RESUMEN

AIM: To investigate the mechanisms of Lactobacillus plantarum (L. plantarum) action on gut barrier in preoperative and postoperative experimental obstructive jaundice in rats. METHODS: Forty rats were randomly divided into groups of sham-operation, bile duct ligation (BDL), BDL + L. plantarum, BDL + internal biliary drainage (IBD), and BDL + IBD + L. plantarum. Ten days after L. plantarum administration, blood and ileal samples were collected from the rats for morphological examination, and intestinal barrier function, liver function, intestinal oxidative stress and protein kinase C (PKC) activity measurement. The distribution and expression of the PKC and tight junction (TJ) proteins, such as occludin, zonula occludens-1, claudin-1, claudin-4, junction adhesion molecule-A and F-actin, were examined by confocal laser scanning microscopy, immunohistochemistry, Western blotting, real-time fluorescent quantitative polymerase chain reaction assay. RESULTS: L. plantarum administration substantially restored gut barrier, decreased enterocyte apoptosis, improved intestinal oxidative stress, promoted the activity and expression of protein kinase (BDL vs BDL + L. plantarum, 0.295 ± 0.007 vs 0.349 ± 0.003, P < 0.05; BDL + IBD vs BDL + IBD + L. plantarum, 0.407 ± 0.046 vs 0.465 ± 0.135, P < 0.05), and particularly enhanced the expression and phosphorylation of TJ proteins in the experimental obstructive jaundice (BDL vs BDL + L. plantarum, 0.266 ± 0.118 vs 0.326 ± 0.009, P < 0.05). The protective effect of L. plantarum was more prominent after internal biliary drainage ( BDL + IBD vs BDL + IBD + L. plantarum, 0.415 ± 0.105 vs 0.494 ± 0.145, P < 0.05). CONCLUSION: L. plantarum can decrease intestinal epithelial cell apoptosis, reduce oxidative stress, and prevent TJ disruption in biliary obstruction by activating the PKC pathway.

Asunto(s)

Mucosa Intestinal/enzimología , Lactobacillus plantarum , Probióticos/uso terapéutico , Proteína Quinasa C/metabolismo , Proteínas de Uniones Estrechas/metabolismo , Uniones Estrechas/metabolismo , Alanina Transaminasa/sangre , Amina Oxidasa (conteniendo Cobre)/sangre , Animales , Apoptosis , Bilirrubina/sangre , Endotoxinas/sangre , Enterocitos/microbiología , Enterocitos/fisiología , Glutatión/sangre , Íleon/patología , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Ictericia Obstructiva/sangre , Ictericia Obstructiva/microbiología , Ictericia Obstructiva/cirugía , Ácido Láctico/sangre , Masculino , Malondialdehído/metabolismo , Estrés Oxidativo , Ratas , Superóxido Dismutasa/metabolismo , Uniones Estrechas/ultraestructura

RESUMEN

Probiotics are efficacious in the treatment of inflammatory bowel disease. However, the precise mechanisms remain unknown. To determine whether probiotic Lactobacillus plantarum (LP) ameliorates colonic epithelial barrier dysfunction present in interleukin-10 knockout (IL-10⁻(/)⁻) mice, IL-10⁻(/)⁻ and wild-type mice received LP or the vehicle for 4 wk. Colitis was assessed by histological scores and clinical manifestation, and gut paracellular permeability was measured by Ussing chamber. Oligopeptide transporter 1 (PepT1)-mediated transepithelial transport was evaluated by measuring the plasma cephalexin concentration. The expression and distribution of apical junctional complex (AJC) proteins and PepT1 were determined by Western blotting and immunofluorescence and their mRNA by reverse transcriptase-PCR. Spontaneous colitis was observed in all IL-10⁻(/)⁻ mice in which paracellular permeability was increased, in conjunction with decreased expression and redistribution of zonula occludens-1, occludin, claudin-1, and ß-catenin. PepT1 expression was increased, accompanied with an enhanced cephalexin transport. Colonic epithelial barrier dysfunction was further confirmed by increased bacterial translocation and proinflammatory cytokine production. Treatment with LP decreased colonic paracellular permeability with restoration of expression and distribution of AJC proteins and partially prevented PepT1 expression and cephalexin transport in IL-10⁻(/)⁻ mice. Moreover, treatment with LP also prevented bacterial translocation and proinflammatory cytokine production in IL-10⁻(/)⁻ mice. Results from this study indicated that treatment with LP may ameliorate colonic epithelial barrier dysfunction in IL-10⁻(/)⁻ mice, by modulating the AJC- and PepT1-mediated transepithelial transport.

