RESUMO
BACKGROUND: Probiotics decrease the risk of necrotizing enterocolitis (NEC). We sought to determine the impact of Bifidobacterium longum subsp. infantis (B. infantis) in the established rat model of NEC. METHODS: Rat pups delivered 1 d prior to term gestation were assigned to one of three groups: dam fed (DF), formula fed (FF), or fed with formula supplemented with 5 × 10(6) CFU B. infantis per day (FF+Binf). Experimental pups were exposed to hypoxia and cold stress. Ileal tissue was examined for pathology and expression of inflammatory mediators, antimicrobial peptides, and goblet-cell products. Ceca were assessed for bacterial composition by analysis of the 16S rRNA sequence. RESULTS: Administration of B. infantis significantly reduced the incidence of NEC, decreased expression of Il6, Cxcl1, Tnfa, Il23, and iNOS, and decreased expression of the antimicrobial peptides Reg3b and Reg3g. There was significant microbial heterogeneity both within groups and between experiments. The cecal microbiota was not significantly different between the FF and FF+Binf groups. Bifidobacteria were not detected in the cecum in significant numbers. CONCLUSION: In the rat model, the inflammation associated with NEC was attenuated by administration of probiotic B. infantis. Dysbiosis was highly variable, precluding determination of the precise role of the microbiota in experimental NEC.
Assuntos
Bifidobacterium/fisiologia , Enterocolite Necrosante/terapia , Imunidade Inata/fisiologia , Inflamação/terapia , Microbiota , Animais , Bifidobacterium/genética , Ceco/microbiologia , RNA Ribossômico 16S/genética , RatosRESUMO
Enterohepatic helicobacter species (EHS) infect the intestinal tract and biliary tree, triggering intestinal and hepatic disorders. Helicobacter hepaticus, the prototypic murine EHS, is also associated with inflammation. Necrotizing enterocolitis (NEC) is a devastating disease of premature infants. The cause of NEC is not fully understood, but anomalies of bacterial colonization (dysbiosis) are thought to play an important role in disease onset. To evaluate the effect of H. hepaticus infection on the development of NEC, premature formula-fed rats were kept either in H. hepaticus-free conditions or colonized with H. hepaticus; both groups were exposed to asphyxia and cold stress. The incidence of NEC, expression of Toll-like receptors (TLRs), production of cytokines and mucins, and presence of autophagy regulators were evaluated at the site of injury. H. hepaticus infection increased the incidence of NEC from 39 to 71% and significantly increased levels of TLR4 receptor, expression of proinflammatory cytokines CXCL1, IL-1ß, IL-12, and IL-23, and altered activation of autophagy. H. hepaticus induces inflammation and increases the incidence and severity of experimental NEC; this is consistent with observations in neonates of blooms of proinflammatory microbes just before the onset of NEC. Future studies using rodent NEC models should include testing for H. hepaticus infection. Further studies in neonates of early identification and/or diminution of proinflammatory microbes may be beneficial in decreasing the incidence of NEC.
Assuntos
Enterocolite Necrosante/microbiologia , Infecções por Helicobacter/microbiologia , Helicobacter hepaticus/fisiologia , Animais , Autofagia , Citocinas/genética , Citocinas/metabolismo , Enterocolite Necrosante/patologia , Feminino , Regulação da Expressão Gênica/fisiologia , Infecções por Helicobacter/patologia , Íleo/metabolismo , Íleo/microbiologia , Íleo/patologia , Inflamação/metabolismo , Inflamação/patologia , Gravidez , RNA Bacteriano/isolamento & purificação , Ratos , Ratos Sprague-DawleyRESUMO
Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency of premature infants and is characterized by an extensive hemorrhagic inflammatory necrosis of the distal ileum and proximal colon. We have previously shown that, during the development of experimental NEC, the liver plays an important role in regulating inflammation in the ileum, and accumulation of ileal bile acids (BA) along with dysregulation of ileal BA transporters contributes to ileal damage. Given these findings, we speculated that hepatic BA transporters would also be altered in experimental NEC. Using both rat and mouse models of NEC, levels of Cyp7a1, Cyp27a1, and the hepatic BA transporters Bsep, Ntcp, Oatp2, Oatp4, Mrp2, and Mrp3 were investigated. In addition, levels of hepatic BA transporters were also determined when the proinflammatory cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-18, which are both elevated in NEC, are neutralized during disease development. Ntcp and Mrp2 were decreased in NEC, but elevated ileal BA levels were not responsible for these reductions. However, neutralization of TNF-α normalized Ntcp, whereas removal of IL-18 normalized Mrp2 levels. These data show that the hepatic transporters Ntcp and Mrp2 are downregulated, whereas Cyp27a1 is increased in rodent models of NEC. Furthermore, increased levels of TNF-α and IL-18 in experimental NEC may play a role in the regulation of Ntcp and Mrp2, respectively. These data suggest the gut-liver axis should be considered when therapeutic modalities for NEC are developed.
