYP7A1, and GPX2) of these nuclear receptors and Nrf2 were confirmed using quantitative reverse transcription polymerase chain reaction. Next, we performed transactivation assays using COS-7 and HEK293 cells to investigate whether these signaling-pathways were activated by the direct effects of PFOA on human PPARα, CAR, PXR, FXR and Nrf2. PFOA concentration-dependently activated PPARα, but not CAR, PXR, FXR, or Nrf2. Taken together, these results suggest that PFOA affects the hepatic transcriptomic responses of HepaRG cells through the direct activation of PPARα and indirect activation of CAR, PXR, FXR, and Nrf2. Our finding indicates that PPARα activation in the “Nuclear receptors-meta pathways” functions as a molecular initiating event for PFOA, and indirect activation of alternative nuclear receptors and Nrf2 also induce important molecular mechanisms in PFOA-induced human hepatotoxicity.

Comprehensive in vitro analysis evaluating the variable drug–drug interaction risk of rifampicin compared to rifabutin

Compared to rifampicin (600 mg/day), standard doses of rifabutin (300 mg/day) have a lower risk of drug–drug interactions due to induction of cytochrome P450 3A4 (CYP3A4) or P-glycoprotein (Pgp/ABCB1) mediated by the pregnane X receptor (PXR). However, clinical comparisons with equal rifamycin doses or in vitro experiments respecting actual intracellular concentrations are lacking. Thus, the genuine pharmacological differences and the potential molecular mechanisms of the discordant perpetrator effects are unknown. Consequently, the cellular uptake kinetics (mass spectrometry), PXR activation (luciferase reporter gene assays), and impact on CYP3A4 and Pgp/ABCB1 expression and activity (polymerase chain reaction, enzymatic assays, flow cytometry) were evaluated in LS180 cells after treatment with different rifampicin or rifabutin concentrations for variable exposure times and eventually normalized to actual intracellular concentrations. In addition, inhibitory effects on CYP3A4 and Pgp activities were investigated. While rifampicin is poorly taken up by LS180 cells, it strongly activates PXR and leads to enhanced expression and activity of CYP3A4 and Pgp. In contrast, rifabutin is a significantly less potent and less efficient PXR activator and gene inducer, despite sixfold to eightfold higher intracellular accumulation. Finally, rifabutin is a potent inhibitor of Pgp (IC50 = 0.3 µM) compared to rifampicin (IC50 = 12.9 µM). Together, rifampicin and rifabutin significantly differ by their effects on the regulation and function of CYP3A4 and Pgp, even when controlled for intracellular concentrations. Rifabutin’s concurrent Pgp inhibitory action might partly compensate the inducing effects, explaining its weaker clinical perpetrator characteristics.

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.

Panaxytriol upregulates CYP3A4 expression through the interaction between nuclear regulators and DNA response elements.

Cytochrome P3A4 (CYP3A4) is a crucial drug-metabolizing enzyme, and its expression is regulated by the pregnane X receptor (PXR), constitutive androstane receptor (CAR), steroid receptor coactivator 1 (SRC-1), and acetyltransferase P300. Panaxytriol is a naturally derived active substance extracted from the roots of Panax ginseng C. A. Mey. which is widely used clinically. Our previous studies have shown that panaxytriol induces CYP3A4 expression through PXR activation, which is antagonized by high CAR expression. However, the underlying mechanism remains unclear. This study aimed to investigate the mechanism of panaxytriol in inducing CYP3A4 expression via interactions between nuclear regulators and DNA response elements. Immunoprecipitation technique was used to assess the binding levels of PXR and CAR with the coactivators SRC-1 and P300 in HepG2 and Huh-7cells. Furthermore, chromatin immunoprecipitation assay was used to investigate the PXR and CAR interaction with the CYP3A4 promoter response element ER-6/DR-3. The binding of PXR to SRC-1, P300, and the response elements ER-6 and DR-3 was improved with an increase in panaxytriol concentration (10-80μM), and the binding affinity was further enhanced upon CAR silencing. The binding of CAR to SRC-1 and the response elements ER-6 and DR-3 was significantly higher at 80μM panaxytriol, whereas no significant binding was observed between CAR and P300. Panaxytriol promoted the recruitment of PXR to SRC-1 and P300, binding to ER-6 and DR-3, and upregulating CYP3A4 expression. Furthermore, an interactive dialogue regulatory mechanism between PXR and CAR was observed.

Hepatic ZBTB22-mediated detoxification ameliorates acetaminophen-induced liver injury by inhibiting pregnane X receptor signaling.

Overdose acetaminophen (APAP) can cause acute liver injury (ALI), but the underlying mechanism remains undetermined. This study explored the role of hepatic Zinc Finger And BTB Domain Containing 22 (ZBTB22) in defense against APAP-mediated hepatotoxicity. The results showed that hepatic ZBTB22 expression was significantly reduced in patients with ALI and mice. In mouse primary hepatocytes (MPHs), ZBTB22 deletion aggravated APAP overdose-induced ALI, whereas ZBTB22 overexpression attenuated that pathological progression. The results were further verified in ZBTB22 over-express or knockout mice models. In parallel, hepatocyte-specific ZBTB22 knockout also enhanced ALI. Furthermore, ZBTB22 decreased pregnane X receptor (PXR) expression, and the PXR activator pregnane-16α-carbonitrile suppressed the protective effect of ZBTB22 in APAP-induced ZBTB22-overexpressing mice. Collectively, our findings highlight the protective effect of ZBTB22 against APAP-induced ALI and unravel PXR signaling as the potential mechanism. Strategies to increase hepatic ZBTB22 expression represent a promising therapeutic approach for APAP overdose-induced ALI.