Asunto(s)

Colon/fisiología , Interleucina-10/genética , Interleucina-10/metabolismo , Mucosa Intestinal/fisiopatología , Simportadores/genética , Simportadores/metabolismo , Animales , Transporte Biológico , Colitis/prevención & control , Regulación de la Expresión Génica/fisiología , Inflamación/metabolismo , Lactobacillus plantarum , Ratones , Ratones Noqueados , Transportador de Péptidos 1

RESUMEN

Although probiotic consumption has generally been shown to have many beneficial effects for the prevention and treatment of inflammatory bowel disease, the effects of Lactobacillus plantarum (LP) on intestinal nutrient absorption, particularly oligopeptide transporter 1 (PepT1)-mediated absorption of dietary protein under inflammatory conditions, has not yet been characterized. In this study, we first investigated the effects of LP consumption on plasma amino acid concentrations and PepT1-mediated absorption of cephalexin in the small intestine of wild-type (WT) mice and interleukin-10 knockout (IL-10(-/-)) mice, a model of spontaneous colitis. We then analyzed expression and distribution of PepT1 and protein kinase C (PKC) activity in the jejunum of these mice. LP consumption (10(9) colony-forming units/0.5 mL) delivered by gavage once per day for 4 wk increased the total plasma amino acid concentration and the concentration of plasma cephalexin through enhancement of PepT1-mediated uptake in LP treated IL-10(-/-) mice compared with IL-10(-/-) mice. However, Western blotting and quantitative PCR analysis revealed no significant differences in PepT1 protein and mRNA expression between LP-treated and untreated mice. Additionally, immunofluorescence analysis showed that PepT1 did not appear to be mislocalized in IL-10(-/-) mice. Interestingly, IL-10(-/-) mice had significantly lower PKC activity and expression of phosphorylated PKC compared with WT mice, and these decreases could be prevented by LP treatment. These data suggest that consumption of LP enhances PepT1-mediated amino acid absorption, likely through alterations in PKC activity, as opposed to changes in expression or distribution of PepT1 in the small intestine of IL-10(-/-) mice.

Asunto(s)

Aminoácidos/metabolismo , Colitis/metabolismo , Lactobacillus plantarum/metabolismo , Proteína Quinasa C/metabolismo , Simportadores/fisiología , Animales , Colitis/enzimología , Colitis/microbiología , Interleucina-10/genética , Interleucina-10/fisiología , Yeyuno/enzimología , Yeyuno/metabolismo , Ratones , Ratones Noqueados , Transportador de Péptidos 1

RESUMEN

UNLABELLED: 5-Fluorouracil has been the chemotherapy agent of first-choice for colorectal cancer for many years, but since there are no proven predictors of a patient's response to therapy, all patients receive similar treatment. Consequently, identification of biomarkers for therapeutic effect is crucial for the development of novel therapeutic strategies. Two human colorectal cancer cell lines of different metastatic potential (LoVo and SW480) were studied. IC50 of 5-FU for both cell lines were measured by 3-(4,5-dimethy-lthiazol-2-yl)-2,5-diphenyltetrazolium assay and validated by cell cycle analysis. Then the cell lines were treated with 5-FU at IC50 concentration and protein was extracted for 2-DE. Differential protein spots were examined by MALDI-TOF/TOF MS. The expression levels of the different proteins were further confirmed by Western blot and immunofluorescence analyses. Eleven proteins were identified. Expression of heterogeneous nuclear ribonucleoprotein K (hnRNP K) in LoVo cells was higher than in SW480 cells, while protein disulfide isomerase (PDI) displayed the opposite trend. After treatment with 5-FU, the expression of hnRNP K in LoVo decreased more significantly than in SW480, while PDI in SW480 increased more significantly than in LoVo cells. CONCLUSION: hnRNP K and PDI in the two cell lines have different expression characteristics. The sensitivity to 5-FU is not consistent in tumor progression. It may assist in development of novel treatment strategies for colorectal cancer metastasis.