Assuntos
Enterocolite Necrosante/metabolismo , Fígado/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/biossíntese , Transportadores de Ânions Orgânicos Dependentes de Sódio/biossíntese , Simportadores/biossíntese , Animais , Ácidos e Sais Biliares/metabolismo , Western Blotting , Proteínas de Transporte/metabolismo , Colestanotriol 26-Mono-Oxigenase/metabolismo , DNA/biossíntese , DNA/genética , Regulação para Baixo , Enterocolite Necrosante/patologia , Enterócitos/metabolismo , Enterócitos/patologia , Interleucina-18/genética , Interleucina-18/metabolismo , Fígado/patologia , Camundongos , Camundongos Knockout , Proteína 2 Associada à Farmacorresistência Múltipla , RNA/biossíntese , RNA/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
Pomegranate seed oil (PSO), which is the major source of conjugated linolenic acids such as punicic acid (PuA), exhibits strong anti-inflammatory properties. Necrotizing enterocolitis (NEC) is a devastating disease associated with severe and excessive intestinal inflammation. The aim of this study was to evaluate the effects of orally administered PSO on the development of NEC, intestinal epithelial proliferation, and cytokine regulation in a rat model of NEC. Premature rats were divided into three groups: dam fed (DF), formula-fed rats (FF), or rats fed with formula supplemented with 1.5% of PSO (FF + PSO). All groups were exposed to asphyxia/cold stress to induce NEC. Intestinal injury, epithelial cell proliferation, cytokine production, and trefoil factor 3 (Tff3) production were evaluated in the terminal ileum. Oral administration of PSO (FF+PSO) decreased the incidence of NEC from 61 to 26%. Feeding formula with PSO improved enterocyte proliferation in the site of injury. Increased levels of proinflammatory IL-6, IL-8, IL-12, IL-23, and TNF-α in the ileum of FF rats were normalized in PSO-treated animals. Tff3 production in the FF rats was reduced compared with DF but not further affected by the PSO. In conclusion, administration of PSO protects against NEC in the neonatal rat model. This protective effect is associated with an improvement of intestinal epithelial homeostasis and a strong anti-inflammatory effect of PSO on the developing intestinal mucosa.
Assuntos
Enterocolite Necrosante/tratamento farmacológico , Lythraceae/química , Óleos de Plantas/farmacologia , Sementes/química , Animais , Animais Recém-Nascidos , Dieta , Enterocolite Necrosante/patologia , Feminino , Regulação da Expressão Gênica , Íleo/patologia , Lipídeos/química , Mucina-2/genética , Mucina-2/metabolismo , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Óleos de Plantas/química , Gravidez , RNA/genética , RNA/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Fator Trefoil-3RESUMO
INTRODUCTION: Necrotizing enterocolitis (NEC) is a devastating disease of premature infants. Probiotics decrease the risk of NEC in clinical and experimental studies. Antimicrobial peptides protect the gut against noxious microbes and shape the commensal microbiota, but their role in NEC remains unclear. METHODS: To investigate the expression of antimicrobial peptides in experimental NEC and the impact of probiotics on their expression, premature rats were divided into three groups: dam fed (DF), hand fed with formula (FF), or hand fed with formula containing Bifidobacterium bifidum (FF + BIF). All groups were exposed to asphyxia and cold stress. RESULTS: Like in human ontogeny, the rat pup has low expression of Paneth cell antimicrobials, which increases rapidly during normal development. The expression of lysozyme, secretory phospholipase A(2) (sPLA(2)), pancreatic-associated proteins 1 and 3 mRNA was elevated in the FF group with a high incidence of NEC, as compared with the DF and FF + BIF groups where the disease was attenuated. DISCUSSION: We conclude that induction of antimicrobial peptides occurs in experimental NEC similar to that reported in human disease and is attenuated when disease is averted by probiotic B. bifidum. The induction of antimicrobial peptides is likely an adaptive mucosal response that is often not sufficient to prevent disease in the premature gut.
