Cholestatic liver diseases are characterized by intrahepatic accumulation of bile acids (BAs), exacerbating liver inflammation, and fibrosis. Dimethyl fumarate (DMF) is a clinically approved antiinflammatory drug that demonstrated protective effects in several experimental models of liver injury. Still, its effect on BA homeostasis and liver fibrosis has not been thoroughly studied. Herein, we hypothesized that DMF could improve BA homeostasis and mitigate the progression of cholestasis-induced liver fibrosis. The DMF was administered to mice with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-induced cholestasis for four weeks. The content of individual BAs in the plasma, liver, bile, intestine, and feces was measured using the LC-MS method alongside the analysis of liver phenotype and related executive and regulatory pathways. The DMF slowed down the progression of DDC-induced liver fibrosis by suppressing hepatic stellate cell (HSC) and macrophage activation, and by reducing c-Jun N-terminal kinase (JNK) phosphorylation. Notably, DMF reduced BA cumulation in the plasma and liver of cholestatic mice by increasing BA fecal excretion via their reduced Bacteroidetes phyla-mediated deconjugation in the intestine. In addition, DMF was identified as the antagonist of the mouse FXR receptor in enterocytes. In conclusion, DMF alleviates DDC-induced cholestatic liver injury through pleiotropic action leading to significant anti-inflammatory and antifibrotic activity of the agent. Additionally, DMF mitigates BA retention in the liver and plasma by increasing their fecal excretion in cholestatic mice. These findings suggest that DMF warrants further investigation as a potential therapeutic agent for human chronic fibrosing cholestatic liver disorders.

• Klíčová slova
• 3,5-Diethoxycarbonyl-1,4-Dihydrocollidine (DDC), Bile acids, Cholestasis, Dimethyl fumarate, Liver fibrosis,
• Publikační typ
• časopisecké články MeSH

The Takeda G protein-coupled receptor 5 (TGR5), also known as GPBAR1 (G protein-coupled bile acid receptor), is a membrane-type bile acid receptor that regulates blood glucose levels and energy expenditure. These essential functions make TGR5 a promising target for the treatment of type 2 diabetes and metabolic disorders. Currently, most research on developing TGR5 agonists focuses on modifying the structure of bile acids, which are the endogenous ligands of TGR5. However, TGR5 agonists with nonsteroidal structures have not been widely explored. This study aimed at discovering new TGR5 agonists using bile acid derivatives as a basis for a computational approach. We applied a combination of pharmacophore-based, molecular docking, and molecular dynamic (MD) simulation to identify potential compounds as new TGR5 agonists. Through pharmacophore screening and molecular docking, we identified 41 candidate compounds. From these, five candidates were selected based on criteria including pharmacophore features, a docking score of less than 9.2 kcal/mol, and similarity in essential interaction patterns with a reference ligand. Biological assays of the five hits confirmed that Hit-3 activates TGR5 similarly to the bile acid control. This was supported by MD simulation results, which indicated that a hydrogen bond interaction with Tyr240 is involved in TGR5 activation. Hit-3 (CSC089939231) represents a new nonsteroidal lead that can be further optimized to design potent TGR5 agonists.

• Klíčová slova
• INT‐777, TGR5, molecular docking, nonbile acid, pharmacophore,
• MeSH
• lidé MeSH
• ligandy MeSH
• molekulární struktura MeSH
• objevování léků MeSH
• receptory spřažené s G-proteiny * agonisté   metabolismus   MeSH
• simulace molekulární dynamiky * MeSH
• simulace molekulového dockingu * MeSH
• vztahy mezi strukturou a aktivitou MeSH
• žlučové kyseliny a soli chemie   metabolismus   farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• GPBAR1 protein, human MeSH Prohlížeč
• ligandy MeSH
• receptory spřažené s G-proteiny * MeSH
• žlučové kyseliny a soli MeSH

