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Background and study aims Endoscope reprocessing has been associated with a variable failure rate. Our aim was to present an overview on current practices for reprocessing in Italian facilities and discuss the principle critical points. Methods In 2014 the Italian Society for Digestive Diseases implemented an accreditation program in collaboration with an independent organization for certification and with the Italian Association for Endoscopy Technical Operators. During a 1-day site visit of the endoscopy center, two endoscopists, one nurse, and the representative of the certification body evaluated the endoscope reprocessing. Results As of July 1, 2020, 28 endoscopy centers had been accredited. Ten centers are completing the measures to correct deficiencies found at the visit. Three centers withdrew from the program. The accreditation program has found variations between the various centers, confirming the poor compliance with guidelines. Major deviations from the standards, established by the model before the site visit according to national and international guidelines, concerned instrument cleaning (44.7â% of the centers), instrument storage (23.7â%), and microbiological tests (31.6â%). Conclusions Our overview demonstrated the lack of many reprocessing phases, which are important to prevent endoscopy-associated infections. Accreditation can achieve a transformation in quality and safety of reprocessing with the Italian centrally-led approach.
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Bloqueadores del Receptor Tipo 1 de Angiotensina II/efectos adversos , Enfermedades del Colon/inducido químicamente , Imidazoles/efectos adversos , Tetrazoles/efectos adversos , Anciano , Bloqueadores del Receptor Tipo 1 de Angiotensina II/administración & dosificación , Enfermedades del Colon/patología , Diarrea/inducido químicamente , Humanos , Hipertensión/tratamiento farmacológico , Imidazoles/administración & dosificación , Masculino , Tetrazoles/administración & dosificaciónRESUMEN
Background and study aims Accreditation of endoscopy services, using valid quality indicators, may address failures to comply with quality standards between endoscopy services. The aim of this work was to present the Italian Society for Digestive Endoscopy (SIED) accreditation model and its effectiveness. Methods A team of eight endoscopists identified quality indicators derived from international guidelines and assessed them in each center voluntarily requesting accreditation. During a 1-day site visit, two expert endoscopists, the representative of the independent and international administrative certification body and a professional nurse evaluated the endoscopy center, by direct observation of the endoscopy team and examination of the medical records Results In all centers we noted shortcomings in instrument reprocessing. In 30 of 40 centers (75â%) the information in the nursing charts was incomplete. Sampling for Helicobacter pylori had not been done in 12 of 40 centers (30â%). In six of 40 centers (15â%) the adenoma detection rate for each endoscopist had not been evaluated. Post-polypectomy intervals were inappropriate in 12 of 40 centers (30â%). We noted a statistically significant difference ( P â<â0.001) between the answers to the SIED checklist of indicators submitted to the inspection team for accreditation before the site visit and the situation found for colonoscopy on site. As of June 30, 2018, 18 endoscopy centers had been accredited and 10 centers had not yet being accredited because they had not completed the measures to correct points raised at the visits. Conclusions Numerous Italian endoscopy centers fail to meet important quality indicators. Our accreditation program can provide means for detecting these problems and correcting them by implementing SIED standards.
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UNLABELLED: The regulatory factors governing adult mesenchymal stem cell (MSC) physiology and their tumorigenic potential are still largely unknown, which substantially delays the identification of effective therapeutic approaches for the treatment of aggressive and lethal forms of MSC-derived mesenchymal tumors, such as undifferentiated sarcomas. Here, we have developed a novel platform to screen and quickly identify genes and pathways responsible for adult MSC transformation, modeled undifferentiated sarcoma in vivo, and, ultimately, tested the efficacy of targeting the identified oncopathways. Importantly, by taking advantage of this new platform, we demonstrate the key role of an aberrant LRF-DLK1-SOX9 pathway in the pathogenesis of undifferentiated sarcoma, with important therapeutic implications. SIGNIFICANCE: The paucity of therapeutic options for the treatment of sarcoma calls for a rapid and effective preclinical assessment of new therapeutic modalities. We have here developed a new platform to deconstruct the molecular genetics underlying the pathogenesis of sarcoma and to evaluate in vivo the efficacy of novel targeted therapies.