Assuntos
Bifidobacterium , Modelos Animais de Doenças , Enterocolite Necrosante/microbiologia , Peptídeos/metabolismo , Probióticos , Proteínas/metabolismo , Animais , Animais Recém-Nascidos , Anti-Infecciosos , Sequência de Bases , Primers do DNA , Enterocolite Necrosante/metabolismo , Enterocolite Necrosante/terapia , Expressão Gênica , Imuno-Histoquímica , Peptídeos/genética , Reação em Cadeia da Polimerase , Proteínas/genética , Ratos , Ratos Sprague-DawleyRESUMO
PURPOSE OF REVIEW: This review will summarize the clinical and experimental studies evaluating the role of epidermal growth factor (EGF) in prophylaxis and treatment of necrotizing enterocolitis (NEC). RECENT FINDINGS: Clinical studies have suggested the importance of EGF in protection of the intestine against NEC, as well as its safety for infants suffering from NEC. The recent experimental studies identified the molecular mechanisms EGF uses for intestinal protection, which involves regulation of intestinal epithelial homeostasis and barrier function. Further studies are necessary to identify the optimal dose, timing, and route of administration of EGF to NEC patients. No clinical studies are currently underway. SUMMARY: NEC is a devastating problem for preterm neonates, but the exact disease pathogenesis remains unclear. Growing clinical evidence supports the use of EGF as a predictive marker of NEC and its use for prevention and treatment of NEC. In addition, experimental data indicate potential mechanisms of EGF prevention against NEC. These include reduction of inflammation, improvement of barrier function, and regulation of epithelial apoptosis and autophagy.
Assuntos
Enterocolite Necrosante/prevenção & controle , Fator de Crescimento Epidérmico/uso terapêutico , Doenças do Prematuro/prevenção & controle , Animais , Apoptose/fisiologia , Autofagia/fisiologia , Biomarcadores/metabolismo , Enterocolite Necrosante/metabolismo , Enterocolite Necrosante/fisiopatologia , Fator de Crescimento Epidérmico/metabolismo , Humanos , Recém-Nascido , Recém-Nascido Prematuro , Doenças do Prematuro/metabolismo , Doenças do Prematuro/fisiopatologia , Absorção Intestinal/fisiologia , Mucosa Intestinal/patologia , Fígado/fisiopatologiaRESUMO
Barrett's esophagus (BE) is a premalignant condition, where normal squamous epithelium is replaced by intestinal epithelium. BE is associated with an increased risk of developing esophageal adenocarcinoma (EAC). However, the BE cell of origin is not clear. We hypothesize that BE tissue originates from esophageal squamous cells, which can differentiate to columnar cells as a result of repeated exposure to gastric acid and bile acids, two components of refluxate implicated in BE pathology. To test this hypothesis, we repeatedly exposed squamous esophageal HET1A cells to 0.2 mM bile acid (BA) cocktail at pH 5.5 and developed an HET1AR-resistant cell line. These cells are able to survive and proliferate after repeated 2-h treatments with BA at pH 5.5. HET1AR cells are resistant to acidification and express markers of columnar differentiation, villin, CDX2, and cytokeratin 8/18. HET1AR cells have increased amounts of reactive oxygen species, concomitant with a decreased level and activity of manganese superoxide dismutase compared with parental cells. Furthermore, HET1AR cells express proteins and activate signaling pathways associated with inflammation, cell survival, and tumorigenesis that are thought to contribute to BE and EAC development. These include STAT3, NF-κB, epidermal growth factor receptor (EGFR), cyclooxygenase-2, interleukin-6, phosphorylated mammalian target of rapamycin (p-mTOR), and Mcl-1. The expression of prosurvival and inflammatory proteins and resistance to cell death could be partially modified by inhibition of STAT3 signaling. In summary, our study shows that long-term exposure of squamous cells to BA at acidic pH causes the cells to display the same characteristics and markers as BE.
Assuntos
Esôfago de Barrett/etiologia , Ácidos e Sais Biliares/farmacologia , Esôfago/efeitos dos fármacos , Ácidos/farmacologia , Adenocarcinoma/metabolismo , Biomarcadores/metabolismo , Linhagem Celular Transformada , Regulação para Baixo , Resistência a Medicamentos , Epitélio/metabolismo , Neoplasias Esofágicas/metabolismo , Esôfago/citologia , Esôfago/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Interleucina-6/metabolismo , Metaplasia/metabolismo , NF-kappa B/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Superóxido Dismutase/metabolismo , Regulação para CimaRESUMO
The aim of this study was to develop and validate fast and easily applicable GC/MS assay for the quantification of the substance that increases cetane number in diesel fuel (2-ethylhexylnitrate, 2-EHN). These requirements were fulfilled best by a headspace GC-MS assay with negative chemical ionization with methane (HS-GC/MS). Chromatographic separation is achieved using a DB5-MS capillary column after the addition of known amount of internal standard (o-nitrotoluene). The limit of detection was 0.009% v/v for 2-EHN and the limit of quantification was 0.03% v/v. The HS-GC/MS method was applied for the quantification of cetane improver in spiked diesel fuel and real diesel fuel. The method is linear over the studied range (0.03-0.3%, v/v), with satisfactory intra- and inter-assay precision, and the relative standard deviations are lower than 10%. Good accuracy is achieved with bias <10% at all levels tested.
