Digestive Diseases and Sciences

, Volume 62, Issue 2, pp 372–386 | Cite as

The Anti-Inflammatory Effect and Intestinal Barrier Protection of HU210 Differentially Depend on TLR4 Signaling in Dextran Sulfate Sodium-Induced Murine Colitis

  • Sisi Lin
  • Yongyu LiEmail author
  • Li Shen
  • Ruiqin Zhang
  • Lizhi Yang
  • Min Li
  • Kun Li
  • Jakub Fichna
Original Article



Ulcerative colitis (UC) is strongly associated with inflammation and intestinal barrier disorder. The nonselective cannabinoid receptor agonist HU210 has been shown to ameliorate inflamed colon in colitis, but its effects on intestinal barrier function and extraintestinal inflammation are unclear.


To investigate the effects and the underlying mechanism of HU210 action on the UC in relation to a role of TLR4 and MAP kinase signaling.


Wild-type (WT) and TLR4 knockout (Tlr4 /) mice were exposed to 4% dextran sulfate sodium (DSS) for 7 days. The effects of HU210 on inflammation and intestinal barrier were explored.


Upon DSS challenge, mice suffered from bloody stool, colon shortening, intestinal mucosa edema, pro-inflammatory cytokine increase and intestinal barrier destruction with goblet cell depletion, increased intestinal microflora accompanied with elevated plasma lipopolysaccharide, reduced mRNA expression of the intestinal tight junction proteins, and abnormal ratio of CD4+/CD8+ T cells in the intestinal Peyer’s patches. Pro-inflammatory cytokines in the plasma and the lung, as well as pulmonary myeloperoxidase activity, indicators of extraintestinal inflammation were increased. Protein expression of p38α and pp38 was up-regulated in the colon of WT mice. Tlr4 / mice showed milder colitis. HU210 reversed the intestinal barrier changes in both strains of mice, but alleviated inflammation only in WT mice.


Our study indicates that in experimental colitis, HU210 displays a protective effect on the intestinal barrier function independently of the TLR4 signaling pathway; however, in the extraintestinal tissues, the anti-inflammatory action seems through affecting TLR4-mediated p38 mitogen-activated protein kinase pathway.


Ulcerative colitis HU210 Intestinal barrier function Toll-like receptor 4 p38 mitogen-activated protein kinase 