Carvedilol is a widely used beta-adrenoreceptor antagonist for multiple cardiovascular indications; however, it may induce cholestasis in patients, but the mechanism for this effect is unclear. Carvedilol also prevents the development of various forms of experimental liver injury, but its effect on nonalcoholic steatohepatitis (NASH) is largely unknown. In this study, we determined the effect of carvedilol (10 mg/kg/day p.o.) on bile formation and bile acid (BA) turnover in male C57BL/6 mice consuming either a chow diet or a western-type NASH-inducing diet. BAs were profiled by liquid chromatography-mass spectrometry and BA-related enzymes, transporters, and regulators were evaluated by western blot analysis and qRT-PCR. In chow diet-fed mice, carvedilol increased plasma concentrations of BAs resulting from reduced BA uptake to hepatocytes via Ntcp transporter downregulation. Inhibition of the β-adrenoreceptor-cAMP-Epac1-Ntcp pathway by carvedilol may be the post-transcriptional mechanism underlying this effect. In contrast, carvedilol did not worsen the deterioration of BA homeostasis accompanying NASH; however, it shifted the spectra of BAs toward more hydrophilic and less toxic α-muricholic and hyocholic acids. This positive effect of carvedilol was associated with a significant attenuation of liver steatosis, inflammation, and fibrosis in NASH mice. In conclusion, our results indicate that carvedilol may increase BAs in plasma by modifying their liver transport. In addition, carvedilol provided significant hepatoprotection in a NASH murine model without worsening BA accumulation. These data suggest beneficial effects of carvedilol in patients at high risk for developing NASH.

• Klíčová slova
• bile acids, carvedilol, nonalcoholic steatohepatitis,
• MeSH
• homeostáza MeSH
• játra MeSH
• karvedilol farmakologie   metabolismus   MeSH
• lidé MeSH
• membránové transportní proteiny metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nealkoholová steatóza jater * metabolismus   MeSH
• žlučové kyseliny a soli metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• karvedilol MeSH
• membránové transportní proteiny MeSH
• žlučové kyseliny a soli MeSH

The nuclear constitutive androstane receptor (CAR, NR1I3) plays significant roles in many hepatic functions, such as fatty acid oxidation, biotransformation, liver regeneration, as well as clearance of steroid hormones, cholesterol, and bilirubin. CAR has been proposed as a hypothetical target receptor for metabolic or liver disease therapy. Currently known prototype high-affinity human CAR agonists such as CITCO (6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime) have limited selectivity, activating the pregnane X receptor (PXR) receptor, a related receptor of the NR1I subfamily. We have discovered several derivatives of 3-(1H-1,2,3-triazol-4-yl)imidazo[1,2-a]pyridine that directly activate human CAR in nanomolar concentrations. While compound 39 regulates CAR target genes in humanized CAR mice as well as human hepatocytes, it does not activate other nuclear receptors and is nontoxic in cellular and genotoxic assays as well as in rodent toxicity studies. Our findings concerning potent human CAR agonists with in vivo activity reinforce the role of CAR as a possible therapeutic target.

• MeSH
• hepatocyty účinky léků   metabolismus   MeSH
• konstitutivní androstanový receptor * agonisté   chemie   MeSH
• lidé MeSH
• myši MeSH
• pyridiny farmakologie   MeSH
• receptory cytoplazmatické a nukleární metabolismus   MeSH
• steroidní receptory * agonisté   chemie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• konstitutivní androstanový receptor * MeSH
• pyridiny MeSH
• receptory cytoplazmatické a nukleární MeSH
• steroidní receptory * MeSH