RESUMO
Necrotizing enterocolitis (NEC) is a devastating intestinal disease of neonates, and clinical studies suggest the beneficial effect of probiotics in NEC prevention. Recently, we have shown that administration of Bifidobacterium bifidum protects against NEC in a rat model. Intestinal apoptosis can be suppressed by activation of cyclooxygenase-2 (COX-2) and increased production of prostaglandin E(2) (PGE(2)). The present study investigates the effect of B. bifidum on intestinal apoptosis in the rat NEC model and in an intestinal epithelial cell line (IEC-6), as a mechanism of protection against mucosal injury. Premature rats were divided into the following three groups: dam fed, hand fed with formula (NEC), or hand fed with formula supplemented with B. bifidum (NEC + B. bifidum). Intestinal Toll-like receptor-2 (TLR-2), COX-2, PGE(2), and apoptotic regulators were measured. The effect of B. bifidum was verified in IEC-6 cells using a model of cytokine-induced apoptosis. Administration of B. bifidum increased expression of TLR-2, COX-2, and PGE(2) and significantly reduced apoptosis in the intestinal epithelium of both in vivo and in vitro models. The Bax-to-Bcl-w ratio was shifted toward cell survival, and the number of cleaved caspase-3 positive cells was markedly decreased in B. bifidum-treated rats. Experiments in IEC-6 cells showed anti-apoptotic effect of B. bifidum. Inhibition of COX-2 signaling blocked the protective effect of B. bifidum treatment in both in vivo and in vitro models. In conclusion, oral administration of B. bifidum activates TLR-2 in the intestinal epithelium. B. bifidum increases expression of COX-2, which leads to higher production of PGE(2) in the ileum and protects against intestinal apoptosis associated with NEC. This study indicates the ability of B. bifidum to downregulate apoptosis in the rat NEC model and in IEC-6 cells by a COX-2-dependent matter and suggests a molecular mechanism by which this probiotic reduces mucosal injury and preserves intestinal integrity.
Assuntos
Apoptose , Bifidobacterium , Enterocolite Necrosante/microbiologia , Íleo/microbiologia , Mucosa Intestinal/microbiologia , Análise de Variância , Animais , Bifidobacterium/metabolismo , Western Blotting , Caspase 3/genética , Caspase 3/metabolismo , Linhagem Celular , Proliferação de Células , Células Cultivadas , Distribuição de Qui-Quadrado , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/genética , Dinoprostona/metabolismo , Enterocolite Necrosante/metabolismo , Enterocolite Necrosante/patologia , Citometria de Fluxo , Íleo/metabolismo , Íleo/patologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency of premature infants. Previously, we showed that luminal bile acids (BAs) are increased and correlated with disease development and that the apical sodium-dependent BA transporter (ASBT), which transports BAs from the ileal lumen into enterocytes, is upregulated in rats with NEC. We hypothesized that intraenterocyte, rather than luminal, BAs are associated with NEC and that upregulation of ASBT may be a mechanism by which this occurs. Neonatal rats with or without the ASBT inhibitor SC-435, mice in which ASBT was knocked out, and mice that overproduce BAs were subjected to the NEC protocol. Disease development, ASBT, and the farnesoid X receptor protein, along with luminal and intraenterocyte BA levels, were assessed. In addition, ileal sections from premature infants with and without NEC were examined for ASBT via immunohistology and real-time PCR. When BAs were not transported into enterocytes (rats given SC-435 and ASBT knockout mice), severity and incidence of NEC were reduced. In contrast, in mice that overproduce BAs, ASBT was elevated, intraenterocyte BAs were increased, and disease development was increased. ASBT staining was more intense on the apical membrane of ileal enterocytes from premature infants with NEC than premature infants with non-NEC diagnoses. In addition, ASBT mRNA levels were significantly higher in infants with NEC. These data show that accumulation of intraenterocyte BAs contributes to disease development, elevated ASBT increases disease severity in experimental models of NEC, and ASBT is elevated in human NEC. These data confirm that BAs and upregulation of ASBT play a crucial role in NEC pathogenesis and suggest that inhibition of ASBT could be utilized as a therapeutic modality against this disease.