We thank Professor Peilin Zhao for providing the technical help in pathology. This work was supported by Grants from the National Science Foundation of China (Nos. 31571181 and 81270477 to Dr. Yongyu Li).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Haskell H, Andrews CW Jr, Reddy SI, et al. Pathologic features and clinical significance of “backwash” ileitis in ulcerative colitis. Am J Surg Pathol. 2005;29:1472–1481.CrossRefPubMedGoogle Scholar
  2. 2.
    Geier MS, Smith CL, Butler RN, Howarth GS. Small-intestinal manifestations of dextran sulfate sodium consumption in rats and assessment of the effects of Lactobacillus fermentum BR11. Dig Dis Sci. 2009;54:1222–1228.CrossRefPubMedGoogle Scholar
  3. 3.
    Yazbeck R, Howarth GS, Butler RN, Geier MS, Abbott CA. Biochemical and histological changes in the small intestine of mice with dextran sulfate sodium colitis. J Cell Physiol. 2011;226:3219–3224.CrossRefPubMedGoogle Scholar
  4. 4.
    Vavricka SR, Schoepfer A, Scharl M, Lakatos PL, Navarini A, Rogler G. Extraintestinal manifestations of inflammatory bowel disease. Inflamm Bowel Dis. 2015;21:1982–1992.CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Liu Y, Wang XY, Yang X, Jing S, Zhu L, Gao SH. Lung and intestine: a specific link in an ulcerative colitis rat model. Gastroenterol Res Pract. 2013;2013:124530.PubMedPubMedCentralGoogle Scholar
  6. 6.
    Danese S, Fiocchi C. Ulcerative colitis. N Engl J Med. 2011;365:1713–1725.CrossRefPubMedGoogle Scholar
  7. 7.
    Molodecky NA, Soon IS, Rabi DM, et al. Increasing incidence and prevalence of the inflammatory bowel diseases with time, based on systematic review. Gastroenterology. 2012;142:46–54.CrossRefPubMedGoogle Scholar
  8. 8.
    Engel MA, Neurath MF. New pathophysiological insights and modern treatment of IBD. J Gastroenterol. 2010;45:571–583.CrossRefPubMedGoogle Scholar
  9. 9.
    Zhao J, Shi P, Sun Y, et al. DHA protects against experimental colitis in IL-10-deficient mice associated with the modulation of intestinal epithelial barrier function. Br J Nutr. 2015;114:181–188.CrossRefPubMedGoogle Scholar
  10. 10.
    MacDonald TT, Biancheri P, Sarra M, Monteleone G. What’s the next best cytokine target in IBD? Inflamm Bowel Dis. 2012;18:2180–2189.CrossRefPubMedGoogle Scholar
  11. 11.
    Salim SY, Söderholm JD. Importance of disrupted intestinal barrier in inflammatory bowel diseases. Inflamm Bowel Dis. 2011;17:362–381.CrossRefPubMedGoogle Scholar
  12. 12.
    Andersen K, Kesper MS, Marschner JA, et al. Intestinal dysbiosis, barrier dysfunction, and bacterial translocation account for CKD-related systemic inflammation. J Am Soc Nephrol. 2016. doi: 10.1681/ASN.2015111285.PubMedGoogle Scholar
  13. 13.
    Sánchez de Medina F, Romero-Calvo I, Mascaraque C, Martinez-Augustin O. Intestinal inflammation and mucosal barrier function. Inflamm Bowel Dis. 2014;20:2394–2404.CrossRefPubMedGoogle Scholar
  14. 14.
    Alhouayek M, Muccioli GG. The endocannabinoid system in inflammatory bowel diseases: from pathophysiology to therapeutic opportunity. Trends Mol Med. 2012;18:615–625.CrossRefPubMedGoogle Scholar
  15. 15.
    Cao MH, Li YY, Xu J, et al. Cannabinoid HU210 protects isolated rat stomach against impairment caused by serum of rats with experimental acute pancreatitis. PLoS ONE. 2012;7:e52921.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Massa F, Marsicano G, Hermann H, et al. The endogenous cannabinoid system protects against colonic inflammation. J Clin Invest. 2004;113:1202–1209.CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Ke P, Shao BZ, Xu ZQ, et al. Activation of cannabinoid receptor 2 ameliorates DSS-induced colitis through inhibiting NLRP3 inflammasome in macrophages. PLoS ONE. 2016;11:e0155076.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Storr M, Emmerdinger D, Diegelmann J, et al. The cannabinoid receptor 1 (CNR1)1359 G/A polymorphism modulates susceptibility to ulcerative colitis and the phenotype in Crohn’s disease. PLoS ONE. 2010;5:e9453.CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Engel MA, Kellermann CA, Burnat G, Hahn EG, Rau T, Konturek PC. Mice lacking cannabinoid CB1-, CB2-receptors or both receptors show increased susceptibility to trinitrobenzene sulfonic acid (TNBS)-induced colitis. J Physiol Pharmacol. 2010;61:89–97.PubMedGoogle Scholar