Farnesoid X receptor (FXR) is a nuclear receptor with an essential role in regulating bile acid synthesis and cholesterol homeostasis. FXR activation by agonists is explained by an αAF-2-trapping mechanism; however, antagonism mechanisms are diverse. We discuss microsecond molecular dynamics (MD) simulations investigating our recently reported FXR antagonists 2a and 2 h. We study the antagonist-induced conformational changes in the FXR ligand-binding domain, when compared to the synthetic (GW4064) or steroidal (chenodeoxycholic acid, CDCA) FXR agonists in the FXR monomer or FXR/RXR heterodimer r, and in the presence and absence of the coactivator. Our MD data suggest ligand-specific influence on conformations of different FXR-LBD regions, including the α5/α6 region, αAF-2, and α9-11. Changes in the heterodimerization interface induced by antagonists seem to be associated with αAF-2 destabilization, which prevents both co-activator and co-repressor recruitment. Our results provide new insights into the conformational behaviour of FXR, suggesting that FXR antagonism/agonism shift requires a deeper assessment than originally proposed by crystal structures.

• Klíčová slova
• bile acids, farnesoid X receptor, molecular dynamics simulations, nuclear receptors,
• MeSH
• DNA vazebné proteiny * chemie   MeSH
• kyselina chenodeoxycholová farmakologie   MeSH
• ligandy MeSH
• receptory cytoplazmatické a nukleární MeSH
• transkripční faktory * metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA vazebné proteiny * MeSH
• kyselina chenodeoxycholová MeSH
• ligandy MeSH
• receptory cytoplazmatické a nukleární MeSH
• transkripční faktory * MeSH

Bile acids (BAs) are key signaling steroidal molecules that regulate glucose, lipid, and energy homeostasis via interactions with the farnesoid X receptor (FXR) and G-protein bile acid receptor 1 (GPBAR1). Extensive medicinal chemistry modifications of the BA scaffold led to the discovery of potent selective or dual FXR and GPBAR1 agonists. Herein, we discovered 7-ethylidene-lithocholic acid (7-ELCA) as a novel combined FXR antagonist/GPBAR1 agonist (IC50 = 15 μM/EC50 = 26 nM) with no off-target activation in a library of 7-alkyl substituted derivatives of BAs. 7-ELCA significantly suppressed the effect of the FXR agonist obeticholic acid in BSEP and SHP regulation in human hepatocytes. Importantly, 7-ELCA significantly stimulated the production of glucagon-like peptide-1 (GLP-1), an incretin with insulinotropic effect in postprandial glucose utilization, in intestinal enteroendocrine cells. We can suggest that 7-ELCA may be a prospective approach to the treatment of type II diabetes as the dual modulation of GPBAR1 and FXR has been supposed to be effective in the synergistic regulation of glucose homeostasis in the intestine.

• Klíčová slova
• G protein-coupled bile acid receptor 1, bile acids, farnesoid X receptor, metabolism, steroid,
• Publikační typ
• časopisecké články MeSH

Bile acids (BA) play a significant role in the pathophysiology of nonalcoholic steatohepatitis (NASH). The present study evaluates the modulation of bile acid metabolomics by atorvastatin, a cholesterol-lowering agent commonly used to treat cardiovascular complications accompanying NASH. NASH was induced in mice by 24 weeks of consuming a high-saturated fat, high-fructose, and high-cholesterol diet (F), with atorvastatin administered orally (20 mg/kg/day) during the last three weeks. Biochemical and histological analyses confirmed the effectiveness of the F diet in inducing NASH. Untreated NASH animals had significantly reduced biliary secretion of BA and increased fecal excretion of BA via decreased apical sodium-dependent bile salt transporter (Asbt)-mediated reabsorption. Atorvastatin decreased liver steatosis and inflammation in NASH animals consistently with a reduction in crucial lipogenic enzyme stearoyl-coenzyme A (CoA) desaturase-1 and nuclear factor kappa light chain enhancer of activated B-cell pro-inflammatory signaling, respectively. In this group, atorvastatin also uniformly enhanced plasma concentration, biliary secretion and fecal excretion of the secondary BA, deoxycholic acid (DCA). However, in the chow diet-fed animals, atorvastatin decreased plasma concentrations of BA, and reduced BA biliary secretions. These changes stemmed primarily from the increased fecal excretion of BA resulting from the reduced Asbt-mediated BA reabsorption in the ileum and suppression of synthesis in the liver. In conclusion, our results reveal that atorvastatin significantly modulates BA metabolomics by altering their intestinal processing and liver synthesis in control and NASH mice.

