Inorganic nitrate prevents the loss of tight junction proteins and modulates inflammatory events induced by broad-spectrum antibiotics: A role for intestinal microbiota?

Upon consumption, dietary nitrate 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. However, the impact of nitrate on gut microbiota, a pleiotropic organism essential to maintain gastrointestinal and systemic welfare, remains elusive. This study investigates the impact of nitrate on gut microbiota profile and ensued mucosal effects during dysbiosis. Male Wistar rats were randomly distributed in 4 groups and the drinking water was supplemented for 7 days as follows: 1) antibiotic cocktail (neomycin, bacitracin and imipenem), 2) antibiotic cocktail + sodium nitrate, 3) sodium nitrate and 4) regular drinking water. Animals were weighted daily and feces were collected before and after the treatment. The stomach was 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. Nitrate prevented antibiotic-induced body weight loss (1.9 ± 1.8% vs 8.9 ±â€¯1.8%, p < 0.05) and cecamegalia (7.1 ±â€¯0.5% vs 5.6 ±â€¯0.4%, p < 0.05). Gastric expression of occludin and claudin-5 tended to decrease during dysbiosis but both protein levels were recovered following nitrate consumption (p < 0.05). Similarly, nitrate inhibited the overexpression of myeloperoxidase and iNOS observed under dysbiosis (p < 0.05). Broad spectrum antibiotics significantly decreased microbiota richness and diversity in comparison to controls (p = 0.0016). After 7 days of treatment, whereas antibiotics reduced microbiota richness by 56%, it was observed that nitrate was able to prevent such microbial loss to only 48%, although without statistical differences (p = 0.068). This data suggests that dietary nitrate may be envisaged as a key component of functional foods with beneficial impact on gastric mucosal integrity during antibiotherapy but further studies are mandatory to better ascertain as to whether it modulates intestinal microbiota in terms of taxonomic and functional levels.

Dietary nitrite induces occludin nitration in the stomach.

The clinical implications of the nitrate-nitrite-nitric oxide pathway have been extensively studied in recent years. However, the physiological impact of bioactive nitrogen oxides produced from dietary nitrate has remained largely elusive. Here, we report a hitherto unrecognized nitrite-dependent nitrating pathway that targets tight junction proteins in the stomach. Inorganic nitrate, nitrite or saliva obtained after the consumption of lettuce were administered by oral gavage to Wistar rats. The enterosalivary circulation of nitrate was allowed to occur for 4 h after which the animals were euthanized and the stomach collected. Nitrated occludin was detected by immunoprecipitation in the gastric epithelium upon inorganic nitrite administration (p < .05) but was not observed in the case of inorganic nitrate or human saliva administration. This observation, along with differences in •NO production rates from inorganic and salivary nitrite under simulated gastric conditions, suggests that competing reactions at acidic pH determine the production of nitrating agents (•NO2) or other, more stable, oxides. Accordingly, it is shown in vitro that salivary nitrite yields higher steady state concentrations of •NO (0.37 ± 0.01 µM) than sodium nitrite (0.12 ± 0.03 µM). Dietary-dependent reactions involving the production of nitrogen oxides should be further investigated as, in the context of occludin nitration, the consumption of green leafy vegetables (with high nitrate content), if able to modulate gut barrier function, may have important implications in the context of leaky gut disorders.