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Transformación Celular Neoplásica/genética , Células Madre Mesenquimatosas/metabolismo , Modelos Biológicos , Sarcoma/genética , Sarcoma/patología , Adulto , Animales , Proteínas de Unión al Calcio , Técnicas de Cultivo de Célula , Diferenciación Celular , Línea Celular , Proliferación Celular , Transformación Celular Neoplásica/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunofenotipificación , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Células Madre Mesenquimatosas/citología , Ratones , Ratones Noqueados , Ratones Transgénicos , Fenotipo , Sarcoma/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
BACKGROUND: Peroxisome proliferator-activated receptors (PPARs) are ligand activated transcription factors belonging to the nuclear receptor superfamily. PPARs activation has a profound impact on the local immune response with consequences affecting the progression of chronic inflammatory diseases. Relatively little is known on the role of PPAR-ß/δ in the regulation of inflammatory responses. The aim of the present study was to evaluate the influence of PPAR-ß/δ receptor in a model of edematous pancreatitis induced in mice by administration of cerulein at supramaximal doses, as well as in necrohemorrhagic model induced by intraductal administration of sodium taurocholate (STC). MEASUREMENTS: Mice were treated with cerulein (50 µg/kg) or STC (5%). GW0742 (0.3 mg/kg) was intraperitoneally administered 1 and 6 hours after cerulein injection or was injected 2 hours before STC infusion. The pancreas and exopancreatic organs were carefully removed for microscopic examination. Pancreatic weight, serum amylase, lipase, tumor necrosis factor-α and interleukin-1ß levels, as well as cytokines, adhesion molecules, nitrotyrosine, poly (ADP-ribose), inducible nitric oxide, FAS ligand, Bax, Bcl-2 expression by immunohistochemistry, and myeloperoxidase activity of the pancreas were assayed. Moreover, the involvement of nuclear factor-κB pathway was investigated by Western blot analysis. RESULTS: Intraperitoneal injection of cerulein in mice resulted in severe, acute pancreatitis characterized by edema, neutrophil infiltration and apoptosis, and elevated serum levels of amylase and lipase. Taurocholate challenge caused a clear increase in serum amylase, neutrophil infiltration, and tissue damage in the pancreas. Tissue and inflammatory changes in the pancreata were significantly less in GW0742 group than in cerulein or STC groups. In addition, the pancreatic water content was reduced in mice treated with PPAR-ß/δ agonist. In the mild pancreatitis, GW0742 was also able to decrease the expression of proinflammatory cytokines and enzymes, as well as of proteins involved in apoptosis and nuclear factor-Kappa B pathway. CONCLUSION: GW0742 attenuated pancreatic damage in 2 different experimental models of pancreatitis in mice.
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Ceruletida/efectos adversos , PPAR delta/agonistas , PPAR-beta/agonistas , Pancreatitis/inducido químicamente , Pancreatitis/prevención & control , Ácido Taurocólico/efectos adversos , Tiazoles/uso terapéutico , Enfermedad Aguda , Amilasas/sangre , Animales , Moléculas de Adhesión Celular/metabolismo , Movimiento Celular , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Lipasa/sangre , Masculino , Ratones , Ratones Endogámicos , Neutrófilos/patología , PPAR delta/metabolismo , PPAR-beta/metabolismo , Pancreatitis/metabolismo , Factores de TiempoRESUMEN
OBJECTIVES: Microvascular thrombosis occurs in severe acute pancreatitis (AP). Exploiting this knowledge, we used human recombinant activated protein C (Xigris; Eli Lilly, Indianapolis, Ind) known to preserve microvascular patency, in evaluating the role of Xigris in experimental AP. METHODS: In accordance with European union experimentation regulations, AP was induced by hourly injection of cerulein 50 µg/kg body weight over 6 hours. Male rats of median weight of 231 g (range, 176-312 g) were allocated at random into groups: group 1, control; group 2, vehicle; group 3, AP; group 4, cerulein + Xigris at induction of AP and killing at 24 h; and group 5, cerulein + Xigris 24 hours after induction and killing at 48 hours. In addition to enzymatic and histological markers of pancreatic injury, apoptosis, nuclear factor κB (NF-κB) p65/IκB, cytokine response, and endothelial injury were assessed. Western blot quantified by densitometry was used to assess marker of apoptosis and endothelial injury. RESULTS: Cerulein injection resulted in acute necrotizing pancreatitis. Intervention with recombinant human activated protein C did not modify coagulation parameters or lead to hemorrhage but ameliorated pancreatic injury with preservation of IκB and reduction of NF-κB p65 and modulation of apoptosis. CONCLUSIONS: Our study indicates that recombinant human activated protein C ameliorates experimental cerulein-induced pancreatitis through apoptotic and NF-κB pathways without causing pancreatic hemorrhage.