• Klíčová slova
• apical sodium-dependent bile acid transporter, atorvastatin, bile acids, deoxycholic acid, nonalcoholic steatohepatitis,
• MeSH
• atorvastatin farmakologie   MeSH
• biologické markery MeSH
• biologické modely MeSH
• dieta s vysokým obsahem tuků MeSH
• homeostáza * MeSH
• játra metabolismus   MeSH
• modely nemocí na zvířatech MeSH
• myši MeSH
• nealkoholová steatóza jater farmakoterapie   etiologie   metabolismus   patologie   MeSH
• statiny farmakologie   MeSH
• triglyceridy biosyntéza   MeSH
• žlučové kyseliny a soli metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• atorvastatin MeSH
• biologické markery MeSH
• statiny MeSH
• triglyceridy MeSH
• žlučové kyseliny a soli MeSH

Macrophages possess an innate ability to scavenge heterogenous objects from the systemic circulation and to regulate inflammatory diseases in various organs via cytokine production. That makes them attractive targets for nanomedicine-based therapeutic approaches to inflammatory diseases. In the present study, we have prepared several different poly(lactic-co-glycolic acid) (PLGA) polymer nanospheres for macrophage-targeted drug delivery using both nanoprecipitation and emulsification solvent evaporation methods. Two experimental linear PLGA polymers with relatively low molar weight, one experimental branched PLGA with unique star-like molecular architecture, and a commercially available PLGA, were used for nanosphere formulation and compared to their macrophage uptake capacity. The nanosphere formulations labelled with loaded fluorescent dye Rhodamine B were further tested in mouse bone marrow-derived macrophages and in hepatocyte cell lines AML-12, HepG2. We found that nanospheres larger than 100 nm prepared using nanoprecipitation significantly enhanced distribution of fluorescent dye selectively into macrophages. No effects of nanospheres on cellular viability were observed. Additionally, no significant proinflammatory effect after macrophage exposure to nanospheres was detected as assessed by a determination of proinflammatory cytokines Il-1β and Tnfα mRNA. All experimental PLGA nanoformulations surpassed the nanospheres obtained with the commercially available polymer taken as a control in their capacity as macrophage-specific carriers.

• Klíčová slova
• PLGA, drug delivery, drug targeting, hepatic disease, inflammation, liver, macrophages, nanoparticles, nanoprecipitation, nanospheres,
• Publikační typ
• časopisecké články MeSH

The constitutive androstane receptor (CAR) is the essential regulator of genes involved both in xenobiotic and endobiotic metabolism. Diazepam has been shown as a potent stimulator of CAR nuclear translocation and is assumed as an indirect CAR activator not interacting with the CAR cavity. In this study, we sought to determine if diazepam is a ligand directly interacting with the CAR ligand binding domain (LBD) and if it regulates its target genes in a therapeutically relevant concentration. We used different CAR constructs in translocation and luciferase reporter assays, recombinant CAR-LBD in a TR-FRET assay, and target genes induction studied in primary human hepatocytes (PHHs), HepaRG cells, and in CAR humanized mice. We also used in silico docking and CAR-LBD mutants to characterize the interaction of diazepam and its metabolites with the CAR cavity. Diazepam and its metabolites such as nordazepam, temazepam, and oxazepam are activators of CAR+Ala in translocation and two-hybrid assays and fit the CAR cavity in docking experiments. In gene reporter assays with CAR3 and in the TR-FRET assay, only diazepam significantly interacts with CAR-LBD. Diazepam also promotes up-regulation of CYP2B6 in PHHs and in HepaRG cells. However, in humanized CAR mice, diazepam significantly induces neither CYP2B6 nor Cyp2b10 genes nor does it regulate critical genes involved in glucose and lipids metabolism and liver proliferation. Thus, we demonstrate that diazepam interacts with human CAR-LBD as a weak ligand, but it does not significantly affect expression of tested CAR target genes in CAR humanized mice.

