RESUMO
The farnesoid-X-receptor (FXR) is validated target in the cholestatic disorders treatment. Obeticholic acid (OCA), the first in class of FXR agonist approved for clinical use, causes side effects including acute liver decompensation when administered to cirrhotic patients with primary biliary cholangitis at higher than recommended doses. The V-Maf avian-musculoaponeurotic-fibrosarcoma-oncogene-homolog-G (Mafg) and nuclear factor-erythroid-2-related-factor-2 (Nrf2) mediates some of the downstream effects of FXR. In the present study we have investigated the role of FXR/MafG/NRF2 pathway in the development of liver toxicity caused by OCA in rodent models of cholestasis. Cholestasis was induced by bile duct ligation (BDL) or administration of α-naphtyl-isothiocyanate (ANIT) to male Wistar rats and FXR-/- and FXR+/+ mice. Treating BDL and ANIT rats with OCA exacerbated the severity of cholestasis, hepatocytes injury and severely downregulated the expression of basolateral transporters. In mice, genetic ablation FXR or its pharmacological inhibition by 3-(naphthalen-2-yl)-5-(piperidin-4-yl)-1,2,4-oxadiazole rescued from negative regulation of MRP4 and protected against liver injury caused by ANIT. By RNAseq analysis we found that FXR antagonism effectively reversed the transcription of over 2100 genes modulated by OCA/ANIT treatment, including Mafg and Nrf2 and their target genes Cyp7a1, Cyp8b1, Mat1a, Mat2a, Gss. Genetic and pharmacological Mafg inhibition by liver delivery of siRNA antisense or S-adenosylmethionine effectively rescued from damage caused by ANIT/OCA. In contrast, Nrf2 induction by sulforaphane was protective. CONCLUSIONS: Liver injury caused by FXR agonism in cholestasis is FXR-dependent and is reversed by FXR and Mafg antagonism or Nrf2 induction.
Assuntos
Ácido Quenodesoxicólico/análogos & derivados , Colestase/metabolismo , Hepatopatias/metabolismo , Receptores Citoplasmáticos e Nucleares/agonistas , Animais , Ácido Quenodesoxicólico/farmacologia , Colestase/complicações , Colestase/genética , Células Hep G2 , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Hepatopatias/etiologia , Hepatopatias/genética , Fator de Transcrição MafG/antagonistas & inibidores , Fator de Transcrição MafG/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 2 Relacionado a NF-E2/agonistas , Fator 2 Relacionado a NF-E2/genética , Ratos Wistar , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/genéticaRESUMO
The cysteinyl leukotrienes (CysLTs), i.e. LTC4, LTD4 and LTE4, are a family of proinflammatory agents synthesized from the arachidonic acid. In target cells, these lipid mediators bind to the cysteinyl leukotriene receptors (CysLTR), a family of seven transmembrane G-protein coupled receptors. The CysLT1R is a validated target for treatment of pulmonary diseases and several selective antagonists for this receptor, including montelukast, zafirlukast and pranlukast, have shown effective in the management of asthma. Nevertheless, others CysLT1R antagonists, such as the alpha-pentyl-3-[2-quinolinylmethoxy] benzyl alcohol (REV5901), have been extensively characterized without reaching sufficient priority for clinical development. Since drug reposition is an efficient approach for maximizing investment in drug discovery, we have investigated whether CysLT1R antagonists might exert off-target effects. In the report we demonstrate that REV5901 interacts with GPBAR1, a well characterized cell membrane receptor for secondary bile acids. REV5901 transactivates GPBAR1 in GPBAR1-transfected cells with an EC50 of 2.5 µM and accommodates the GPBAR1 binding site as shown by in silico analysis. Exposure of macrophages to REV5901 abrogates the inflammatory response elicited by bacterial endotoxin in a GPBAR1-dependent manner. In vivo, in contrast to montelukast, REV5901 attenuates inflammation and immune dysfunction in rodent models of colitis. The beneficial effects exerted by REV5901 in these models were abrogated by GPBAR1 gene ablation, confirming that REV5901, a shelved CysLT1R antagonist, is a GPBAR1 ligand. These data ground the basis for the development of novel hybrid ligands designed for simultaneous modulation of CysTL1R and GPBAR1.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Colite/tratamento farmacológico , Antagonistas de Leucotrienos/farmacologia , Quinolinas/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Leucotrienos/metabolismo , Acetatos/farmacologia , Animais , Ácidos e Sais Biliares/farmacologia , Colite/genética , Colite/metabolismo , Colite/patologia , Ciclopropanos , Modelos Animais de Doenças , Expressão Gênica , Genes Reporter , Células HEK293 , Células Hep G2 , Humanos , Leucotrieno C4/metabolismo , Leucotrieno D4/metabolismo , Leucotrieno E4/metabolismo , Luciferases/genética , Luciferases/metabolismo , Camundongos , Camundongos Knockout , Simulação de Acoplamento Molecular , Células RAW 264.7 , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Receptores de Leucotrienos/química , Receptores de Leucotrienos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , SulfetosRESUMO