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Apoptosis/efectos de los fármacos , Fibrinolíticos/uso terapéutico , FN-kappa B/metabolismo , Páncreas/efectos de los fármacos , Pancreatitis Aguda Necrotizante/tratamiento farmacológico , Proteína C/uso terapéutico , Animales , Biomarcadores/metabolismo , Western Blotting , Ceruletida/administración & dosificación , Citocinas/metabolismo , Fibrinolíticos/farmacología , Etiquetado Corte-Fin in Situ , Masculino , Malondialdehído/metabolismo , Páncreas/metabolismo , Páncreas/patología , Pancreatitis Aguda Necrotizante/inducido químicamente , Peroxidasa/metabolismo , Proteína C/farmacología , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéuticoRESUMEN
Multiple organ dysfunction syndrome (MODS) is a systemic inflammatory event that can result in organ damage, failure, and high risk of mortality. The aim of this study was to evaluate the possible role of glucocorticoid-induced TNFR-related (GITR) on zymosan-induced MODS. Mice were allocated into one GITR knockout (GITR-KO) and two GITR wild-type (GITR-WT) experimental groups. All the animals were treated with zymosan (500 mg/kg, suspended in saline solution, i.p.), and animals of one GITR-WT group received GITR-Fc (6.25 µg/mouse; 3 h after zymosan injection) by mini-osmotic pump. Moreover, three control groups were performed (one GITR-KO and two GITR-WT experimental groups), administering saline instead of zymosan and treating one of the GITR-WT group with GITR-Fc (6.25 µg/mouse; 3 h after saline injection) by mini-osmotic pump. A number of inflammatory parameters such as edema formation, histological damage, adhesion molecules expression, neutrophil infiltration, proinflammatory cytokines, nitrotyrosine, and iNOS production are significantly reduced in GITR-KO as compared with GITR-WT mice as well as in GITR-WT mice treated with GITR-Fc. We here show that GITR plays a role in the modulation of experimental MODS. In particular, we show that genetic inhibition of GITR expression, in GITR-KO mice, or administration of soluble GITR-Fc receptor in GITR-WT mice, reduces inflammation, organ tissue damage, and mortality. Results, while confirming the proinflammatory role of GITR, extend our observations indicating that GITR plays a role in zymosan-induced inflammation and MODS.
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Proteína Relacionada con TNFR Inducida por Glucocorticoide/metabolismo , Insuficiencia Multiorgánica/prevención & control , Proteínas Recombinantes de Fusión/uso terapéutico , Zimosan/toxicidad , Animales , Moléculas de Adhesión Celular , Eliminación de Gen , Proteína Relacionada con TNFR Inducida por Glucocorticoide/genética , Inmunohistoquímica , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Masculino , Ratones , Ratones Noqueados , Insuficiencia Multiorgánica/inducido químicamente , Insuficiencia Multiorgánica/genética , Insuficiencia Multiorgánica/metabolismo , Nitratos/metabolismo , Nitritos/metabolismo , Peroxidasa/metabolismo , Proteínas Recombinantes de Fusión/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a tumor suppressor that antagonizes signaling through the phosphatidylinositol 3-kinase-Akt pathway. We have demonstrated that subtle decreases in PTEN abundance can have critical consequences for tumorigenesis. Here, we used a computational approach to identify miR-22, miR-25, and miR-302 as three PTEN-targeting microRNA (miRNA) families found within nine genomic loci. We showed that miR-22 and the miR-106b~25 cluster are aberrantly overexpressed in human prostate cancer, correlate with abundance of the miRNA processing enzyme DICER, and potentiate cellular transformation both in vitro and in vivo. We demonstrated that the intronic miR-106b~25 cluster cooperates with its host gene MCM7 in cellular transformation both in vitro and in vivo, so that the concomitant overexpression of MCM7 and the miRNA cluster triggers prostatic intraepithelial neoplasia in transgenic mice. Therefore, the MCM7 gene locus delivers two simultaneous oncogenic insults when amplified or overexpressed in human cancer. Thus, we have uncovered a proto-oncogenic miRNA-dependent network for PTEN regulation and defined the MCM7 locus as a critical factor in initiating prostate tumorigenesis.