Assuntos
Enterocolite Necrosante/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Simportadores/metabolismo , Regulação para Cima/fisiologia , Animais , Animais Recém-Nascidos , Óxidos N-Cíclicos , Enterócitos/metabolismo , Humanos , Íleo/patologia , Recém-Nascido , Recém-Nascido Prematuro , Camundongos , Camundongos Knockout , Transportadores de Ânions Orgânicos Dependentes de Sódio/antagonistas & inibidores , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Simportadores/antagonistas & inibidores , TropanosRESUMO
Necrotizing enterocolitis (NEC) is a devastating intestinal disease of premature infants. Epidermal growth factor (EGF) is one of the most promising candidates in NEC prophylaxis. Autophagy regulates cell homeostasis, but uncontrolled activation of autophagy may lead to cellular injury. The aim was to evaluate the effects of EGF on intestinal autophagy in epithelial cells and in the rat NEC model and measure autophagy in NEC patients. Intestinal epithelial cells (IEC-6) and the rat NEC model were used to study the effect of EGF on intestinal autophagy. Protein levels of Beclin 1 and LC3II were measured in the intestinal epithelium in both in vivo and in vitro models. Ultrastructural changes in intestinal epithelium were studied by electron microscopy. Expression of Beclin 1, LC3II, and p62 protein was evaluated in biopsies from NEC patients. Autophagy was induced in IEC-6 cells and inhibited by adding EGF into the culture. In the rat NEC model, EGF treatment of NEC reduced expression of Beclin 1 and LC3II in ileal epithelium. Morphologically, typical signs of autophagy were observed in the epithelium of the NEC group, but not in the EGF group. A strong signal for Beclin 1 and LC3II was detected in the intestine from patients with NEC. Autophagy is activated in the intestinal epithelium of NEC patients and in the ileum of NEC rats. Supplementation of EGF blocks intestinal autophagy in both in vivo and in vitro conditions. Results from this study indicate that EGF-mediated protection against NEC injury is associated with regulation of intestinal autophagy.
Assuntos
Autofagia , Enterocolite Necrosante/tratamento farmacológico , Fator de Crescimento Epidérmico/farmacologia , Fator de Crescimento Epidérmico/uso terapêutico , Mucosa Intestinal/patologia , Administração Oral , Animais , Animais Recém-Nascidos , Proteínas Reguladoras de Apoptose/metabolismo , Proteína Beclina-1 , Linhagem Celular , Modelos Animais de Doenças , Enterocolite Necrosante/patologia , Fator de Crescimento Epidérmico/administração & dosagem , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Incidência , Mucosa Intestinal/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Maternal milk is a complex fluid, with multifunctional roles within the developing gastrointestinal tract. Epidermal growth factor (EGF) and heparin-binding EGF-like growth factor (HB-EGF) are members of the family of EGF-related peptides. Biological actions of these growth factors are mediated via interaction with the EGF-receptor (EGF-R). In the early postnatal period, breast milk is the major source of EGF for the developing intestinal mucosa. HB-EGF is also detected in breast milk, but in concentrations 2 to 3 times lower than EGF. With normal physiological conditions, the intestinal epithelium undergoes a continuing process of cell proliferation, differentiation, and maturation. EGF plays an important role in these processes. In pathophysiologic situations, EGF contributes to epithelial protection from injury and post-injury mucosal repair. Necrotizing enterocolitis (NEC) is a devastating disease affecting infants born prematurely. The pathogenesis of NEC is not known, and there is no effective treatment for this disease. In an experimental NEC model, oral administration of a physiological dose of EGF significantly reduces the incidence and severity of NEC. HB-EGF provides similar protection against NEC, but only when pharmacological doses are used. Further studies are necessary before EGF can be introduced as an efficient therapeutic approach of intestinal injury.
Assuntos
Fator de Crescimento Epidérmico/fisiologia , Trato Gastrointestinal/crescimento & desenvolvimento , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Leite Humano/química , Animais , Enterocolite Necrosante/fisiopatologia , Enterocolite Necrosante/terapia , Fator de Crescimento Epidérmico/uso terapêutico , Feminino , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Humanos , Lactente , Recém-Nascido , Mucosa Intestinal/fisiologiaRESUMO
The reaction of α-amino-ω-methoxypoly(ethylene glycol) [M = 5000] or star α-amino-poly(ethylene glycol) [M = 20 000] with hemiesters of prednisolone dicarboxylic acids (succinic, glutaric, adipic, phthalic acid) has been used to prepare the corresponding conjugates. The rate of esterase catalyzed hydrolysis of the conjugates is controlled by the molecular mass of poly(ethylene glycol) and the length of the linker between prednisolone and poly(ethylene glycol) (τ(1/2)â¼ 5-0.5 h). The enzymatic hydrolysis proceeds most rapidly at conjugates with linkers derived from adipic and phthalic acids. The synthesized conjugates form polypseudorotaxanes with α-cyclodextrin which were characterized by 2D NOESY NMR spectra, powder X-ray diffraction patterns and in one case also by STM microscopy. In the case of the polypseudorotaxane having the linker derived from adipic acid, the enzymatic release proceeds ca. five times slower in comparison with the rate of prednisolone release from the corresponding conjugate. The rate of prednisolone release from the carrier can be controlled by three factors: character of the linker between the polymeric carrier and prednisolone, the molecular mass of poly(ethylene glycol) and complex formation with α-cyclodextrin. The synthesized polypseudorotaxanes represent new promising transport systems intended for targeted release of prednisolone in transplanted liver.