Drug-induced liver injury caused by acetaminophen (acetyl-para-aminophenol [APAP]) is the main cause of acute liver failure and liver transplantation in several Western countries. Whereas direct toxicity exerted by APAP metabolites is a key determinant for early hepatocytes injury, the recruitment of cells of innate immunity exerts a mechanistic role in disease progression, determining the clinical outcomes. GPBAR1 is a G protein-coupled receptor for secondary bile acids placed at the interface between liver sinusoidal cells and innate immunity. In this report, using genetic and pharmacological approaches, we demonstrate that whereas Gpbar1 gene deletion worsens the severity of liver injury, its pharmacological activation by 6ß-ethyl-3a,7b-dihydroxy-5b-cholan-24-ol rescues mice from liver injury caused by APAP. This protective effect was supported by a robust attenuation of liver recruitment of monocyte-derived macrophages and their repolarization toward an anti-inflammatory phenotype. Macrophage depletion by gadolinium chloride pretreatment abrogated disease development, whereas their reconstitution by spleen-derived macrophage transplantation restored the sensitivity to APAP in a GPBAR1-dependent manner. RNA sequencing analyses demonstrated that GPBAR1 agonism modulated the expression of multiple pathways, including the chemokine CCL2 and its receptor, CCR2. Treating wild-type mice with an anti-CCL2 mAb attenuated the severity of liver injury. We demonstrated that negative regulation of CCL2 production by GPBAR1 agonism was promoter dependent and involved FOXO1. In conclusion, we have shown that GPBAR1 is an upstream modulator of CCL2/CCR2 axis at the sinusoidal cell/macrophage interface, providing a novel target in the treatment of liver damage caused by APAP.
Assuntos
Capilares/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Quimiocina CCL2/metabolismo , Fígado/metabolismo , Macrófagos/metabolismo , Receptores CCR2/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Acetaminofen/farmacologia , Animais , Ácidos e Sais Biliares/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Proteína Forkhead Box O1/metabolismo , Células Hep G2 , Humanos , Fígado/efeitos dos fármacos , Camundongos , Regiões Promotoras Genéticas/fisiologia , Células RAW 264.7 , Transdução de Sinais/fisiologia , Baço/efeitos dos fármacos , Baço/metabolismo , Células THP-1RESUMO
Pelargonidins are anthocyanidins thought to be beneficial for the human health, although controversies exist over the doses needed and the unclear mechanism of action, along with poor systemic bioavailability. One putative target of pelargonidins is the aryl hydrocarbon receptor (AhR). A synthetic pelargonidin (Mt-P) was synthesized by the methylation of the pelargonidin (the natural compound indicated as P). Mt-P transactivated the AhR with an EC50 of 1.97 µM and was ~2-fold more potent than the natural compound. In vitro Mt-P attenuated pro-inflammatory activities of Raw264.7 macrophage cells in an AhR-dependent manner. In vivo, administration of the Mt-P in Balb/c mice resulted in a dose-dependent attenuation of signs and symptoms of colitis induced by TNBS. A dose of 5 mg/kg Mt-P, but not the natural compound P, reversed intestinal inflammation and increased expression of Tnf-α, Ifn-Æ´, and Il-6, while promoted the expansion of regulatory T cells and M2 macrophages. In C57BL/6J mice fed a high fat diet (HFD), Mt-P attenuated body weight gain, intestinal and liver inflammation, and ameliorated insulin sensitivity, while worsened liver steatosis by up-regulating the liver expression of Cd36 and Apo100b. These effects were abrogated by AhR gene ablation. Mt-P is a synthetic pelargonidin endowed with robust AhR agonist activity that exerts beneficial effects in murine models of inflammation and metabolic dysfunction.