• Klíčová slova
• CAR, NR1I3, cytochrome P450, diazepam, drug interaction, gene regulation,
• MeSH
• buněčné linie MeSH
• diazepam farmakologie   MeSH
• dospělí MeSH
• hepatocyty účinky léků   MeSH
• játra účinky léků   MeSH
• konstitutivní androstanový receptor MeSH
• lidé středního věku MeSH
• lidé MeSH
• ligandy MeSH
• myši MeSH
• proliferace buněk účinky léků   MeSH
• proteinové domény účinky léků   MeSH
• receptory cytoplazmatické a nukleární metabolismus   MeSH
• reportérové geny účinky léků   genetika   MeSH
• transport proteinů účinky léků   MeSH
• zvířata MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• diazepam MeSH
• konstitutivní androstanový receptor MeSH
• ligandy MeSH
• NR1I3 protein, human MeSH Prohlížeč
• Nr1i3 protein, mouse MeSH Prohlížeč
• receptory cytoplazmatické a nukleární MeSH

Bile acids (BAs) are important signaling molecules acting via the farnesoid X nuclear receptor (FXR) and the membrane G protein-coupled bile acid receptor 1 (GPBAR1). Besides deconjugation of BAs, the oxidoreductive enzymes of colonic bacteria and hepatocytes enable the conversion of BAs into their epimers or dehydrogenated forms. Obeticholic acid (OCA) is the first-in-class BA-derived FXR agonist approved for the treatment of primary biliary cholangitis. Herein, a library of OCA derivatives, including 7-keto, 6-ethylidene derivatives and 3β-epimers, was synthetized and investigated in terms of interactions with FXR and GPBAR1 in transaction assays and evaluated for FXR target genes expression in human hepatocytes and C57BL/6 mice. The derivatives were further subjected to cell-free analysis employing in silico molecular docking and a TR-FRET assay. The conversion of the 3βhydroxy epimer and its pharmacokinetics in mice were studied using LC-MS. We found that only the 3β-hydroxy epimer of OCA (3β-isoOCA) possesses significant activity to FXR in hepatic cells and mice. However, in a cell-free assay, 3β-isoOCA had about 9-times lower affinity to FXR than did OCA. We observed that 3β-isoOCA readily epimerizes to OCA in hepatocytes and murine liver. This conversion was significantly inhibited by the hydroxy-Δ5-steroid dehydrogenase inhibitor trilostane. In addition, we found that 3,7-dehydroobeticholic acid is a potent GPBAR1 agonist. We conclude that 3β-isoOCA significantly activates FXR due to its epimerization to the more active OCA by hepatic metabolism. Other modifications as well as epimerization on the C3/C7 positions and the introduction of 6-ethylidene in the CDCA scaffold abrogate FXR agonism and alleviate GPBAR1 activation.

• Klíčová slova
• Bile acids, Gene regulation, Metabolism, Nuclear receptors, Pharmacokinetics,
• MeSH
• buněčné linie MeSH
• Cercopithecus aethiops MeSH
• isomerie MeSH
• játra metabolismus   MeSH
• kyselina chenodeoxycholová analogy a deriváty   farmakologie   MeSH
• lidé MeSH
• myši inbrední C57BL MeSH
• receptory cytoplazmatické a nukleární metabolismus   MeSH
• receptory spřažené s G-proteiny genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• farnesoid X-activated receptor MeSH Prohlížeč
• GPBAR1 protein, human MeSH Prohlížeč
• kyselina chenodeoxycholová MeSH
• receptory cytoplazmatické a nukleární MeSH
• receptory spřažené s G-proteiny MeSH