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Proteínas de Ciclo Celular/genética , Transformación Celular Neoplásica , Proteínas de Unión al ADN/genética , Intrones , MicroARNs/genética , Familia de Multigenes , Proteínas Nucleares/genética , Fosfohidrolasa PTEN/genética , Proto-Oncogenes , Animales , Humanos , Masculino , Ratones , Ratones Transgénicos , Componente 7 del Complejo de Mantenimiento de Minicromosoma , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patologíaRESUMEN
The aim of the present study was to examine the effects of verbascoside (VB) in rats subjected to experimental colitis. Colitis was induced in rats by intracolonic instillation of 2,4 dinitrobenzene sulfonic acid (DNBS; 25 mg/rat). VB was administered daily per os (0.2 and 2 mg/kg) 4 days after DNBS administration in the colon. Treatment with VB significantly (P < 0.01) reduced macroscopic damage score, loss of body weight, myeloperoxidase activity and thiobarbituric acid-reactant substances. Moreover, the intensity of the positive staining for tumor necrosis factor-alpha, interleukin-1beta, intercellular adhesion molecule-1, P-selectin, inducible nitric oxide synthase, and poly(ADP ribose) was also significantly (P < 0.01) reduced by VB treatment. Therefore, VB treatment significantly (P < 0.01) reduced the degree of NF-kappaB p65 and activation of the pro-active form metalloproteinase (MMP)-2 and pro-MMP-9 activity. The results of this study suggested that VB functions as an intracellular radical scavenger and so reduces the microscopic and macroscopic signs of colitis in the rat. Therefore, administration of VB may be beneficial for the treatment of inflammatory bowel disease.
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Antioxidantes/uso terapéutico , Colitis/tratamiento farmacológico , Glucósidos/uso terapéutico , Fenoles/uso terapéutico , Syringa/química , Animales , Antioxidantes/administración & dosificación , Antioxidantes/aislamiento & purificación , Peso Corporal/efectos de los fármacos , Células Cultivadas , Colitis/fisiopatología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Precursores Enzimáticos/efectos de los fármacos , Precursores Enzimáticos/metabolismo , Gelatinasas/efectos de los fármacos , Gelatinasas/metabolismo , Glucósidos/administración & dosificación , Glucósidos/aislamiento & purificación , Masculino , Metaloproteinasa 9 de la Matriz/efectos de los fármacos , Metaloproteinasa 9 de la Matriz/metabolismo , Peroxidasa/efectos de los fármacos , Peroxidasa/metabolismo , Fenoles/administración & dosificación , Fenoles/aislamiento & purificación , Ratas , Ratas Sprague-Dawley , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo , Factor de Transcripción ReIA/efectos de los fármacos , Factor de Transcripción ReIA/metabolismoRESUMEN
The aim of the present study was to examine the effects of phenylpropanoid glycoside, teupolioside, biotechnologically produced by IRBN22 Ajuga reptans cell line, in rats subjected to experimental colitis. Colitis was induced in rats by intracolonic instillation of dinitrobenzene sulfonic acid (DNBS). Teupolioside was administered daily orally (0.2 or 2mgkg(-1)). On Day 4, animals were sacrificed and tissues were taken for histological and biochemical analysis. Four days after DNBS administration, colon TNF-alpha and IL-1beta productions were increased, associated with colon damage. Neutrophil infiltration, by myeloperoxidase activity, in the mucosa was associated with up-regulation of ICAM-1 and P-selectin and high levels of malondialdehyde. Immunohistochemistry for nitrotyrosine and poly (ADP-ribose) polymerase (PARP) showed an intense staining in the inflamed colon. Biochemical methods and zymography were used to analyze MMP-9 and -2 activities in colon tissues from DNBS-injured rats. Treatment with teupolioside significantly reduced the appearance of diarrhoea and the loss of body weight. This was associated with a remarkable amelioration in the disruption of the colonic architecture and a significant reduction in colonic myeloperoxidase activity and malondialdehyde levels. Teupolioside also reduced the pro-inflammatory cytokines release, the appearance of nitrotyrosine and PARP immunoreactivity in the colon and reduced the up-regulation of ICAM-1 and the expression of P-selectin. Therefore, teupolioside also reduced proMMP-9 and -2 activity induced in the colon by DNBS administration. The results of this study suggested that administration of teupolioside may be beneficial for treatment of inflammatory bowel disease.
