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. 2019 Feb 20;14(2):e0211436.
doi: 10.1371/journal.pone.0211436. eCollection 2019.

Involvement of Gliadin, a Component of Wheat Gluten, in Increased Intestinal Permeability Leading to Non-Steroidal Anti-Inflammatory Drug-Induced Small-Intestinal Damage

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Involvement of Gliadin, a Component of Wheat Gluten, in Increased Intestinal Permeability Leading to Non-Steroidal Anti-Inflammatory Drug-Induced Small-Intestinal Damage

Sunao Shimada et al. PLoS One. .
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Abstract

Gliadin, a component of wheat gluten known to be an important factor in the etiology of celiac disease, is related to several other diseases through its enhancing effect on intestinal paracellular permeability. We investigated the significance of gliadin in non-steroidal anti-inflammatory drug (NSAID)-induced small-intestinal damage in mice. 7-week-old C57BL/6 male mice were divided into the following groups: standard diet group, in which mice were fed with wheat-containing standard rodent diet (CE-2); gluten-free diet group, in which mice were fed with gluten-free diet (AIN-76A); and gliadin-administered group, in which mice fed with gluten-free diet were administered with gliadin (~250 mg/kg BW). Each group was subdivided into negative, healthy control group and NSAID-treated group. To some mice fed with gluten-free diet and administered with gliadin, epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor was administered for clarification of the significance of EGFR in NSAID-induced small intestinal damage and intestinal permeability. In mice fed with a gluten-free diet, indomethacin or diclofenac induced very mild mucosal damage in the small intestine compared with that in mice fed with a wheat-containing standard diet. Gliadin exacerbated the NSAID-induced small-intestinal damage in mice fed with a gluten-free diet. With the administration of indomethacin, MPO activity, a marker of neutrophil infiltration into the mucosa and mRNA expression level of tumor necrosis factor α and interleukin-1β in the small intestine were higher in the gliadin-administered mice. Gliadin increased the intestinal paracellular permeability without indomethacin administration (4.3-fold) and further increased the permeability after indomethacin administration (2.1-fold). Gliadin induced phosphorylation of epidermal growth factor receptor (EGFR) in small-intestinal tissues, and erlotinib (an EGFR tyrosine kinase inhibitor) attenuated the indomethacin-induced intestinal damage and permeability exacerbated by gliadin, accompanied by inhibition of EGFR phosphorylation. These results suggest that gliadin plays an important role in the induction and exacerbation of NSAID-induced small-intestinal damage, and that increase in intestinal permeability via the EGFR signalling pathway is involved in its mechanism.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Effect of gliadin on small-intestinal damage and inflammation induced by non-steroidal anti-inflammatory drugs (NSAIDs).
(a,b) Lesion indices in the small intestine after NSAID administration (a: indomethacin, b: diclofenac) in mice fed with a standard diet (SD) or a gluten-free diet (GFD). In mice fed with the GFD, we examined the effect of gliadin administration on the lesion indices. Lesion index was defined as the total area of deep-dyeing concavities of the mucosa delineated by intravenous injection of Evans blue dye. n = 4–6. (c) Typical macroscopic and microscopic images of indomethacin-induced small-intestinal damage and the effect of gliadin on the damage. The lesion is macroscopically visualized as areas of the deep-dyeing concavities of the mucosa delineated by intravenous injection of Evans blue dye (arrows). Bars in histological images: 200 μm. (d) Histological scores of the small intestine after indomethacin administration. Histological scores were estimated on a scale from 0 to 13 according to microscopic findings. n = 5. The photo images are typical high-power field images of mucosal break and infiltration of inflammatory cells into the mucosa. (e) Dose-dependent effect of gliadin on lesion indices after indomethacin administration. n = 7. (f) Lesion indices in the small intestine after 10, 20 or 30 mg/kg BW indomethacin administration in mice fed with GFD and administered orally with low dose gliadin (0 (vehicle) or 12.5 mg/kg BW per time). Lesion index was defined as the total area of deep-dyeing concavities of the mucosa delineated by intravenous injection of Evans blue dye. n = 5–6. (g) Myeloperoxidase (MPO) activities of small-intestinal tissue after indomethacin administration. n = 8. (h, i) mRNA expression levels of interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα) in small-intestinal tissues as determined using quantitative reverse transcription polymerase chain reaction. The mRNA expression levels were shown as ratios relative to the mean value for small-intestinal tissues from untreated mice in each group. n = 8. All data were expressed as mean ± standard error of mean. * p < 0.05, ** p < 0.01.
Fig 2
Fig 2. Effect of gliadin on small-intestinal permeability without and with indomethacin administration determined according to relative ratios of the concentration of serum fluorescein isothiocyanate (FITC)-dextran.
The fluorescence was measured using a spectrophotofluorometer, and the concentrations were determined through comparison with a standard curve of serially diluted FITC-dextran. The concentrations of serum FITC-dextran were expressed as ratios relative to the mean value from vehicle-administered mice. n = 5–8. All data were expressed as mean ± standard error of mean. * p < 0.05, ** p < 0.01.
Fig 3
Fig 3. Role of phosphorylation of epidermal growth factor receptor (EGFR) on the exacerbating effect of gliadin on indomethacin-induced small-intestinal damage.
(a) Time course of phosphorylation of EGFR assessed with Western blotting. The phosphorylation levels of EGFR were normalized to those of EGFR, and relative expression levels were expressed as ratios relative to the mean value for small-intestinal tissues from vehicle-treated mice in each experimental group. n = 5–6. (b) Effect of gliadin administration on phosphorylation of EGFR of small-intestinal tissue assessed with Western blotting. Small-intestinal tissues were obtained after 4 h of gliadin administration. The phosphorylation levels of EGFR were normalized to those of EGFR, and relative expression levels were expressed as ratios relative to the mean value for small-intestinal tissues from vehicle-treated mice in each experimental group. n = 5–6. (c) Lesion indices after indomethacin administration with and without intraperitoneal administration of erlotinib. n = 4–6. (d) Histological scores of the small intestine after 24 h of indomethacin administration. Histological scores were estimated on a scale from 0 to 13 according to microscopic findings. n = 5. The photo images are typical high-power filed image of mucosal break and infiltration of inflammatory cells into the mucosa. (e) Effect of erlotinib administration on indomethacin. The concentrations of serum FITC-dextran were expressed as ratios relative to the mean value from indomethacin-and-vehicle-administered mice in each experiment group. n = 5–8. (f) Macroscopic and microscopic photos of small-intestinal lesions after 24 h of indomethacin administration. Lesions were recognized macroscopically as deep-dying blue spots in the small intestine (red arrows). Bars in histological images: 200 μm. All data were expressed as mean ± standard error of mean, *p < 0.05, **p < 0.01.

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References

    1. Graham DY, Opekun AR, Willingham FF, Qureshi WA. Visible small-intestinal mucosal injury in chronic NSAID users. Clin Gastroenterol Hepatol. 2005;3(1):55–9. Epub 2005/01/13. . - PubMed
    1. Sugimori S, Watanabe T, Tabuchi M, Kameda N, Machida H, Okazaki H, et al. Evaluation of small bowel injury in patients with rheumatoid arthritis by capsule endoscopy: effects of anti-rheumatoid arthritis drugs. Digestion. 2008;78(4):208–13. Epub 2009/01/15. 10.1159/000190403 . - DOI - PubMed
    1. Goldstein JL, Eisen GM, Lewis B, Gralnek IM, Zlotnick S, Fort JG. Video capsule endoscopy to prospectively assess small bowel injury with celecoxib, naproxen plus omeprazole, and placebo. Clin Gastroenterol Hepatol. 2005;3(2):133–41. Epub 2005/02/11. . - PubMed
    1. Maiden L, Thjodleifsson B, Theodors A, Gonzalez J, Bjarnason I. A quantitative analysis of NSAID-induced small bowel pathology by capsule enteroscopy. Gastroenterology. 2005;128(5):1172–8. Epub 2005/05/12. . - PubMed
    1. Watanabe T, Tanigawa T, Nadatani Y, Nagami Y, Sugimori S, Okazaki H, et al. Risk factors for severe nonsteroidal anti-inflammatory drug-induced small intestinal damage. Dig Liver Dis. 2013;45(5):390–5. 10.1016/j.dld.2012.12.005 . - DOI - PubMed

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