RESUMEN
Liver fibrosis (LF) is a dangerous clinical condition with no available treatment. Inflammation plays a critical role in LF progression. Glucocorticoid-induced leucine zipper (GILZ, encoded in mice by the Tsc22d3 gene) mimics many of the anti-inflammatory effects of glucocorticoids, but its role in LF has not been directly addressed. Here, we found that GILZ deficiency in mice was associated with elevated CCL2 production and pro-inflammatory leukocyte infiltration at the early LF stage, resulting in enhanced LF development. RNA interference-mediated in vivo silencing of the CCL2 receptor CCR2 abolished the increased leukocyte recruitment and the associated hepatic stellate cell activation in the livers of GILZ knockout mice. To highlight the clinical relevance of these findings, we found that TSC22D3 mRNA expression was significantly downregulated and was inversely correlated with that of CCL2 in the liver samples of patients with LF. Altogether, these data demonstrate a protective role of GILZ in LF and uncover the mechanism, which can be targeted therapeutically. Therefore, modulating GILZ expression and its downstream targets represents a novel avenue for pharmacological intervention for treating LF and possibly other liver inflammatory disorders.
Asunto(s)
Quimiocina CCL2/metabolismo , Leucocitos/metabolismo , Cirrosis Hepática/metabolismo , Factores de Transcripción/metabolismo , Animales , Humanos , Leucocitos/patología , Cirrosis Hepática/patología , Masculino , Ratones , Ratones NoqueadosRESUMEN
Autophagy is a highly conserved catabolic process activated by fasting and caloric restriction. FXR, a receptor for primary bile acids, reverses the activity of cAMP-response element binding protein (CREB) on autophagy-related genes (Atg)s and terminates autophagy in the fed state. GPBAR1, a receptor for secondary bile acids, exerts its genomic effects via cAMP-CREB pathway. By genetic and pharmacological approaches, we have obtained evidence that GPBAR1 functions as a positive modulator of autophagy in liver and white adipose tissue (WAT) in fasting. Mechanistically, we found that Gpbar1-/- mice lack the expression of Cyp2c70 a gene essential for generation of muricholic acids which are FXR antagonists, and have an FXR-biased bile acid pool. Because FXR represses autophagy, Gpbar1-/- mice show a defective regulation of autophagy in fasting. BAR501, a selective GPBAR1 agonist, induces autophagy in fed mice. Defective regulation of autophagy in Gpbar1-/- could be reversed by FXR antagonism, while repression of autophagy by feeding was partially abrogated by FXR gene ablation, and FXR activation repressed Atgs in the fast state. BAR501 reversed the negative regulatory effects of feeding and FXR agonism on autophagy and promoted the recruitment of CREB to a CRE on the LC3 promoter. In mice exposed to chronic high caloric intake, GPBAR1 agonism ameliorated insulin sensitivity and induced Atgs expression in the liver and WAT. In summary, GPBAR1 is required for positive regulation of autophagy in fasting and its ligands reverse the repressive effects exerted on liver and WAT autophagy flow by FXR in fed.
Asunto(s)
Tejido Adiposo Blanco/metabolismo , Autofagia/efectos de los fármacos , Ácidos Cólicos/farmacología , Hígado/metabolismo , Receptores Citoplasmáticos y Nucleares , Receptores Acoplados a Proteínas G , Animales , Autofagia/genética , Ratones , Ratones Noqueados , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
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.
Asunto(s)
Ácido Quenodesoxicólico/análogos & derivados , Colestasis/metabolismo , Hepatopatías/metabolismo , Receptores Citoplasmáticos y Nucleares/agonistas , Animales , Ácido Quenodesoxicólico/farmacología , Colestasis/complicaciones , Colestasis/genética , Células Hep G2 , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Hepatopatías/etiología , Hepatopatías/genética , Factor de Transcripción MafG/antagonistas & inhibidores , Factor de Transcripción MafG/genética , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 2 Relacionado con NF-E2/agonistas , Factor 2 Relacionado con NF-E2/genética , Ratas Wistar , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/genéticaRESUMEN
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.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Colitis/tratamiento farmacológico , Antagonistas de Leucotrieno/farmacología , Quinolinas/farmacología , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Leucotrienos/metabolismo , Acetatos/farmacología , Animales , Ácidos y Sales Biliares/farmacología , Colitis/genética , Colitis/metabolismo , Colitis/patología , Ciclopropanos , Modelos Animales de Enfermedad , Expresión Génica , Genes Reporteros , Células HEK293 , Células Hep G2 , Humanos , Leucotrieno C4/metabolismo , Leucotrieno D4/metabolismo , Leucotrieno E4/metabolismo , Luciferasas/genética , Luciferasas/metabolismo , Ratones , Ratones Noqueados , Simulación del Acoplamiento Molecular , Células RAW 264.7 , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Receptores de Leucotrienos/química , Receptores de Leucotrienos/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , SulfurosRESUMEN
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.
