RESUMO
BACKGROUND: Enteroaggregative Escherichia coli (EAEC) is an important pathogen causing enteric infections worldwide. This pathotype is linked to malnutrition in children from developing countries. Alanyl-glutamine (Ala-Gln) is an immune modulator nutrient that acts during intestinal damage and/or inflammation. This study investigated the effect of EAEC infection and Ala-Gln on cell viability, cell death, and inflammation of intestinal epithelium cells (IEC-6). METHODS: Cells were infected with an EAEC prototype 042 strain, an EAEC wild-type strain isolated from a Brazilian malnourished child, and a commensal E coli HS. Gene transcription and protein levels of caspases-3, -8, and -9 and cytokine-induced neutrophil chemoattractant 1 (CINC-1/CXCL1) were evaluated using RT-qPCR, western blot analysis, and ELISA. RESULTS: Infections with both EAEC strains decreased cell viability and induced apoptosis and necrosis after 24âhours. Ala-Gln supplementation increased cell proliferation and reduced cell death in infected cells. Likewise, EAEC strain 042 significantly increased the transcript levels of caspases-3, -8, and -9 when compared to the control group, and Ala-Gln treatment reversed this effect. Furthermore, EAEC induced CXCL1 protein levels, which were also reduced by Ala-Gln supplementation. CONCLUSION: These findings suggest that EAEC infection promotes apoptosis, necrosis, and intestinal inflammation with involvement of caspases. Supplementation of Ala-Gln inhibits cell death, increases cell proliferation, attenuates mediators associated with cell death, and inflammatory pathways in infected cells.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Dipeptídeos/farmacologia , Infecções por Escherichia coli/terapia , Escherichia coli/metabolismo , Substâncias Protetoras/farmacologia , Quimiocina CXCL1/metabolismo , Criança , Suplementos Nutricionais , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/microbiologiaRESUMO
Viperidae venom has several local and systemic effects, such as pain, edema, inflammation, kidney failure and coagulopathy. Additionally, bothropic venom and its isolated components directly interfere on cellular metabolism, causing alterations such as cell death and proliferation. Inflammatory cells are particularly involved in pathological envenomation mechanisms due to their capacity of releasing many mediators, such as nitric oxide (NO). NO has many effects on cell viability and it is associated to the development of inflammation and tissue damage caused by Bothrops and Bothropoides venom. Bothropoides insularis is a snake found only in Queimada Grande Island, which has markedly toxic venom. Thus, the aim of this work was to evaluate the biological effects of Bothropoides insularis venom (BiV) on RAW 264.7 cells and assess NO involvement. The venom was submitted to colorimetric assays to identify the presence of some enzymatic components. We observed that BiV induced H2O2 production and showed proteolytic and phospholipasic activities. RAW 264.7 murine macrophages were incubated with different concentrations of BiV and then cell viability was assessed by MTT reduction assay after 2, 6, 12 and 24 hours of incubation. A time- and concentration-dependent effect was observed, with a tendency to cell proliferation at lower BiV concentrations and cell death at higher concentrations. The cytotoxic effect was confirmed after lactate dehydrogenase (LDH) measurement in the supernatant from the experimental groups. Flow cytometry analyses revealed that necrosis is the main cell death pathway caused by BiV. Also, BiV induced NO release. The inhibition of both proliferative and cytotoxic effects with L-NAME were demonstrated, indicating that NO is important for these effects. Finally, BiV induced an increase in iNOS expression. Altogether, these results demonstrate that B. insularis venom have proliferative and cytotoxic effects on macrophages, with necrosis participation. We also suggest that BiV acts by inducing iNOS expression and causing NO release.
Assuntos
Venenos de Crotalídeos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Macrófagos/metabolismo , Óxido Nítrico Sintase Tipo II/biossíntese , Óxido Nítrico/biossíntese , Viperidae , Animais , Linhagem Celular , Peróxido de Hidrogênio/metabolismo , Camundongos , NG-Nitroarginina Metil Éster/farmacologia , Fatores de TempoRESUMO
PURPOSE: Intestinal mucositis (IM) is a common side effect of anticancer agents. Despite polychemotherapy use in clinical practice, the pathogenesis of IM has been investigated in single drug injection animal models. However, the progression of IM could vary according to drug regimens. Thus, we aimed to develop a new experimental mucositis model induced by combining irinotecan and 5-fluorouracil (5-FU) treatments. METHODS: IM was induced in male C57BL/6 mice by the intraperitoneal administration of either 0.9 % saline (5 mL/kg), irinotecan (IRI, 30 or 45 mg/kg), 5-FU (25, 37.5, or 50 mg/kg), or the combination of these doses (IRI + 5-FU) for 4 days. Animal survival, body mass variation, and diarrhea scores were evaluated daily. On the 7th day, the mice were euthanized, and intestinal samples were collected for histopathology and morphometric analysis, as well as for the determination of myeloperoxidase activity and cytokine dosage (TNF-α and IL-6). RESULTS: The optimal dose combination that induced IM and presented no substantial mortality on the 7th day was IRI (45 mg/kg) + 5-FU (37.5 mg/kg), which was used for subsequent studies. IRI and 5-FU in combination induced significant diarrhea, body weight loss, intestinal damage, inflammatory cell infiltration, and increased levels of cytokines when compared with other groups (P < 0.05). Neither IRI nor 5-FU alone induced IM. CONCLUSIONS: We developed a new experimental model of IM induced by combining irinotecan and 5-FU treatments, which will allow us to gain a better knowledge concerning the pathogenesis of this disease through the pharmacological modulation of key inflammatory mediators.