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Ajuga/química , Ácidos Cafeicos/uso terapéutico , Colitis/tratamiento farmacológico , Modelos Animales de Enfermedad , Trisacáridos/uso terapéutico , Ajuga/citología , Animales , Bencenosulfonatos/administración & dosificación , Ácidos Cafeicos/aislamiento & purificación , Línea Celular , Cromatografía Líquida de Alta Presión , Colitis/inducido químicamente , Colitis/metabolismo , Colon/enzimología , Colon/metabolismo , Inmunohistoquímica , Molécula 1 de Adhesión Intercelular/metabolismo , Interleucina-1beta/biosíntesis , Peroxidación de Lípido/efectos de los fármacos , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Selectina-P/metabolismo , Ratas , Ratas Sprague-Dawley , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo , Trisacáridos/aislamiento & purificación , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Glucocorticoids (GCs) are effective anti-inflammatory agents widely used in therapeutic approach to treatment of inflammatory bowel disease (IBD). Previous results suggest that peroxisome proliferator-activated receptor [alpha] (PPAR-[alpha]), an intracellular transcription factor activated by fatty acids, plays a role in control of inflammation. With the aim to characterize the role of PPAR-[alpha] in GC-mediated anti-inflammatory activity, we tested the efficacy of dexamethasone (DEX), a synthetic GC specific for GR, in an experimental model of IBD induced by dinitrobenzene sulfonic acid, comparing mice lacking PPAR-[alpha] (PPAR-[alpha]KO) with wild-type (WT) mice. Results indicate that DEX-mediated anti-inflammatory activity is weakened in PPAR-[alpha]KO mice as compared with WT controls. In particular, DEX was less effective in PPAR-[alpha]KO compared with WT mice, as evaluated by inhibition of proinflammatory cytokines production, cell migration, oxidative stress, apoptosis, and colon injury. These results indicate that PPAR-[alpha] can contribute to the anti-inflammatory activity of GCs in IBD.
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Dexametasona/farmacología , Glucocorticoides/farmacología , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , PPAR alfa/agonistas , Animales , Antiinflamatorios/farmacología , Apoptosis/efectos de los fármacos , Bencenosulfonatos/toxicidad , Movimiento Celular/efectos de los fármacos , Citocinas/metabolismo , Mediadores de Inflamación/metabolismo , Enfermedades Inflamatorias del Intestino/inducido químicamente , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/metabolismo , Ratones , Ratones Noqueados , Estrés Oxidativo/efectos de los fármacos , PPAR alfa/genética , PPAR alfa/metabolismoRESUMEN
MicroRNAs (miRNAs) are short 20-22 nucleotide RNA molecules that act as negative regulators of gene expression via translational repression: they have been shown to play a role in development, proliferation, stress response, and apoptosis. The transcriptional regulator LRF (Leukemia/lymphoma Related Factor) has been shown to prevent p19ARF transcription and consequently to inhibit senescence in mouse embryonic fibroblasts (MEF). Here we report, for the first time, that LRF is post-transcriptionally regulated by miR-20a. Using a gene reporter assay, direct interaction of miR-20a with the LRF 3'UTR is demonstrated. To validate the interaction miR-20a/3'UTR LRF miR-20a was over-expressed, either by transient transfection or retroviral infection, in wild type mouse embryo fibroblasts and in LRF-null MEF derived from LRF knock-out mice. We observed LRF decrease, p19ARF increase, inhibition of cell proliferation and induction of senescence. The comparison of miR-20a activity in wt and LRF-null MEF indicates that LRF is the main mediator of the miR-20a-induced senescence and that other targets are cooperating. As LRF down-regulation/p19ARF induction is always accompanied by E2F1 down-regulation and increase of p16, we propose that all these events act in synergy to accomplish miR-20a-induced senescence in MEF. Senescence has been recently revaluated as a tumor suppressor mechanism, alternative to apoptosis; from this point of view the discovery of new physiological "senescence inducer" appears to be promising as this molecule could be used as anticancer drug.