Asunto(s)
Capilares/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Quimiocina CCL2/metabolismo , Hígado/metabolismo , Macrófagos/metabolismo , Receptores CCR2/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Acetaminofén/farmacología , Animales , Ácidos y Sales Biliares/metabolismo , Línea Celular , Línea Celular Tumoral , Proteína Forkhead Box O1/metabolismo , Células Hep G2 , Humanos , Hígado/efectos de los fármacos , Ratones , Regiones Promotoras Genéticas/fisiología , Células RAW 264.7 , Transducción de Señal/fisiología , Bazo/efectos de los fármacos , Bazo/metabolismo , Células THP-1RESUMEN
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.
Asunto(s)
Antocianinas/farmacología , Antiinflamatorios/farmacología , Inflamación/tratamiento farmacológico , Receptores de Hidrocarburo de Aril/fisiología , Animales , Antocianinas/química , Células CACO-2 , Colitis/inducido químicamente , Colitis/tratamiento farmacológico , Hígado Graso/tratamiento farmacológico , Células Hep G2 , Humanos , Macrófagos/efectos de los fármacos , Macrófagos/fisiología , Metilación , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Células RAW 264.7 , Receptores de Hidrocarburo de Aril/efectos de los fármacos , Receptores de Hidrocarburo de Aril/genéticaRESUMEN
Obeticholic acid (OCA) is a farnesoid-X-receptor (FXR) ligand, shown effective in reducing steatosis and fibrosis in NASH patients. However, OCA causes major side effects including pruritus, while increases the risk for liver decompensation in cirrhotic patients. Ursodeoxycholic acid (UDCA), is a safe and unexpensive bile acid used in the treatment of liver disorders whose mechanism of action is poorly defined. Here we have compared the effects of OCA and UDCA in a mouse model of NASH. In mice exposed to a diet rich in fat/cholesterol and fructose (HFD-F), treatment with OCA or UDCA effectively prevented body weight gain, insulin resistance, as demonstrated by OGTT, and AST plasma levels. After 12â¯weeks HFD-F mice developed liver microvesicular steatosis, inflammation and mild fibrosis, increased expression of inflammatory (TNFα, IL6, F4/80) and fibrosis (αSma, Col1α1, Tgfß) markers, reduced liver expression of FXR, dysregulated liver FXR signaling and elevated levels of Tauro-α and ß-muricholic acid (T-α and ßMCA), two FXR antagonists in mice. Both compounds prevented these changes and improved liver histopathology. OCA reduced primary bile acid synthesis worsening the T-CA/T-ßMCA ratio. UDCA effectively transactivated GPBAR1 in vitro. By RNAseq analysis we found that among over 2400 genes modulated by the HFD-F, only 32 and 60 genes were modulated by OCA and UDCA, with only 3 genes (Dbp, Adh7, Osgin1) being modulated by both agents. Both agents partially prevented the intestinal dysbiosis. CONCLUSIONS: UDCA is a GPBAR1 ligand and exerts beneficial effects in a rodent model of NASH by activating non-overlapping pathway with OCA.