  20. 20.
    Ottani A, Giuliani D. Hu 210: a potent tool for investigations of the cannabinoid system. CNS Drug Rev. 2001;7:131–145.CrossRefPubMedGoogle Scholar
  21. 21.
    Lax P, Esquiva G, Altavilla C, Cuenca N. Neuroprotective effects of the cannabinoid agonist HU210 on retinal degeneration. Exp Eye Res. 2014;120:175–185.CrossRefPubMedGoogle Scholar
  22. 22.
    Maslov LN, Krylatov AV, Lishmanov YB. Role of cyclic nucleotides and NO synthase in mechanisms of cardioprotective effects of cannabinoid HU-210. Bull Exp Biol Med. 2014;157:588–591.CrossRefPubMedGoogle Scholar
  23. 23.
    Lucas K, Maes M. Role of the toll like receptor (TLR) radical cycle in chronic inflammation: possible treatments targeting the TLR4 pathway. Mol Neurobiol. 2013;48:190–204.CrossRefPubMedGoogle Scholar
  24. 24.
    Wang W, Xia T, Yu X. Wogonin suppresses inflammatory response and maintains intestinal barrier function via TLR4–MyD88–TAK1-mediated NF-κB pathway in vitro. Inflamm Res. 2015;64:423–431.CrossRefPubMedGoogle Scholar
  25. 25.
    Rakoff-Nahoum S, Paglino J, Eslami-Varzaneh F, Edberg S, Medzhitov R. Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis. Cell. 2004;118:229–241.CrossRefPubMedGoogle Scholar
  26. 26.
    Tan Y, Zou KF, Qian W, Chen S, Hou XH. Expression and implication of toll-like receptors TLR2, TLR4 and TLR9 in colonic mucosa of patients with ulcerative colitis. J Huazhong Univ Sci Technol Med Sci. 2014;34:785–790.CrossRefPubMedGoogle Scholar
  27. 27.
    Feng J, Guo C, Zhu Y, et al. Baicalin down regulates the expression of TLR4 and NFkB-p65 in colon tissue in mice with colitis induced by dextran sulfate sodium. Int J Clin Exp Med. 2014;7:4063–4072.PubMedPubMedCentralGoogle Scholar
  28. 28.
    Li YY, Yuece B, Cao HM, et al. Inhibition of p38/Mk2 signaling pathway improves the anti-inflammatory effect of WIN55 on mouse experimental colitis. Lab Invest. 2013;93:322–333.CrossRefPubMedGoogle Scholar
  29. 29.
    Duncan M, Galic MA, Wang A, et al. Cannabinoid 1 receptors are critical for the innate immune response to TLR4 stimulation. Am J Physiol Regul Integr Comp Physiol. 2013;305:R224–R231.CrossRefPubMedGoogle Scholar
  30. 30.
    Yan Y, Kolachala V, Dalmasso G, et al. Temporal and spatial analysis of clinical and molecular parameters in dextran sodium sulfate induced colitis. PLoS ONE. 2009;4:e6073.CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Melgar S, Karlsson L, Rehnstrom E, et al. Validation of murine dextran sulfate sodium-induced colitis using four therapeutic agents for human inflammatory bowel disease. Int Immunopharmacol. 2008;8:836–844.CrossRefPubMedGoogle Scholar
  32. 32.
    Engel MA, Kellermann CA, Rau T, Burnat G, Hahn EG, Konturek PC. Ulcerative colitis in AKR mice is attenuated by intraperitoneally administered anandamide. J Physiol Pharmacol. 2008;59:673–689.PubMedGoogle Scholar
  33. 33.
    Takagi T, Naito Y, Uchiyama K, et al. Carbon monoxide liberated from carbon monoxide-releasing molecule exerts an anti-inflammatory effect on dextran sulfate sodium-induced colitis in mice. Dig Dis Sci. 2011;56:1663–1671.CrossRefPubMedGoogle Scholar
  34. 34.
    Randall L, Heinrich K, Horton R, et al. Detection of antibiotic residues and association of cefquinome residues with the occurrence of extended-spectrum β-lactamase (ESBL)-producing bacteria in waste milk samples from dairy farms in England and Wales in 2011. Res Vet Sci. 2014;96:15–24.CrossRefPubMedGoogle Scholar
  35. 35.
    Cottell JL, Kanwar N, Castillo-Courtade L, et al. blaCTX-M-32 on an IncN plasmid in Escherichia coli from beef cattle in the United States. Antimicrob Agents Chemother. 2013;57:1096–1097.CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Wang S, Zhu H, Lu C, et al. Fermented milk supplemented with probiotics and prebiotics can effectively alter the intestinal microbiota and immunity of host animals. J Dairy Sci. 2012;95:4813–4822.CrossRefPubMedGoogle Scholar
  37. 37.