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Senescencia Celular/genética , Proteínas de Unión al ADN/genética , MicroARNs/metabolismo , Proto-Oncogenes , Interferencia de ARN , Factores de Transcripción/genética , Animales , Secuencia de Bases , Proliferación Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo , Factor de Transcripción E2F1/genética , Factor de Transcripción E2F1/metabolismo , Fibroblastos/metabolismo , Ratones , Datos de Secuencia Molecular , Factores de Transcripción/metabolismoRESUMEN
Matrix metalloproteinases (MMPs) are associated with matrix turnover in both physiological and pathological conditions. Several data indicate that MMPs play an important role in the pathogenesis of colitis. Various evidence has documented that the pineal secretory product melatonin exerts an important anti-inflammatory effect in different experimental models including colitis. However, no reports are available on the relationship between the activity and expression of MMPs and anti-inflammatory effect of melatonin. The aim of the present study was to evaluate whether melatonin prevents the experimental colitis in rats by regulating MMP-9 and MMP-2 activity and expression. Colitis was induced by intracolonic instillation of dinitrobenzene sulphonic acid (DNBS). Four days after DNBS administration, colon TNF-alpha production was associated with colon damage. Biochemical methods and zymography were used to analyse MMP-9 and MMP-2 activities in colon tissues from DNBS-injured rats. Our studies reveal that melatonin prevented colon injury and lipid peroxidation in rats at 4 days after DNBS-induced colitis. Melatonin also reduced proMMP-9 and MMP-2 activities that were induced in the colon tissues by DNBS administration. Reduced MMP-9 and MMP-2 activities were associated with reduced expression of TNF-alpha. We conclude that melatonin's ability to reduce DNBS-induced colon injury in rats is related to a reduction in proMMP-9 and MMP-2 activities and expression.
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Colitis/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Melatonina/farmacología , Animales , Antioxidantes/farmacología , Western Blotting , Colitis/inducido químicamente , Dinitrofluorobenceno/análogos & derivados , Peroxidación de Lípido/efectos de los fármacos , Masculino , Ratas , Ratas Sprague-Dawley , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Various evidences have documented that the pineal secretory product melatonin exerts an important anti-inflammatory effect in different experimental models including colitis. The aim of the present study was to evaluate whether melatonin regulates the inflammatory response of experimental colitis in rats at the level of signal transduction pathway. Colitis was induced by intracolonic instillation of dinitrobenzene sulfonic acid (DNBS). Four days after DNBS administration, a substantial increase of colon TNF-alpha production was associated with the colon damage. In DNBS-treated rats, the colon injury correlated with a significant rise of apoptosis (evaluated by TUNEL coloration) which was associated with a significant increased expression of proapoptotic Bax and decreased colon content of antiapoptotic Bcl-2. This inflammatory response was also related to activation of nuclear factor-kappaB (NF-kappaB) and phosphorylation of c-Jun as well as FAS ligand expression in the colon. Treatment with melatonin (15 mg/kg daily i.p.) was associated with a remarkable amelioration of colonic disrupted architecture as well as a significant reduction of TNF-alpha. Melatonin also reduced the NF-kappaB activation and phosphorylation of c-Jun as well as the Fas ligand expression in the colon. Furthermore, melatonin reduced the expression of Bax and prevented the loss of Bcl-2 proteins as well as the presence of apoptotic cells caused by DNBS. The results of this study show that melatonin administration exerts beneficial effects in inflammatory bowel disease by modulating signal transduction pathways.
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Apoptosis/efectos de los fármacos , Colitis/tratamiento farmacológico , Colitis/patología , Melatonina/uso terapéutico , Transducción de Señal/efectos de los fármacos , Animales , Colitis/inducido químicamente , Colitis/metabolismo , Citocinas/metabolismo , Dinitrofluorobenceno/análogos & derivados , Dinitrofluorobenceno/farmacología , Modelos Animales de Enfermedad , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Masculino , Fosfoserina/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas , Ratas Sprague-Dawley , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The nuclear receptors farnesoid X receptor (FXR) and peroxisome proliferator-activated receptor (PPAR)gamma exert counter-regulatory effects on hepatic stellate cells (HSCs) and protect against liver fibrosis development in rodents. Here, we investigated whether FXR ligands regulate PPARgamma expression in HSCs and models of liver fibrosis induced in rats by porcine serum and carbon tetrachloride administration and bile duct ligation. Our results demonstrate that HSCs trans-differentiation associated with suppression of PPARgamma mRNA expression, whereas FXR mRNA was unchanged. Exposure of cells to natural and synthetic ligands of FXR, including 6-ethyl chenodeoxycholic acid (6-ECDCA), a synthetic derivative of chenodeoxycholic acid, reversed this effect and increased PPARgamma mRNA by approximately 40-fold. Submaximally effective concentrations of FXR and PPARgamma ligands were additive in inhibiting alpha1(I) collagen mRNA accumulation induced by transforming growth factor (TGF)beta1. Administration of 6-ECDCA in rats rendered cirrhotic by porcine serum and carbon tetrachloride administration or bile duct ligation reverted down-regulation of PPARgamma mRNA expression in HSCs. Cotreatment with 6-ECDCA potentiates the antifibrotic activity of rosiglitazone, a PPARgamma ligand, in the porcine serum model as measured by morphometric analysis of liver collagen content, hydroxyproline, and liver expression of alpha1(I) collagen mRNA, alpha-smooth muscle actin, fibronectin, TGFbeta1, and tissue inhibitor of metalloprotease 1 and 2, whereas it enhanced the expression of PPARgamma and uncoupling protein 2, a PPARgamma-regulated gene, by 2-fold. In conclusion, by using an in vitro and in vivo approach, we demonstrated that FXR ligands up-regulate PPARgamma mRNA in HSCs and in rodent models of liver fibrosis. A FXR-PPARgamma cascade exerts counter-regulatory effects in HSCs activation.
Asunto(s)
Ácido Quenodesoxicólico/análogos & derivados , Proteínas de Unión al ADN/agonistas , Cirrosis Hepática Experimental/tratamiento farmacológico , PPAR gamma/genética , Tiazolidinedionas/farmacología , Factores de Transcripción/agonistas , Animales , Células Cultivadas , Ácido Quenodesoxicólico/farmacología , Proteínas de Unión al ADN/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Hígado/citología , Hígado/metabolismo , Cirrosis Hepática Experimental/etiología , Cirrosis Hepática Experimental/metabolismo , ARN Mensajero/análisis , Ratas , Receptores Citoplasmáticos y Nucleares , Rosiglitazona , Factores de Transcripción/fisiologíaRESUMEN
The farnesoid X receptor (FXR) is expressed by and regulates hepatic stellate cells (HSCs). In the present study, we investigated whether 6-ethyl chenodeoxycholic acid (6-ECDCA or INT-747), a semisynthetic derivative of chenodeoxycholic acid (CDCA), modulates tissue metalloproteinase inhibitor (TIMP)-1 and matrix metalloprotease (MMP)-2 expression/activity in HSCs and in the liver of rats rendered cirrhotic by 4-week administration of CCl(4). Exposure of HSCs to FXR ligands increases small heterodimer partner (SHP) mRNA by 3-fold and reduces basal and thrombin-stimulated expression of alpha1(I)collagen, alpha-smooth