Asunto(s)
Ácido Quenodesoxicólico/análogos & derivados , Disbiosis/tratamiento farmacológico , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Ácido Ursodesoxicólico/uso terapéutico , Animales , Ácido Quenodesoxicólico/uso terapéutico , Dieta/efectos adversos , Disbiosis/etiología , Disbiosis/metabolismo , Humanos , Masculino , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/etiología , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
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.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Células T Asesinas Naturales/metabolismo , Receptores Acoplados a Proteínas G/genética , Animales , Línea Celular , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Colestanoles/efectos adversos , Concanavalina A/efectos adversos , Modelos Animales de Enfermedad , Galactosilceramidas/efectos adversos , Células Hep G2 , Hepatitis , Humanos , Interleucina-10/metabolismo , Masculino , Ratones , Células T Asesinas Naturales/citología , Células RAW 264.7 , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
Non-alcoholic steatohepatitis (NASH) is a progressive, chronic, liver disease whose prevalence is growing worldwide. Despite several agents being under development for treating NASH, there are no drugs currently approved. The Farnesoid-x-receptor (FXR) and the G-protein coupled bile acid receptor 1 (GPBAR1), two bile acid activated receptors, have been investigated for their potential in treating NASH. Here we report that BAR502, a steroidal dual ligand for FXR/GPBAR1, attenuates development of clinical and liver histopathology features of NASH in mice fed a high fat diet (HFD) and fructose (F). By RNAseq analysis of liver transcriptome we found that BAR502 restores FXR signaling in the liver of mice feed HFD-F, and negatively regulates a cluster of genes including Srebf1 (Srepb1c) and its target genes-fatty acid synthase (Fasn) and Cell death-inducing DFF45-like effector (CIDE) genes, Cidea and Cidec-involved in lipid droplets formation and triglycerides storage in hepatocytes. Additionally, BAR502 increased the intestinal expression of Fgf15 and Glp1 and energy expenditure by white adipose tissues. Finally, exposure to BAR502 reshaped the intestinal microbiota by increasing the amount of Bacteroidaceae. In conclusion, we have shown that dual FXR/GPBAR1 agonism might have utility in treatment of NASH.
Asunto(s)
Colanos/uso terapéutico , Gotas Lipídicas/efectos de los fármacos , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Ácidos y Sales Biliares/metabolismo , Colanos/farmacología , Dieta Alta en Grasa/efectos adversos , Heces , Microbioma Gastrointestinal , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Ligandos , Gotas Lipídicas/metabolismo , Metabolismo de los Lípidos , Masculino , Ratones , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/etiología , Enfermedad del Hígado Graso no Alcohólico/patología , Receptores Citoplasmáticos y Nucleares/genética , Receptores Acoplados a Proteínas G/genéticaRESUMEN
Acetaminophen misuse is a leading cause of acute liver failure and liver transplantation for which therapy is poorly effective. FXR ligands have shown effective in reducing liver injury in several experimental and clinical settings. In this Letter, we have elaborated on the structure of GW4064, the first nonsteroidal agonist for FXR, to identify novel isoxazoles endowed with FXR agonistic activity and improved ADME properties. The pharmacological characterization and molecular docking studies for the structure-activity rationalization allowed the identification of several FXR agonists with nanomolar potency in transactivation and SRC-1 recruitment assays. This characterization resulted in the identification of a potent FXR agonist, compound 20 that was orally active, and rescued mice from acute liver failure caused by acetaminophen overdose in a FXR-dependent manner.
RESUMEN
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.
