RESUMEN
BACKGROUND: Intestinal hyperpermeability has been reported in several intestinal and non-intestinal disorders. We aimed to investigate the role of the ubiquitin proteasome system in gut barrier regulation in two mice models: the water avoidance stress model (WAS) and a post-inflammatory model (post-TNBS). METHODS: Both models were applied in C57BL/6 male mice (n=7-8/group); Proteasome was targeted by injection of a selective proteasome inhibitor or by using knock-out mice for ß2i proteasome subunit. Finally, glutamine supplementation was evaluated. RESULTS: In both models (WAS at day 10, post-TNBS at day 28), we observed an increase in proteasome trypsin-like activity and in inducible ß2/constitutive ß2 subunit protein expression ratio, associated with an increase in intestinal permeability. Moreover, intestinal hyperpermeability was blunted by intraperitoneal injection of selective proteasome inhibitor in WAS and post-TNBS mice. Of note, knock-out mice for the ß2i subunit exhibited a significant decrease in intestinal permeability and fecal pellet output during WAS. Glutamine supplementation also improved colonic permeability in both models. CONCLUSIONS: In conclusion, the proteasome system is altered in the colonic mucosa of WAS and post-TNBS mice with increased trypsin-like activity. Associated intestinal hyperpermeability was blunted by immunoproteasome inhibition.
Asunto(s)
Suplementos Dietéticos , Glutamina/farmacología , Intestinos/fisiopatología , Complejo de la Endopetidasa Proteasomal/inmunología , Animales , Reacción de Prevención/efectos de los fármacos , Colon/efectos de los fármacos , Colon/fisiopatología , Modelos Animales de Enfermedad , Inflamación/patología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/patología , Mucosa Intestinal/fisiopatología , Intestinos/efectos de los fármacos , Intestinos/patología , Masculino , Ratones Endogámicos C57BL , Ocludina/metabolismo , Permeabilidad/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/farmacología , Ácido TrinitrobencenosulfónicoRESUMEN
Ubiquitin proteasome system contributes to the regulation of intestinal inflammatory response as its inhibition is associated with tissue damage improvement. We aimed to evaluate whether glutamine is able to limit inflammation by targeting ubiquitin proteasome system in experimental colitis. Colitis was induced in male rats by intrarectal instillation of 2-4-6-trinitrobenzen sulfonic acid (TNBS) at day 1. From day 2 to day 6, rats daily received either an intrarectal instillation of PBS (TNBS/PBS group) or glutamine (TNBS/Gln). Rats were euthanized at day 7 and colonic samples were taken to evaluate ubiqutinated proteins by proteomic approach combining 2D electrophoresis and immunoblots directed against ubiquitin. Results were then confirmed by evaluating total expression of proteins and mRNA levels. Survival rate, TNFα, and IL-1ß mRNA were improved in TNBS/Gln compared with TNBS/PBS (p < 0.05). Proteasome activities were affected by TNBS but not by glutamine. We identified eight proteins that were less ubiquitinated in TNBS/PBS compared with controls with no effect of glutamine. Four proteins were more ubiquitinated in TNBS/PBS group and restored in TNBS/Gln group. Finally, 12 ubiquitinated proteins were only affected by glutamine. Among proteins affected by glutamine, eight proteins (GFPT1, Gapdh, Pkm2, LDH, Bcat2, ATP5a1, Vdac1, and Vdac2) were involved in metabolic pathways. In conclusion, glutamine may regulate ubiquitination process during intestinal inflammation.
Asunto(s)
Colitis/metabolismo , Enema , Glutamina/uso terapéutico , Proteómica/métodos , Animales , Western Blotting , Peso Corporal/fisiología , Inmunoprecipitación , Masculino , Mitocondrias/metabolismo , Ratas , Ratas Sprague-Dawley , Espectrometría de Masa por Ionización de Electrospray , UbiquitinaciónRESUMEN
BACKGROUND & AIMS: Chemotherapy induces an increase of intestinal permeability that is partially related to an alteration of tight junction proteins, occludin and zonula occludens-1 (ZO-1). Protective effects of glutamine on intestinal barrier function have been previously shown but the effects of other amino acids remained poorly documented. Thus, we aimed to evaluate the effects of nine amino acids on intestinal permeability during methotrexate (MTX) treatment in Caco-2 cells. METHODS: Caco-2 cells were incubated in culture medium supplemented with glutamine, arginine, glutamate, leucine, taurine, citrulline, glycine, histidine or cysteine during 24 h and then treated with MTX (100 ng/ml). The dose of each amino acid was 16.6 fold the physiological plasma concentrations. Barrier function was assessed by transepithelial electrical resistance (TEER), FITC-dextran paracellular flux, occludin and ZO-1 expression and localization. Signaling pathways were also studied. RESULTS: Only glutamine, glutamate, arginine and leucine reversed the decrease of TEER observed after MTX treatment (P < 0.05). Interestingly, the addition of 6-diazo-5-oxo-1-norleucine, an inhibitor of glutaminase, blunted the effect of glutamine on MTX-treated cells (P < 0.05). Glutamine and arginine combination restored TEER and FITC-dextran flux to a similar extent than glutamine alone. In addition, pretreatment of Caco-2 cells with glutamine and arginine, alone or combined, differently limited the decrease of ZO-1 and occludin expression (P < 0.05) and the alteration of their cellular distribution, through c-Jun N-terminal kinase (JNK), Extracellular signal-regulated kinase (ERK) and nuclear factor kappa B (NF-κB) pathways. CONCLUSIONS: Glutamine prevented MTX-induced barrier disruption in Caco-2 cells. Arginine also had protective effects but in a lesser extent. The effect of glutamine and arginine should be evaluated in vivo.
Asunto(s)
Arginina/metabolismo , Enterocitos/efectos de los fármacos , Glutamina/metabolismo , Metotrexato/antagonistas & inhibidores , Ocludina/biosíntesis , Regulación hacia Arriba , Proteína de la Zonula Occludens-1/biosíntesis , Antimetabolitos Antineoplásicos/efectos adversos , Antimetabolitos Antineoplásicos/química , Arginina/uso terapéutico , Células CACO-2 , Suplementos Dietéticos , Enterocitos/metabolismo , Enterocitos/patología , Antagonistas del Ácido Fólico/efectos adversos , Antagonistas del Ácido Fólico/química , Fármacos Gastrointestinales/metabolismo , Fármacos Gastrointestinales/uso terapéutico , Glutamina/uso terapéutico , Humanos , Mucosa Intestinal/citología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Metotrexato/efectos adversos , Mucositis/inducido químicamente , Mucositis/metabolismo , Mucositis/patología , Mucositis/prevención & control , Inhibidores de la Síntesis del Ácido Nucleico/efectos adversos , Inhibidores de la Síntesis del Ácido Nucleico/química , Ocludina/metabolismo , Permeabilidad/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismo , Uniones Estrechas/patología , Regulación hacia Arriba/efectos de los fármacos , Proteína de la Zonula Occludens-1/metabolismoRESUMEN
BACKGROUND & AIMS: Gastric electrical stimulation (GES) is an effective therapy to treat patients with chronic dyspepsia refractory to medical management. However, its mechanisms of action remain poorly understood. METHODS: Gastric pain was induced by performing gastric distension (GD) in anesthetized rats. Pain response was monitored by measuring the pseudo-affective reflex (e.g., blood pressure variation), while neuronal activation was determined using c-fos immunochemistry in the central nervous system. Involvement of primary afferents was assessed by measuring phosphorylation of ERK1/2 in dorsal root ganglia. RESULTS: GES decreased blood pressure variation induced by GD, and prevented GD-induced neuronal activation in the dorsal horn of the spinal cord (T9-T10), the nucleus of the solitary tract and in CRF neurons of the hypothalamic paraventricular nucleus. This effect remained unaltered within the spinal cord when sectioning the medulla at the T5 level. Furthermore, GES prevented GD-induced phosphorylation of ERK1/2 in dorsal root ganglia. CONCLUSIONS: GES decreases GD-induced pain and/or discomfort likely through a direct modulation of gastric spinal afferents reducing central processing of visceral nociception.