muscle actin (alpha-SMA), TIMP-1, and TIMP-2 by approximately 60 to 70%, whereas it increased matrix metalloprotease (MMP)-2 activity by 2-fold. In coimmunoprecipitation, electromobility shift, and transactivation experiments, FXR activation/overexpression caused a SHP-dependent inhibition of JunD binding to its consensus element in the TIMP-1 promoter. Inhibition of TIMP-1 expression by SHP overexpression enhanced the sensitivity of HSCs to proapoptogenic stimuli. Administration of 3 mg/kg 6-ECDCA, but not 15 mg/kg ursodeoxycholic acid, resulted in early (3-5-day) induction of SHP and prevention of early up-regulation of TIMP-1 mRNA induced by CCl(4). In the prevention protocol, 4-week administration of 6-ECDCA reduced alpha1(I)collagen, alpha-SMA, and TIMP-1 mRNA by 60 to 80%, whereas it increased MMP-2 activity by 5-fold. In the resolution protocol, administration of 3 mg/kg 6-ECDCA promoted liver fibrosis resolution and increased the apoptosis of nonparenchyma liver cells. By demonstrating that a FXR-SHP regulatory cascade promotes the development of a quiescent phenotype and increases apoptosis of HSCs, this study establishes that FXR ligands may be beneficial in treatment of liver fibrosis.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Regulación de la Expresión Génica/fisiología , Hepatocitos/metabolismo , Cirrosis Hepática/patología , Metaloproteinasas de la Matriz/biosíntesis , Inhibidor Tisular de Metaloproteinasa-1/biosíntesis , Factores de Transcripción/fisiología , Actinas/metabolismo , Animales , Apoptosis/fisiología , Western Blotting , Intoxicación por Tetracloruro de Carbono/patología , Separación Celular , Ensayo de Cambio de Movilidad Electroforética , Hepatocitos/enzimología , Humanos , Inmunohistoquímica , Cirrosis Hepática/inducido químicamente , Cirrosis Hepática/metabolismo , Metaloproteinasa 2 de la Matriz/biosíntesis , Plásmidos/genética , Proteínas Proto-Oncogénicas c-jun/metabolismo , Ratas , Receptores Citoplasmáticos y Nucleares , Factor de Transcripción AP-1/metabolismoRESUMEN
BACKGROUND AND AIMS: The farnesoid X receptor (FXR) is an endogenous sensor for bile acids and inhibits bile acid synthesis by inducing small heterodimer partner (SHP) gene expression. The aim of this study was to investigate whether FXR is expressed by and modulates function of hepatic stellate cells (HSCs). METHODS: The antifibrotic activity of FXR ligand was tested in 2 rodent models: the porcine serum and bile duct ligation (BDL). RESULTS: Twelve-week administration of 1-10 mg/kg 6-ethyl chenodeoxycholic acid (6-ECDCA), a synthetic FXR ligand, to porcine serum-treated rats prevented liver fibrosis development and reduced liver expression of alpha1(I) collagen, TGF-beta1 and alpha-SMA mRNA by approximately 90%. Therapeutic administration of 6-ECDCA, 3 mg/kg, to BDL rats reduced liver fibrosis and alpha1(I) collagen, transforming growth factor (TGF)-beta1, alpha-SMA, and tissue metalloproteinase inhibitor (TIMP)-1 and 2 messenger RNA (mRNA) by 70%-80%. No protection was observed in BDL rats treated with CDCA, 3 mg/kg, and ursodeoxycholic acid, 15 mg/kg. FXR expression was detected in HSCs. Exposure of HSCs to FXR ligands caused a 3-fold increase of SHP, reduced alpha1(I)collagen and TGF-beta1 by approximately 60%-70% and abrogates alpha1(I) collagen mRNA up-regulation induced by thrombin and TGF-beta1. By retrovirus infection and small interference RNA, we generated SHP overexpressing and SHP-deficient HSC-T6. Using these cell lines, we demonstrated that SHP binds JunD and inhibits DNA binding of adaptor protein (AP)-1 induced by thrombin. CONCLUSIONS: By demonstrating that an FXR-SHP regulatory cascade promotes resolution of liver fibrosis, this study establish that FXR ligands might represent a novel therapeutic option to treat liver fibrosis.