Assuntos
Ciclodextrinas/química , Poloxâmero/química , Polietilenoglicóis/química , Prednisolona/química , Rotaxanos/química , alfa-Ciclodextrinas/química , Microscopia de Tunelamento , Estrutura MolecularRESUMO
Neonatal necrotizing enterocolitis (NEC) is a major cause of morbidity and mortality in premature infants. Oral administration of probiotics has been suggested as a promising strategy for prevention of NEC. However, little is known about the mechanism(s) of probiotic-mediated protection against NEC. The aim of this study was to evaluate the effects of Bifidobacterium bifidum treatment on development of NEC, cytokine regulation, and intestinal integrity in a rat model of NEC. Premature rats were divided into three groups: dam fed (DF), hand fed with formula (NEC), or hand fed with formula supplemented with 5 x 10(6) CFU B. bifidum per day (B. bifidum). All groups were exposed to asphyxia and cold stress to develop NEC. Intestinal injury, mucin and trefoil factor 3 (Tff3) production, cytokine levels, and composition of tight junction (TJ) and adherens junction (AJ) proteins were evaluated in the terminal ileum. B. bifidum decreased the incidence of NEC from 57 to 17%. Increased levels of IL-6, mucin-3, and Tff3 in the ileum of NEC rats was normalized in B. bifidum treated rats. Reduced mucin-2 production in the NEC rats was not affected by B. bifidum. Administration of B. bifidum normalized the expression and localization of TJ and AJ proteins in the ileum compared with animals with NEC. In conclusion, administration of B. bifidum protects against NEC in the neonatal rat model. This protective effect is associated with reduction of inflammatory reaction in the ileum, regulation of main components of mucus layer, and improvement of intestinal integrity.
Assuntos
Bifidobacterium , Enterocolite Necrosante/microbiologia , Enterocolite Necrosante/terapia , Intestinos/microbiologia , Intestinos/patologia , Probióticos/uso terapêutico , Junções Aderentes/metabolismo , Animais , Animais Recém-Nascidos , Asfixia/complicações , Caderinas/metabolismo , Cateninas/metabolismo , Claudina-3 , Temperatura Baixa , Modelos Animais de Doenças , Enterocolite Necrosante/metabolismo , Enterocolite Necrosante/patologia , Enterócitos/metabolismo , Expressão Gênica/genética , Íleo/metabolismo , Íleo/patologia , Incidência , Interleucinas/genética , Mucosa Intestinal/metabolismo , Proteínas de Membrana/metabolismo , Mucina-2/genética , Mucina-2/metabolismo , Mucina-3/genética , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Ocludina , Ratos , Ratos Sprague-Dawley , Estresse Fisiológico , Junções Íntimas/metabolismo , Fator Trefoil-3 , Fator de Necrose Tumoral alfa/genéticaRESUMO
Necrotizing enterocolitis (NEC) is a devastating disease of premature babies. Previously, we have shown that EGF reduces NEC and that overproduction of hepatic TNF-alpha is associated with intestinal damage. Leakage of TNF-alpha may be a consequence of epithelial hepatic cellular junction dysfunction. The aim of this study was to investigate changes in the composition of hepatic tight junctions (TJs) and adherens junctions (AJs). Using an established rat model of NEC, animals were divided into the following groups: dam fed (DF), formula fed (NEC), or fed with formula supplemented with EGF (EGF). Serum EGF and histologic localization of major TJ and AJ proteins were evaluated. Distribution patterns of hepatic TJ and AJ proteins were significantly altered in the NEC group compared with those in DF or EGF groups. Cytoplasmic accumulation of occludin, claudin-2, and ZO-1 with reduction of claudin-3 signal was detected in the liver of NEC rats. Localization of beta-catenin was associated with the hepatocyte membrane in EGF and DF groups, but diffused in the NEC group. These data show that hepatic cellular junctions are significantly altered during NEC pathogenesis. EGF-mediated reduction of experimental NEC is associated with protection of hepatic integrity and structure.
