Microbiota-host interactions during antibiotic therapy: unveiling novel redox signaling pathways with impact on gut homeostasis

Abstract

INTRODUÇÃO. O nitrato, constituinte major de vegetais de folha verde, é reduzido a nitrito na boca e a óxido nítrico (•NO) no estômago. Aqui, o •NO induz vasodilatação, estimula a produção de muco e erradica microorganismos sensíveis. Contudo, o microbiota é também essencial para assegurar a homeostasia local, sendo que alterações da sua diversidade são responsáveis pelo aumento da permeabilidade gastrointestinal e pela activação de vias de sinalização inflamatórias. Neste trabalho, é avaliado o impacto do nitrato no perfil do microbiota e o seu efeito em mecanismos-chave da fisiologia gastrointestinal.METODOLOGIA. Ratos Wistar foram divididos em 4 grupos aos quais foi oferecida água suplementada com: 1) antibióticos (neomicina, bacitracina, imipenem), 2) antibióticos e nitrato, 3) nitrato e 4) água sem suplementação. Os animais foram pesados diariamente e foram colhidas fezes antes e depois da intervenção. Ao 7º dia os animais foram eutanasiados e o cego, estômago e cólon foram isolados. Avaliou-se a expressão de proteínas das tight junctions (ocludina; claudina-5) e de marcadores inflamatórios (mieloperoxidase; iNOS). O DNA bacteriano foi amplificado e resolvido por electroforese em gel de gradiente desnaturante. RESULTADOS. Os antibióticos induziram perda ponderal e cegamegalia em todos os animais mas a suplementação com nitrato atenuou significativamente estes efeitos (p<0.05). Quer no estômago, quer no cólon, a expressão das proteínas das tight junctions diminuiu com a exposição aos antibióticos mas recuperou na presença de nitrato (p<0.05). Pelo contrário, os antibióticos aumentaram a expressão dos marcadores inflamatórios (mieloperoxidase e iNOS), o que também se reverteu na presença de nitrato (p<0.05). No cólon, o nitrato aumentou a expressão de claudina-5 após a suplementação com antibióticos (p<0.01), mas teve o efeito oposto relativamente à expressão da ocludina (p<0.001). Os animais tratados com antibióticos e nitrato apresentaram um microbiota mais abundante e diverso do que aqueles apenas expostos aos antimicrobianos.CONCLUSÃO. Este trabalho sugere que o nitrato da dieta assegura a diversidade do microbiota e portanto o seu consumo pode ser útil durante a terapia antibiótica.

Introduction. Dietary nitrate, found in green-leafy vegetables, is reduced to nitrite in the oral cavity and to nitric oxide (•NO) in the stomach. Here, •NO increases mucosal blood flow, mucus thickness and prevents microbial infections. Gut microbiome is now recognized as a pleiotropic organism essential to maintain gastrointestinal welfare, to assure epithelial fence function and to prevent the activation of inflammatory pathways. Physiological disturbances at this level may elicit symptoms such as heartburn, dyspepsia and diarrhea during antibiotherapy. This study investigates the impact of nitrate on gut microbiome profile and ensued mucosal effects during dysbiosis (altered gut flora). Methods. All animal experiments were performed according to the European Community Council Directive for the Care and Use of Laboratory Animals (86/609/ECC). Male Wistar rats were used in this study. Animals were randomly distributed in 4 groups (n=8, per group) and the drinking water was supplemented for 7 days as follows: 1) antibiotic cocktail (neomycin 5 mg/mL, bacitracin 5 mg/mL, imipenem 1.25 μg/mL), 2) antibiotic cocktail + sodium nitrate (10 mM), 3) sodium nitrate and 4) control (no supplementation). Animals were weighted daily and feces were collected before and after the treatment. Animals were anesthetized and euthanized by cervical dislocation. Ceca were collected and weighted. The stomach and ascending colon were isolated and the expression of occludin, claudin-5 as well as myeloperoxidase and iNOS was studied. Bacterial DNA was analyzed in fecal samples by PCR-DGGE genetic fingerprinting. Results. Nitrate prevented antibiotic-induced body weight loss (1.9±1.8 vs 8.9±1.8, % of increase in respect to day 0, p<0.05) and cecamegalia (7.1±0.5 vs 5.6±0.4, % of total body weight, p<0.05). Gastric expression of occludin and claudin-5 was decreased during dysbiosis but both protein levels were recovered by nitrate (p<0.05). Similarly, nitrate inhibited myeloperoxidase and iNOS overexpression under dysbiosis (p<0.05) in the stomach. In the large intestine, nitrate increased claudin-5 expression under dysbiosis (p<0.01) but had the opposite effect on occludin (p<0.001). Microbial richness was significantly decreased by the antibiotic cocktail but the microflora of rats exposed both to antibiotics and nitrate exhibited more similarities to control rather than to dysbiotic animals.Conclusion. This data suggests that dietary nitrate may be envisaged as a functional food with beneficial impact on gastric mucosal integrity and microbial profile during dysbiosis and therefore its consumption may be useful throughout antibiotherapy.