Asunto(s)

Neoplasias Colorrectales/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo K/metabolismo , Proteína Disulfuro Isomerasas/metabolismo , Western Blotting , Ciclo Celular , Línea Celular Tumoral , Supervivencia Celular , Electroforesis en Gel Bidimensional/métodos , Fluorouracilo/metabolismo , Humanos , Inmunohistoquímica , Proteínas/análisis , Proteínas/metabolismo , Reproducibilidad de los Resultados , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos

RESUMEN

BACKGROUND: Lactobacillus consumption has been shown to attenuate the severity of experimental colitis. Whether the effects of Lactobacillus on colitis are related to modulation of leukocyte recruitment into the inflamed intestine is unclear. AIMS: To investigate the effect of Lactobacillus plantarum daily intragastric administration on lymphocyte homing and intestinal inflammation in interleukin 10 (IL-10) knockout mice, an experimental model of colitis. METHODS: Two groups of ten IL-10 knockout mice were fed phosphate buffered saline containing Lactobacillus plantarum 1258 or unmodified vehicle for 4 weeks. Two groups of ten wild-type mice were used as controls. At killing, the bowels were histologically scored and evaluated by transmission electron microscopy. Mucosal addressin cell adhesion molecule 1 (MAdCAM-1) and intercellular adhesion molecule 1 (ICAM-1) expression were determined by immunohistochemistry. The levels of proinflammatory cytokines, tumor necrosis factor alpha (TNF-alpha), and interferon gamma (IFN-gamma) were determined by ELISA. In addition, levels of CD3, alpha4beta7, ICAM-1, and MAdCAM-1 were determined by reverse-transcription polymerase chain reaction and Western blot. RESULTS: L. plantarum treatment improved the histological damage score in KO mice compared to untreated KO mice. L. plantarum significantly attenuated the expression of MAdCAM-1, ICAM-1, CD3, and alpha4beta7, but did not affect the levels of TNF-alpha and IFN-gamma when treated KO mice were compared to untreated KO mice. CONCLUSIONS: L. plantarum interfered with the upregulation of adhesion molecules observed in IL-10 knockout mice compared to wild-type mice, attenuating the symptoms of colitis.

Asunto(s)

Moléculas de Adhesión Celular/metabolismo , Colitis/terapia , Colon/microbiología , Lactobacillus plantarum/crecimiento & desarrollo , Probióticos , Animales , Western Blotting , Complejo CD3/metabolismo , Moléculas de Adhesión Celular/genética , Colitis/genética , Colitis/inmunología , Colitis/microbiología , Colitis/patología , Colon/inmunología , Colon/patología , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Inmunohistoquímica , Mediadores de Inflamación/metabolismo , Integrinas/metabolismo , Molécula 1 de Adhesión Intercelular/metabolismo , Interferón gamma/metabolismo , Interleucina-10/deficiencia , Interleucina-10/genética , Masculino , Ratones , Ratones Noqueados , Microscopía Electrónica de Transmisión , Mucoproteínas , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Índice de Severidad de la Enfermedad , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia Arriba

RESUMEN

Surgery and infection are prominent risk factors for the development of obstructive cholestasis which in turn is associated with failure of the liver barrier. We studied the effects of oral Lactobacillus plantarum (LP) supplementation on endotoxemia, oxidative stress, apoptosis, and tight junctions of hepatocytes in an experimental model of obstructive jaundice. Fifty male Wistar rats were randomly divided into five groups of 10 each: group I, sham-operated; group II, ligation and division of the common bile duct (BDL); group III, BLD followed by oral LP treatment; group IV, BDL followed by internal biliary drainage (IBD); group V, BDL followed by IBD and oral LP treatment. Hepatocyte apoptosis, plasma reduced glutathione (GSH) and oxidized glutathione (GSSG) levels, and portal blood endotoxin levels were measured and changes in tight junction-associated proteins occludin, claudin-1, claudin-4, and ZO-1 were observed. Compared to the sham-operated group I, significant increases in endotoxemia, apoptosis, and GSSG were observed in group II and significant decreases were observed in group V. Tight junctions were destroyed in group II animals but were not in animals treated with oral LP (groups III and V). An increase in occludin, claudin-1, claudin-4, and ZO-1 mRNA and protein levels were detected in livers in LP-treated animals (group V) compared with group II levels. Oral LP treatment of rats with obstructive jaundice assisted in the return of active hepatic barrier function. These results may lead to treatments to prevent the deleterious effects of obstructive jaundice.