Assuntos
Antocianinas/farmacologia , Anti-Inflamatórios/farmacologia , Inflamação/tratamento farmacológico , Receptores de Hidrocarboneto Arílico/fisiologia , Animais , Antocianinas/química , Células CACO-2 , Colite/induzido quimicamente , Colite/tratamento farmacológico , Fígado Gorduroso/tratamento farmacológico , Células Hep G2 , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/fisiologia , Metilação , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células RAW 264.7 , Receptores de Hidrocarboneto Arílico/efeitos dos fármacos , Receptores de Hidrocarboneto Arílico/genéticaRESUMO
BACKGROUND & AIMS: GPBAR1, also known as TGR5, is a G protein-coupled receptor activated by bile acids. Hepatic innate immune cells are involved in the immunopathogenesis of human liver diseases and in several murine hepatitis models. Here, by using genetic and pharmacological approaches, we provide evidence that GPBAR1 ligation attenuates the inflammation in rodent models of hepatitis. MATERIAL AND METHODS: Hepatitis was induced by concanavalin A (Con A) or α-galactosyl-ceramide (α-GalCer). 6b-Ethyl-3a,7b-dihydroxy-5b-cholan-24-ol (BAR501), a selective agonist of GPBAR1, was administrated by o.s. RESULTS: In the mouse models of hepatitis, the genetic ablation of Gpabar1 worsened the severity of liver injury and resulted in a type I NKT cells phenotype that was biased toward a NKT1, a proinflammatory, IFN-γ producing, NKT cells subtype. Further on, NKT cells from GPBAR1-/- mice were sufficient to cause a severe hepatitis when transferred to naïve mice. In contrast, GPBAR1 agonism rescued wild-type mice from acute liver damage and redirects the NKT cells polarization toward a NKT10, a regulatory, IL-10 secreting, type I NKT cell subset. In addition, GPBAR1 agonism significantly expanded the subset of IL-10 secreting type II NKT cells. RNAseq analysis of both NKT cells type confirmed that IL-10 is a major target for GPABR1. Accordingly, IL-10 gene ablation abrogated protection afforded by GPBAR1 agonism in the Con A model. CONCLUSION: Present results illustrate a role for GPBAR1 in regulating liver NKT ecology. Because NKT cells are an essential component of liver immune system, our data provide a compelling evidence for a GPBAR1-IL-10 axis in regulating of liver immunity.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/genética , Células T Matadoras Naturais/metabolismo , Receptores Acoplados a Proteínas G/genética , Animais , Linhagem Celular , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Colestanóis/efeitos adversos , Concanavalina A/efeitos adversos , Modelos Animais de Doenças , Galactosilceramidas/efeitos adversos , Células Hep G2 , Hepatite , Humanos , Interleucina-10/metabolismo , Masculino , Camundongos , Células T Matadoras Naturais/citologia , Células RAW 264.7 , Receptores Acoplados a Proteínas G/metabolismoRESUMO
A growing body of evidence suggests that probiotic functionality is not accurately predicted by their taxonomy. Here, we have set up a study to investigate the effectiveness of two probiotic formulations containing a blend of seven bacterial species in modulating intestinal inflammation in two rodent models of colitis, induced by treating mice with 2,4,6-Trinitrobenzenesulfonic acid (TNBS) or dextran sodium sulfate (DSS). Despite the taxonomy of the bacterial species in the two probiotic formulations being similar, only one preparation (Blend 2-Vivomixx) effectively attenuated the development of colitis in both models. In the TNBS model of colitis, Blend 2 reduced the expression of pro-inflammatory genes while increasing the production of anti-inflammatory cytokines, promoting the expansion M2 macrophages and the formation of IL-10-producing Treg cells in the colon's lamina propria. In the DSS model of colitis, disease attenuation and Treg formation was observed only in mice administered with Blend 2, and this effect was associated with intestinal microbiota remodeling and increased formation of lactate, butyrate, and propionate. None of these effects were observed in mice administered with Blend 1 (VSL#3). In summary, we have shown that two probiotic mixtures obtained by combining taxonomically similar species produced with different manufacturing methods exert divergent effects in mouse models of colitis.
Assuntos
Bactérias , Colite/microbiologia , Colo/microbiologia , Microbioma Gastrointestinal , Mucosa Intestinal/microbiologia , Probióticos , Animais , Bactérias/genética , Bactérias/crescimento & desenvolvimento , Bactérias/metabolismo , Colite/induzido quimicamente , Colite/metabolismo , Colite/prevenção & controle , Colo/metabolismo , Colo/patologia , Sulfato de Dextrana , Modelos Animais de Doenças , Ácidos Graxos Voláteis/metabolismo , Inflamação/induzido quimicamente , Inflamação/metabolismo , Inflamação/microbiologia , Inflamação/prevenção & controle , Doenças Inflamatórias Intestinais , Interleucina-10/metabolismo , Mucosa Intestinal/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos BALB C , Probióticos/uso terapêutico , Especificidade da Espécie , Linfócitos T Reguladores/metabolismo , Ácido TrinitrobenzenossulfônicoRESUMO
Indole-3-carbinol (I3C), autolysis product of glucosinolates present in cruciferous vegetables, has been indicated as a promising agent in preventing the development and progression of breast cancer. I3C has been shown to inhibit the growth of human cancer cells in vitro and possesses anticarcinogenic activity in vivo. Because I3C is unstable and may be converted into many polymeric products in the digestive tract, it is not yet clear whether the biological activity observed can be attributed to I3C or some of its polymeric products. In this study we synthesized a stable I3C cyclic tetrameric derivative and investigated its effects on a panel of human breast cancer cell lines. The I3C tetramer suppressed the growth of both estrogen receptor (ER) -positive (MCF-7, 734B, and BT474) and ER-negative (BT20, MDA-MB-231, and BT539) human breast cancer cell lines, and it was found to induce G(1) cell cycle arrest in a dose-dependent manner without evidence of apoptosis, suggesting a growth arrest via a cytostatic mechanism. At the molecular level, the tetramer inhibited cyclin-dependent kinase (CDK) 6 expression and activity, induced an increase in the level of p27(kip1), and reduced the level of retinoblastoma protein expression. Contrarily to CDK6, the level of CDK4, the other kinase involved in the G(1) phase of the cell cycle, remains unchanged. Interestingly, the tetramer resulted about five times more active than I3C in suppressing the growth of human breast cancer cells. On the whole, our data suggest that the I3C tetrameric derivative is a novel lead inhibitor of breast cancer cell growth that may be a considered a new, promising therapeutic agent for both ER+ and ER- breast cancer.