Asunto(s)
Bacterias , Colitis/microbiología , Colon/microbiología , Microbioma Gastrointestinal , Mucosa Intestinal/microbiología , Probióticos , Animales , Bacterias/genética , Bacterias/crecimiento & desarrollo , Bacterias/metabolismo , Colitis/inducido químicamente , Colitis/metabolismo , Colitis/prevención & control , Colon/metabolismo , Colon/patología , Sulfato de Dextran , Modelos Animales de Enfermedad , Ácidos Grasos Volátiles/metabolismo , Inflamación/inducido químicamente , Inflamación/metabolismo , Inflamación/microbiología , Inflamación/prevención & control , Enfermedades Inflamatorias del Intestino , Interleucina-10/metabolismo , Mucosa Intestinal/metabolismo , Macrófagos/metabolismo , Masculino , Ratones Endogámicos BALB C , Probióticos/uso terapéutico , Especificidad de la Especie , Linfocitos T Reguladores/metabolismo , Ácido TrinitrobencenosulfónicoRESUMEN
Nonalcoholic steatohepatitis (NASH) is associated with an increased risk of developing cardiovascular complications and mortality, suggesting that treatment of NASH might benefit from combined approaches that target the liver and the cardiovascular components of NASH. Using genetic and pharmacologic approaches, we show that G protein-coupled bile acid-activated receptor 1 (GPBAR1) agonism reverses liver and vascular damage in mouse models of NASH. NASH is associated with accelerated vascular inflammation representing an independent risk factor for development of cardiovascular diseases and cardiovascular-related mortality. GPBAR1, also known as TGR5, is a G protein-coupled receptor for secondary bile acids that reduces inflammation and promotes energy expenditure. Using genetic and pharmacologic approaches, we investigated whether GPBAR1 agonism by 6ß-ethyl-3α,7ß-dihydroxy-5ß-cholan-24-ol (BAR501) reverses liver and vascular damage induced by exposure to a diet enriched in fat and fructose (HFD-F). Treating HFD-F mice with BAR501 reversed liver injury and promoted the browning of white adipose tissue in a Gpbar1-dependent manner. Feeding HFD-F resulted in vascular damage, as shown by the increased aorta intima-media thickness and increased expression of inflammatory genes (IL-6,TNF-α, iNOS, and F4/80) and adhesion molecules (VCAM, intercellular adhesion molecule-1, and endothelial selectin) in the aorta, while reducing the expression of genes involved in NO and hydrogen sulfide generation, severely altering vasomotor activities of aortic rings in an ex vivo assay. BAR501 reversed this pattern in a Gpbar1-dependent manner, highlighting a potential role for GPBAR1 agonism in treating the liver and vascular component of NASH.-Carino, A., Marchianò, S., Biagioli, M., Bucci, M., Vellecco, V., Brancaleone, V., Fiorucci, C., Zampella, A., Monti, M. C., Distrutti, E., Fiorucci, S. Agonism for the bile acid receptor GPBAR1 reverses liver and vascular damage in a mouse model of steatohepatitis.
Asunto(s)
Colestanoles/farmacología , Modelos Animales de Enfermedad , Inflamación/prevención & control , Hepatopatías/prevención & control , Enfermedad del Hígado Graso no Alcohólico/fisiopatología , Receptores Acoplados a Proteínas G/agonistas , Enfermedades Vasculares/prevención & control , Animales , Dieta Alta en Grasa/efectos adversos , Inflamación/etiología , Inflamación/metabolismo , Inflamación/patología , Hepatopatías/etiología , Hepatopatías/metabolismo , Hepatopatías/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Enfermedad del Hígado Graso no Alcohólico/etiología , Receptores Acoplados a Proteínas G/fisiología , Enfermedades Vasculares/etiología , Enfermedades Vasculares/metabolismo , Enfermedades Vasculares/patologíaRESUMEN
Background: The DNAX adaptor protein 12 (DAP12) is a transmembrane adaptor molecule that signals through the activation of Syk (Spleen Tyrosine Kinase) in myeloid cells. The purpose of this study is to investigate the role of DAP12 and Syk pathways in inflammatory bowel diseases (IBDs). Methods: DAP12 deficient and DAP12 transgenic, overexpressing an increased amount of DAP12, mice and Syk deficient mice in the C57/BL6 background were used for these studies. Colitis was induced by administering mice with dextran sulfate sodium (DSS), in drinking water, or 2,4,6-trinitrobenzene sulfonic acid (TNBS), by intrarectal enema. Results: Abundant expression of DAP12 and Syk was detected in colon samples obtained from Crohn's disease patients with expression restricted to immune cells infiltrating the colonic wall. In rodents development of DSS colitis as measured by assessing severity of wasting diseases, global colitis score,and macroscopic and histology scores was robustly attenuated in DAP12-/- and Syk-/- mice. In contrast, DAP12 overexpression resulted in a striking exacerbation of colon damage caused by DSS. Induction of colon expression of proinflammatory cytokines and chemokines in response to DSS administration was attenuated in DAP12-/- and Syk-/- mice, whereas opposite results were observed in DAP12 transgenic mice. Treating wild-type mice with a DAP-12 inhibitor or a Syk inhibitor caused a robust attenuation of colitis induced by DSS and TNBS. Conclusions: DAP12 and Syk are essential mediators in inflammation-driven immune dysfunction in murine colitides. Because DAP12 and Syk expression is upregulated in patients with active disease, present findings suggest a beneficial role for DAP12 and Syk inhibitors in IBD.