Asunto(s)

Hepatocitos/metabolismo , Ictericia Obstructiva/fisiopatología , Lactobacillus plantarum/metabolismo , Uniones Estrechas/metabolismo , Administración Oral , Animales , Bilirrubina/metabolismo , Western Blotting , Endotoxinas/metabolismo , Hepatocitos/ultraestructura , Etiquetado Corte-Fin in Situ , Hígado/metabolismo , Hígado/patología , Hígado/ultraestructura , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Oxidación-Reducción , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Compuestos de Sulfhidrilo/metabolismo , Uniones Estrechas/ultraestructura

RESUMEN

OBJECTIVE: To explore the potential markers of colorectal cancer metastasis and the influence of 5-FU on differentially expressed proteins by using proteomic technology, and to elucidate the mechanism of colorectal cancer metastasis. METHODS: Human colorectal carcinoma cell lines of different metastatic potential, Lovo and SW480 were conventionally cultured, and the protein was extracted. 50% inhibitory concentration (IC(50)) of 5-FU to these two cell lines was measured by MTT assay. Proteins of these two cell lines after intervention by 5-FU at IC(50) were extracted, then 2-dimensional gel electrophoresis was conducted for the proteins. The differential protein spots were examined by mass spectrometry and analyzed by bioinformatics. Difference of expressed proteins in two cell lines before and after the intervention of 5-FU was validated by Western blot and immunofluorescence. RESULTS: Eleven differentially expressed proteins were identified by 2-dimensional gel electrophoresis and mass spectrometry. The hnRNP K protein and PDI were selected to be examined by Western blot and immunofluorescence. Results revealed that the expression of hnRNP K in Lovo was higher than that in SW480, while the expression of PDI was lower in Lovo. After intervention by 5-FU at IC(50), the expression of hnRNP K in Lovo decreased more as compared to SW480, while the expression of PDI in SW480 increased more as compared to Lovo. CONCLUSION: There are significant differences in expression of hnRNP K and PDI proteins between Lovo and SW480 cell lines, and the proteins alter regularly after 5-FU intervention.

Asunto(s)

Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Proteómica , Biomarcadores de Tumor/sangre , Línea Celular Tumoral , Fluorouracilo/farmacología , Humanos , Metástasis de la Neoplasia

RESUMEN

Colorectal cancer (CRC) is the third most common cancer worldwide and has poor prognosis. To identify the proteins involved in colorectal carcinogenesis, we employed 2-DE and MALDI-TOF/TOF-based proteomics approach to study the differentially expressed proteins in tumor and adjacent nontumor tissue samples. Samples from 10 colorectal patients were analyzed. Of the 7 significantly and consistently altered proteins identified, hnRNP A1 was one of the most significantly altered proteins and its overexpression was confirmed using RT-PCR and Western blot analyses. Immunohistochemical examination showed that the enhanced expression of hnRNP A1 was correlated with the increasing severity of colorectal tissue and the progression of the colorectal cancer, as well as UICC (International Union against Cancer) staging, histo-differentiation, recurrence and decreased survival. By developing a highly sensitive immunoassay, hnRNP A1 could be detected in human serum and was significantly elevated in CRC patients compared with healthy volunteers. We proposed that hnRNP A1 could be considered as a novel serum tumor marker for CRC that may have significance in the detection and in the management of patients with this disease. Knockdown of hnRNP A1 expression by RNA interference led to the significant suppression of the cell growth in colorectal cancer SW480 cells in vitro. These data suggested that hnRNP A1 may be a potential biomarker for early diagnosis, prognosis, and monitoring in the therapy of colorectal cancer. Further studies are needed to fully assess the potential clinical value of this biomarker candidate.

Asunto(s)

Biomarcadores de Tumor/metabolismo , Neoplasias Colorrectales/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Proteómica/métodos , Anciano , Análisis de Varianza , Área Bajo la Curva , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/genética , Línea Celular Tumoral , Electroforesis en Gel Bidimensional , Femenino , Silenciador del Gen , Ribonucleoproteína Nuclear Heterogénea A1 , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/sangre , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/genética , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Curva ROC , Reproducibilidad de los Resultados , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción