Digestive Diseases and Sciences

, Volume 63, Issue 1, pp 92–104 | Cite as

Protective Effect of 1,25-Dihydroxy Vitamin D3 on Pepsin–Trypsin-Resistant Gliadin-Induced Tight Junction Injuries

  • Shouquan Dong
  • Tikka Prabhjot Singh
  • Xin Wei
  • Huang Yao
  • Hongling WangEmail author
Original Article



Tight junction (TJ) injuries induced by pepsin–trypsin-resistant gliadin (PT–G) play an important role in the pathogenesis of celiac disease. Previously, 1,25-dihydroxy vitamin D3 (VD3) was reported to be a TJ regulator that attenuates lipopolysaccharide- and alcohol-induced TJ injuries. However, whether VD3 can attenuate PT–G-induced TJ injuries is unknown.


The aim of this study was to evaluate the effects of VD3 on PT–G-induced TJ injuries.


Caco-2 monolayers were used as in vitro models. After being cultured for 21 days, the monolayers were treated with PT–G plus different concentrations of VD3. Then, the changes in trans-epithelial electrical resistance and FITC-dextran 4000 (FD-4) flux were determined to evaluate the monolayer barrier function. TJ protein levels were measured to assess TJ injury severity, and myeloid differentiation factor 88 (MyD88) expression and zonulin release levels were determined to estimate zonulin release signaling pathway activity. Additionally, a gluten-sensitized mouse model was established as an in vivo model. After the mice were treated with VD3 for 7 days, we measured serum FD-4 concentrations, TJ protein levels, MyD88 expression, and zonulin release levels to confirm the effect of VD3.


Both in vitro and in vivo, VD3 significantly attenuated the TJ injury-related increase in intestinal mucosa barrier permeability. Moreover, VD3 treatment up-regulated TJ protein expression levels and significantly decreased MyD88 expression and zonulin release levels.


VD3 has protective effects against PT–G-induced TJ injuries both in vitro and in vivo, which may correlate with the disturbance of the MyD88-dependent zonulin release signaling pathway.


Pepsin–trypsin-resistant gliadin 1,25-Dihydroxy vitamin D3 Tight junction MyD88 Zonulin release 


Author’s contribution

SD performed the majority of the experiments. XW and HY performed the animal experiments and analyzed the data. HW and TPS designed the research. SD, TPS, and HW wrote the paper.

Compliance with ethical standards

Conflict of interest

All the authors declare that there are no conflict of interests for this article.


  1. 1.
    Kang JY, Kang AH, Green A, Gwee KA, Ho KY. Systematic review: Worldwide variation in the frequency of coeliac disease and changes over time. Aliment Pharmacol Ther. 2013;38:226–245.CrossRefPubMedGoogle Scholar
  2. 2.
    Caminero A, Galipeau HJ, McCarville JL, et al. Duodenal bacteria from patients with celiac disease and healthy subjects distinctly affect gluten breakdown and immunogenicity. Gastroenterology. 2016;151:670–683.CrossRefPubMedGoogle Scholar
  3. 3.
    Maiuri L, Ciacci C, Ricciardelli I, et al. Association between innate response to gliadin and activation of pathogenic T cells in coeliac disease. Lancet. 2003;362:30–37.CrossRefPubMedGoogle Scholar
  4. 4.
    Almeida LM, Gandolfi L, Pratesi R, et al. Presence of DQ2.2 associated with DQ2.5 increases the risk for celiac disease. Autoimmune Dis. 2016;2016:5409653.PubMedPubMedCentralGoogle Scholar
  5. 5.
    Lammers KM, Khandelwal S, Chaudhry F, et al. Identification of a novel immunomodulatory gliadin peptide that causes interleukin-8 release in a chemokine receptor CXCR3-dependent manner only in patients with coeliac disease. Immunology. 2011;132:432–440.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Lammers KM, Lu R, Brownley J, et al. Gliadin induces an increase in intestinal permeability and zonulin release by binding to the chemokine receptor CXCR3. Gastroenterology. 2008;135:194.e3–204.e3.Google Scholar
  7. 7.
    Sturgeon C, Fasano A. Zonulin, a regulator of epithelial and endothelial barrier functions, and its involvement in chronic inflammatory diseases. Tissue Barriers. 2016;4:e1251384.CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Fasano A. Intestinal permeability and its regulation by zonulin: diagnostic and therapeutic implications. Ann N Y Acad Sci. 2012;1258:25–33.CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Sapone A, de Magistris L, Pietzak M, et al. Zonulin upregulation is associated with increased gut permeability in subjects with type 1 diabetes and their relatives. Diabetes. 2006;55:1443–1449.CrossRefPubMedGoogle Scholar
  10. 10.
    Vanuytsel T, Vermeire S, Cleynen I. The role of Haptoglobin and its related protein, Zonulin, in inflammatory bowel disease. Tissue Barriers. 2013;1:e27321.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Chiba H, Osanai M, Murata M, Kojima T, Sawada N. Transmembrane proteins of tight junctions. Biochim Biophys Acta. 2008;1778:588–600.CrossRefPubMedGoogle Scholar
  12. 12.
    Thomas KE, Sapone A, Fasano A, Vogel SN. Gliadin stimulation of murine macrophage inflammatory gene expression and intestinal permeability are MyD88-dependent: role of the innate immune response in Celiac disease. J Immunol. 2006;176:2512–2521.CrossRefPubMedGoogle Scholar
  13. 13.
    Chung BH, Kim BM, Doh KC, et al. Suppressive effect of 1α,25-dihydroxyvitamin D3 on Th17-immune responses in kidney transplant recipients with tacrolimus-based immunosuppression. Transplantation. 2016;101:1711.CrossRefGoogle Scholar
  14. 14.
    Li M, Li L, Zhang L, et al. 1,25-Dihydroxyvitamin D3 suppresses gastric cancer cell growth through VDR- and mutant p53-mediated induction of p21. Life Sci. 2017;179:88.CrossRefPubMedGoogle Scholar
  15. 15.
    Kim SH, Pei QM, Jiang P, Yang M, Qian XJ, Liu JB. Effect of active vitamin D3 on VEGF-induced ADAM33 expression and proliferation in human airway smooth muscle cells: implications for asthma treatment. Respir Res. 2017;18:7.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Dai ZH, Tan B, Yang H, Wang O, Qian JM, Lv H. 1,25-Hydroxyvitamin D relieves colitis in rats via down-regulation of toll-like receptor 9 expression. Croat Med J. 2015;56:515–524.CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Zhao H, Zhang H, Wu H, et al. Protective role of 1,25(OH)2 vitamin D3 in the mucosal injury and epithelial barrier disruption in DSS-induced acute colitis in mice. BMC Gastroenterol. 2012;30:57.CrossRefGoogle Scholar
  18. 18.
    Chen S, Zhu J, Chen G, et al. 1,25-Dihydroxyvitamin D3 preserves intestinal epithelial barrier function from TNF-α induced injury via suppression of NF-kB p65 mediated MLCK-P-MLC signaling pathway. Biochem Biophys Res Commun. 2015;460:873–878.CrossRefPubMedGoogle Scholar
  19. 19.
    Chen SW, Ma YY, Zhu J, et al. Protective effect of 1,25-dihydroxyvitamin D3 on ethanol-induced intestinal barrier injury both in vitro and in vivo. Toxicol Lett. 2015;237:79–88.CrossRefPubMedGoogle Scholar
  20. 20.
    Jiang J, Shi D, Zhou XQ, et al. Vitamin D inhibits lipopolysaccharide-induced inflammatory response potentially through the Toll-like receptor 4 signalling pathway in the intestine and enterocytes of juvenile Jian carp (Cyprinus carpio var. Jian). Br J Nutr. 2015;114:1560–1568.CrossRefPubMedGoogle Scholar
  21. 21.
    Li B, Baylink DJ, Deb C, et al. 1,25-Dihydroxyvitamin D3 suppresses TLR8 expression and TLR8-mediated inflammatory responses in monocytes in vitro and experimental autoimmune encephalomyelitis in vivo. PLoS ONE. 2013;8:e58808.CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Equils O, Naiki Y, Shapiro AM, et al. 1,25-Dihydroxyvitamin D inhibits lipopolysaccharide-induced immune activation in human endothelial cells. Clin Exp Immunol. 2006;143:58–64.CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Gujral N, Suh JW, Sunwoo HH. Effect of anti-gliadin IgY antibody on epithelial intestinal integrity and inflammatory response induced by gliadin. BMC Immunol. 2015;9:41.CrossRefGoogle Scholar
  24. 24.
    Guo S, Al-Sadi R, Said HM, Ma TY. Lipopolysaccharide causes an increase in intestinal tight junction permeability in vitro and in vivo by inducing enterocyte membrane expression and localization of TLR-4 and CD14. Am J Pathol. 2013;182:375–387.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Papista C, Gerakopoulos V, Kourelis A, et al. Gluten induces coeliac-like disease in sensitised mice involving IgA, CD71 and transglutaminase 2 interactions that are prevented by probiotics. Lab Invest. 2012;92:625–635.CrossRefPubMedGoogle Scholar
  26. 26.
    Huber M, Baier W, Bessler WG, Heinevetter L. Modulation of the Th1/Th2 bias by lipopeptide and saponin adjuvants in orally immunized mice. Immunobiology. 2002;205:61–73.CrossRefPubMedGoogle Scholar
  27. 27.
    Gourbeyre P, Denery-Papini S, Larré C, Gaudin JC, Brossard C, Bodinier M. Wheat gliadins modified by deamidation are more efficient than native gliadins in inducing a Th2 response in Balb/c mice experimentally sensitized to wheat allergens. Mol Nutr Food Res. 2012;56:336–344.CrossRefPubMedGoogle Scholar
  28. 28.
    Everard A, Geurts L, Caesar R, et al. Intestinal epithelial MyD88 is a sensor switching host metabolism towards obesity according to nutritional status. Nat Commun. 2014;5:5648.CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Gopalakrishnan S, Durai M, Kitchens K, et al. Larazotide acetate regulates epithelial tight junctions in vitro and in vivo. Peptides. 2012;35:86–94.CrossRefPubMedGoogle Scholar
  30. 30.
    Khaleghi S, Ju JM, Lamba A, Murray JA. The potential utility of tight junction regulation in celiac disease: focus on larazotide acetate. Therap Adv Gastroenterol. 2016;9:37–49.CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Leffler DA, Kelly CP, Green PH, et al. Larazotide acetate for persistent symptoms of celiac disease despite a gluten-free diet: a randomized controlled trial. Gastroenterology. 2015;148:1311.e6–1319.e6.CrossRefGoogle Scholar
  32. 32.
    Leffler DA, Kelly CP, Abdallah HZ, et al. A randomized, double-blind study of larazotide acetate to prevent the activation of celiac disease during gluten challenge. Am J Gastroenterol. 2012;107:1554–1562.CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Lange TS, Stuckey AR, Robison K, et al. Effect of a vitamin D3 derivative (B3CD) with postulated anti-cancer activity in an ovarian cancer animal model. Investig New Drugs. 2010;28:543–553.CrossRefGoogle Scholar
  34. 34.
    Gui B, Chen Q, Hu C, Zhu C, He G. Effects of calcitriol (1,25-dihydroxy-vitamin D3) on the inflammatory response induced by H9N2 influenza virus infection in human lung A549 epithelial cells and in mice. Virol J. 2017;14:10.CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Orlando A, Linsalata M, Notarnicola M, Tutino V, Russo F. Lactobacillus GG restoration of the gliadin induced epithelial barrier disruption: The role of cellular polyamines. BMC Microbiol. 2014;14:19.CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Datta P, Weis MT. Calcium glycerophosphate preserves transepithelial integrity in the Caco-2 model of intestinal transport. WJG. 2015;21:9055–9066.CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Barilli A, Rotoli BM, Visigalli R, et al. Gliadin-mediated production of polyamines by RAW264.7 macrophages modulates intestinal epithelial permeability in vitro. Biochim Biophys Acta. 2015;1852:1779–1786.CrossRefPubMedGoogle Scholar
  38. 38.
    Dormoy V, Béraud C, Lindner V, et al. Vitamin D3 triggers antitumor activity through targeting hedgehog signaling in human renal cell carcinoma. Carcinogenesis. 2012;33:2084–2093.CrossRefPubMedGoogle Scholar
  39. 39.
    Chen S, Zhu J, Zuo S, et al. 1,25(OH)2D3 attenuates TGF-β1/β2-induced increased migration and invasion via inhibiting epithelial-mesenchymal transition in colon cancer cells. Biochem Biophys Res Commun. 2015;468:130–135.CrossRefPubMedGoogle Scholar
  40. 40.
    Aggarwal A, Kállay E. Cross talk between the calcium-sensing receptor and the vitamin D system in prevention of cancer. Front Physiol. 2016;7:451. (eCollection 2016).CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Abe R, Shimizu S, Yasuda K, et al. Evaluation of reduced allergenicity of deamidated gliadin in a mouse model of wheat-gliadin allergy using an antibody prepared by a peptide containing three epitopes. J Agric Food Chem. 2014;62:2845–2852.CrossRefPubMedGoogle Scholar
  42. 42.
    Silva MA, Jury J, Sanz Y, et al. Increased bacterial translocation in gluten-sensitive mice is independent of small intestinal paracellular permeability defect. Dig Dis Sci. 2012;57:38–47. doi: 10.1007/s10620-011-1847-z.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2017

Authors and Affiliations

  • Shouquan Dong
    • 1
    • 2
  • Tikka Prabhjot Singh
    • 1
    • 2
  • Xin Wei
    • 1
    • 2
  • Huang Yao
    • 1
    • 2
  • Hongling Wang
    • 1
    • 2
    Email author
  1. 1.Department of Gastroenterology/HepatologyZhongnan Hospital of Wuhan UniversityWuhanChina
  2. 2.The Hubei Clinical Center and Key Laboratory of Intestinal and Colorectal DiseasesWuhanChina

Personalised recommendations