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Inflamación/prevención & control , Enfermedades Inflamatorias del Intestino/fisiopatología , Enfermedades Intestinales/prevención & control , Cetotifen/farmacología , Estilbenos/farmacología , Quinasa Syk/fisiología , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Adulto , Animales , Antipruriginosos/farmacología , Colitis/inducido químicamente , Colitis/genética , Colitis/prevención & control , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Humanos , Inflamación/etiología , Inflamación/genética , Enfermedades Intestinales/etiología , Enfermedades Intestinales/genética , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Quinasa Syk/antagonistas & inhibidoresRESUMEN
AIMS: Spinal cord injury (SCI) occurs following damage to the spinal column. Following trauma, tissue damage is further exacerbated by a secondary damage due to a SCI-activated inflammatory process. Control of leukocytes activity is essential to therapeutic inhibition of the spinal cord damage to ameliorate the patient's conditions. The mechanisms that regulate neuroinflammation following SCI, including T-cell infiltration, have not been completely clarified. Glucocorticoids (GC) are antiinflammatory drugs widely used in therapy, including treatment of SCI. GC efficacy may be linked to many molecular mechanisms that are involved in regulation of leukocytes migration, activation, and differentiation. We have previously shown that the antiinflammatory activity of GC is in part mediated by glucocorticoid-induced leucine zipper (GILZ). Here, we investigated the role of GILZ in inflammation and spinal cord tissue damage following a spinal trauma. METHODS: We address the role of GILZ in SCI-induced inflammation and tissue damage using a model of SCI in gilz knockout (gilz KO) and wild-type (WT) mice. RESULTS: We found that GILZ deficiency is associated with a strong reduction of SCI-induced inflammation and a significantly reduced lesion area following SCI. CONCLUSION: These results demonstrate that GILZ is involved in induction of neuroinflammation and functional outcomes of spinal cord trauma.
Asunto(s)
Antiinflamatorios/uso terapéutico , Regulación de la Expresión Génica/fisiología , Glucocorticoides/uso terapéutico , Inflamación/tratamiento farmacológico , Traumatismos de la Médula Espinal/patología , Factores de Transcripción/metabolismo , Análisis de Varianza , Animales , Antiinflamatorios/farmacología , Antígenos CD/metabolismo , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Inflamación/etiología , Molécula 1 de Adhesión Intercelular/metabolismo , Leucocitos/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Infiltración Neutrófila/genética , Selectina-P/metabolismo , Traumatismos de la Médula Espinal/complicaciones , Traumatismos de la Médula Espinal/tratamiento farmacológico , Factores de Tiempo , Factores de Transcripción/genéticaRESUMEN
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.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Fase G1/efectos de los fármacos , Indoles/farmacología , Neoplasias Hormono-Dependientes/tratamiento farmacológico , Apoptosis/efectos de los fármacos , Apoptosis/genética , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proteínas de Ciclo Celular/metabolismo , Quinasa 6 Dependiente de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/biosíntesis , Inducción Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Moduladores de los Receptores de Estrógeno/síntesis química , Moduladores de los Receptores de Estrógeno/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Indoles/síntesis química , Neoplasias Hormono-Dependientes/enzimología , Neoplasias Hormono-Dependientes/metabolismo , Neoplasias Hormono-Dependientes/patología , Fosforilación/efectos de los fármacos , Receptores de Estrógenos/biosíntesis , Proteína de Retinoblastoma/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
A new, simple device generates accurate nano- and microflow rate gradients from any conventional HPLC system. The core of the new device is represented by an electric-actuated, computer-controlled, multiposition HPLC valve. The valve hosts six reservoirs for as many different mobile-phase compositions of increasing strength. A low flow rate stream pushes the weakest solvent through the column as long as required and at the desired flow rate, until the chromatographic run is started. From this time on, the electric actuation allows one to select which reservoir will be on-line with the column and for how long, thus generating a specific solvent gradient, through a sequence of controlled segments of precise mobile-phase composition. This permits one not only to exactly reproduce the programmed slope but also to achieve different gradient shapes (i.e., linear, convex, concave) for different separation needs. The new device has proven to be reliable and reproducible even at the lowest flow rate tested (250 nL x min(-1)) and in different chromatographic conditions.