Assuntos
Enterocolite Necrosante , Fator de Crescimento Epidérmico , Hepatócitos , Junções Intercelulares , Ração Animal , Animais , Caderinas/metabolismo , Claudina-1 , Claudina-3 , Claudinas , Dieta , Modelos Animais de Doenças , Enterocolite Necrosante/tratamento farmacológico , Enterocolite Necrosante/patologia , Fator de Crescimento Epidérmico/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Fator de Crescimento Epidérmico/uso terapêutico , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Humanos , Junções Intercelulares/química , Junções Intercelulares/efeitos dos fármacos , Junções Intercelulares/ultraestrutura , Proteínas de Membrana/metabolismo , Ocludina , Fosfoproteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/metabolismo , Proteína da Zônula de Oclusão-1 , Proteína da Zônula de Oclusão-2 , alfa Catenina/metabolismo , beta Catenina/metabolismoRESUMO
The multifunctional cytokine interleukin-18 (IL-18) is an important mediator in intestinal inflammatory processes. The aim of this study was to evaluate the constitutive expression of IL-18 and its receptors (IL-18Ralpha and IL-18Rbeta) in intestinal epithelial cells (IEC) stimulated by tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). In addition, cellular proliferation and evaluation of brush border enzymes as differentiation markers were studied. Nontransformed rat intestinal epithelial IEC-6 cells were grown on an extracellular matrix (ECM) in medium with or without TNF-alpha, IFN-gamma, or a combination of both. Gene expression of IL-18, its receptors and apoptotic markers was evaluated using real-time PCR. Expression of IL-18Ralpha protein was demonstrated by flow cytometry and Western blot. Enzymatic activities of brush border enzymes and caspase-1 were determined. The constitutive expression of IL-18, IL-18Ralpha and IL-18Rbeta mRNAs and proteins were detected in IEC-6 cells. The biologically active form of IL-18 was released in response to TNF-alpha and IFN-gamma treatment. Exogenous IL-18 had no effect on cellular proliferation, brush border enzyme activities, and gene expression of apoptotic markers. However, the addition of IL-18 stimulated production and release of the chemokine IL-8. These data suggest that IEC-6 cells may be not only a source of IL-18 but also a target for its action.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Células Epiteliais/citologia , Interferon gama/farmacologia , Subunidade alfa de Receptor de Interleucina-18/genética , Subunidade beta de Receptor de Interleucina-18/genética , Interleucina-18/genética , Fator de Necrose Tumoral alfa/farmacologia , Animais , Apoptose/efeitos dos fármacos , Biomarcadores/metabolismo , Caspase 1/metabolismo , Contagem de Células , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Quimiocina CCL2/metabolismo , Meios de Cultura , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Células Epiteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-18/metabolismo , Subunidade alfa de Receptor de Interleucina-18/metabolismo , Subunidade beta de Receptor de Interleucina-18/metabolismo , Intestinos/citologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Bile acids (BAs) facilitate emulsification, absorption, and transport of fats and sterols in the intestine and liver and are essential for normal digestion. However, accumulation of BAs in the intestine can result in damage to the intestinal epithelium. Using the neonatal rat model of necrotizing enterocolitis (NEC), we have recently shown that BAs accumulate in both the ileal lumen and enterocytes of neonatal rats with NEC and the increased BA levels are positively correlated with disease severity. Importantly, when BAs are not allowed to accumulate, neonatal rat pups develop significantly less disease. In addition, BA transporters are altered during disease development. These data indicate that BAs play an important role in the development of experimental NEC, and suggest that the inability of neonatal rats to adequately regulate BA transporters may be a mechanism by which ileal damage occurs.
Assuntos
Ácidos e Sais Biliares/efeitos adversos , Ácidos e Sais Biliares/metabolismo , Enterocolite Necrosante/patologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Animais , Modelos Animais de Doenças , Enterocolite Necrosante/etiologia , Humanos , Imuno-Histoquímica , Recém-Nascido , Recém-Nascido Prematuro , Mucosa Intestinal/lesões , Sistema Porta , Ratos , Índice de Gravidade de DoençaRESUMO
OBJECTIVES: The molecular factors contributing to the development of Barrett's esophagus (BE) are unclear. Our previous studies showed that BE tissues secrete interleukin-6 (IL-6) and express proteins associated with IL-6 signaling, including IL-6 receptor, activated signal transducer and activators of transcription 3 (STAT3), and antiapoptotic proteins Bcl-x(L) and Mcl-1. Here, we test the hypothesis that bile acids and gastric acids, two components of refluxate associated with gastresophageal reflux disease, activate the IL-6/STAT3 pathway. MATERIALS AND METHODS: Immunohistochemistry was used to assess levels of phosphorylated STAT3 in esophageal tissue samples from BE patients with different grades of dysplasia. Seg-1 esophageal adenocarcinoma cells were evaluated for STAT3 activation and IL-6 and Bcl-x(L) expression by molecular biology techniques, including Western blot, reverse transcription-PCR, and ELISA after exposure to control media (pH 7.4), media supplemented with a 0.1 mmol/L bile acid cocktail with media at pH 4 or media at pH 4 with bile acid cocktail. RESULTS: Immunohistochemical analysis showed that activated, phosphorylated STAT3 is expressed in nuclei of dysplastic BE and cancer tissues. Treatment of Seg-1 cells with media containing bile acid cocktail and acidified to pH 4 resulted in increased activation of STAT3, IL-6 secretion, and increased expression of Bcl-x(L). Inhibition of the STAT3 pathway using STAT3 small interfering RNA or Janus-activated kinase inhibitor resulted in increased apoptosis. CONCLUSIONS: The IL-6/STAT3 antiapoptotic pathway is induced by short exposure to bile acid cocktail and low pH. This alteration, if persistent in vivo, may underlie the development of dysplastic BE and tumor progression.
Assuntos
Esôfago de Barrett/metabolismo , Ácidos e Sais Biliares/metabolismo , Esôfago/metabolismo , Interleucina-6/metabolismo , Fator de Transcrição STAT3/metabolismo , Adenocarcinoma/química , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose , Esôfago de Barrett/patologia , Ácidos e Sais Biliares/farmacologia , Neoplasias Esofágicas/química , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Esôfago/efeitos dos fármacos , Esôfago/patologia , Feminino , Ácido Gástrico/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Interleucina-6/genética , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Fator de Transcrição STAT3/análise , Fator de Transcrição STAT3/genética , Células Tumorais Cultivadas , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
OBJECTIVES: A primary objective was to evaluate whether addition of enteral iron supplementation will facilitate a systemic erythropoietic effect when feeding erythropoietin (Epo) to suckling rats. A secondary objective was to confirm that iron does not alter the previous finding that enteral Epo exerts local trophic effects on the small intestine. METHODS: Four-day-old Sprague-Dawley rats underwent gastrostomy and were fed a cow's milk-based rat milk substitute for 8 days. We studied rats fed rat milk substitute alone (control), enteral Epo 425 U x kg(-1) x day(-1), and enteral Epo 1700 U x kg(-1) x day(-1), and the effects of oral iron sulfate (Fe) therapy (6 mg x kg(-1) x day(-1)). Blood was collected to measure hemoglobin (Hb), reticulocytes, red cell indices, and zinc protoporphyrin/heme. To confirm previous work describing trophic effects of enteral Epo on the intestine, duodenal villous height was measured. RESULTS: Hb levels in control (84 +/- 1 g/L) were similar to Epo 425 (87 +/- 1 g/L). Hb levels in control+Fe (97 +/- 1 g/L), Epo 425+Fe (97 +/- 1 g/L), and Epo 1700 (94 +/- 1 g/L) were higher than control, P < 0.001, but mean Hb level in Epo 1700+Fe was higher (105 +/- 1 g/L) than the other groups, P < 0.003. Mean cell volume was higher in rats receiving iron supplementation, compared with those without iron, P < 0.005. Duodenal villous height was taller in Epo 1700+Fe compared with control + Fe, P < 0.01. CONCLUSIONS: If combined with sufficient iron supplementation, high-dose Epo artificially fed to suckling rats exerted a systemic erythropoietic effect in addition to the previously reported local trophic effects.
Assuntos
Nutrição Enteral , Eritropoese/efeitos dos fármacos , Eritropoetina/administração & dosagem , Ferro da Dieta/administração & dosagem , Animais , Animais Lactentes , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Quimioterapia Combinada , Duodeno/efeitos dos fármacos , Duodeno/crescimento & desenvolvimento , Índices de Eritrócitos , Eritropoese/fisiologia , Eritropoetina/farmacologia , Gastrostomia , Hemoglobinas/análise , Humanos , Recém-Nascido , Recém-Nascido Prematuro/crescimento & desenvolvimento , Doenças do Prematuro/tratamento farmacológico , Ferro da Dieta/farmacologia , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Proteínas RecombinantesRESUMO
BACKGROUND: Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease of prematurely born infants. Epidermal growth factor (EGF) and heparin-binding EGF-like growth factor (HB-EGF) have protective effects against intestinal injury. The aim of this study was to compare the effect of oral administration of HB-EGF, EGF, or both on the incidence of NEC in a neonatal rat model. MATERIALS AND METHODS: Premature rats were fed by hand and exposed to asphyxia and cold stress to develop NEC. Four diets were used: formula (NEC), formula supplemented with 500 ng/mL HB-EGF (HB), 500 ng/mL EGF (EGF), or a combination of both (E+HB). Ileal injury, endogenous HB-EGF production, expression of EGF receptors, goblet cell density, and expression of apoptotic proteins were evaluated. RESULTS: Oral administration of either EGF or HB-EGF significantly reduced the incidence of NEC; however, EGF provided better protection in physiologically relevant doses. Simultaneous administration of both growth factors did not result in any synergistic protective effect against NEC. There were no significant differences between treatment groups in ileal gene expression of EGF receptors or HB-EGF. However, the balance of apoptotic proteins in the ileum was shifted in favor of cell survival in EGF-treated rats. This mechanism may be responsible for the higher efficiency of EGF protection against NEC. CONCLUSIONS: These data suggest that a physiological dosage of EGF or a pharmacological dosage of HB-EGF could be used for prevention of NEC.