    Luo H, Guo P, Zhou Q. Role of TLR4/NF-κB in damage to intestinal mucosa barrier function and bacterial translocation in rats exposed to hypoxia. PLoS ONE. 2012;7:e46291.CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Zhu YH, Li XQ, Zhang W, Zhou D, Liu HY, Wang JF. Dose-dependent effects of Lactobacillus rhamnosus on serum interleukin-17 production and intestinal T-cell responses in pigs challenged with Escherichia coli. Appl Environ Microbiol. 2014;80:1787–1798.CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Hu L, Yang GZ, Zhang Y, et al. Overexpression of SULT2B1b is an independent prognostic indicator and promotes cell growth and invasion in colorectal carcinoma. Lab Invest. 2015;95:1005–1018.CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Mandalasi M, Dorabawila N, Smith DF, Heimburg-Molinaro J, Cummings RD, Nyame AK. Development and characterization of a specific IgG monoclonal antibody toward the Lewis × antigen using splenocytes of Schistosoma mansoni-infected mice. Glycobiology. 2013;23:877–892.CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Gölz G, Karadas G, Alutis ME, et al. Arcobacter butzleri induce colonic, extra-intestinal and systemic inflammatory responses in gnotobiotic IL-10 deficient mice in a strain-dependent manner. PLoS ONE. 2015;10:e0139402.CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Leppkes M, Roulis M, Neurath MF, Kollias G, Becker C. Pleiotropic functions of TNF-α in the regulation of the intestinal epithelial response to inflammation. Int Immunol. 2014;26:509–515.CrossRefPubMedGoogle Scholar
  43. 43.
    Ardesjo B, Portela-Gomes GM, Rorsman F, et al. Immunoreactivity against goblet cells in patients with inflammatory bowel disease. Inflamm Bowel Dis. 2008;14:652–661.CrossRefPubMedGoogle Scholar
  44. 44.
    Hao XP, Lucero CM, Turkbey B, et al. Experimental colitis in SIV-uninfected rhesus macaques recapitulates important features of pathogenic SIV infection. Nat Commun. 2015;6:8020.CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Yang J, Zhao Y, Shao F. Non-canonical activation of inflammatory caspases by cytosolic LPS in innate immunity. Curr Opin Immunol. 2015;32:78–83.CrossRefPubMedGoogle Scholar
  46. 46.
    Kamada N, Seo SU, Chen GY, Nunez G. Role of the gut microbiota in immunity and inflammatory disease. Nat Rev Immunol. 2013;13:321–335.CrossRefPubMedGoogle Scholar
  47. 47.
    Jung C, Hugot JP, Barreau F. Peyer’s patches: the immune sensors of the intestine. Int J Inflam. 2010;2010:823710.CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Gao F, Li M, Liu Y, Gao C, Wen S, Tang L. Intestinal dysbacteriosis induces changes of T lymphocyte subpopulations in Peyer’s patches of mice and orients the immune response towards humoral immunity. Gut Pathog. 2012;4:19.CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Zenewicz LA, Antov A, Flavell RA. CD4 T-cell differentiation and inflammatory bowel disease. Trends Mol Med. 2009;15:199–207.CrossRefPubMedGoogle Scholar
  50. 50.
    Yao Y, Han W, Liang J, et al. Glatiramer acetate ameliorates inflammatory bowel disease in mice through the induction of Qa-1-restricted CD8+ regulatory cells. Eur J Immunol. 2013;43:125–136.CrossRefPubMedGoogle Scholar
  51. 51.
    Van Itallie CM, Anderson JM. Architecture of tight junctions and principles of molecular composition. Semin Cell Dev Biol. 2014;36:157–165.CrossRefPubMedGoogle Scholar
  52. 52.
    Wang Z, Li R, Tan J, et al. Syndecan-1 acts in synergy with tight junction through Stat3 signaling to maintain intestinal mucosal barrier and prevent bacterial translocation. Inflamm Bowel Dis. 2015;21:1894–1907.CrossRefPubMedGoogle Scholar
  53. 53.
    Hwang I, An BS, Yang H, Kang HS, Jung EM, Jeung EB. Tissue-specific expression of occludin, zona occludens-1, and junction adhesion molecule A in the duodenum, ileum, colon, kidney, liver, lung, brain, and skeletal muscle of C57BL mice. J Physiol Pharmacol. 2013;64:11–18.PubMedGoogle Scholar
  54. 54.
    Gong Y, Li H, Li Y. Effects of Bacillus subtilis on epithelial tight junctions of mice with inflammatory bowel disease. J Interferon Cytokine Res. 2016;36:75–85.CrossRefPubMedGoogle Scholar
  55. 55.
    Poritz LS, Harris LR III, Kelly AA, Koltun WA. Increase in the tight junction protein claudin-1 in intestinal inflammation. Dig Dis Sci. 2011;56:2802–2809.CrossRefPubMedPubMedCentralGoogle Scholar
  56. 56.
    Bowie RV, Donatello S, Lyes C, et al. Lipid rafts are disrupted in mildly inflamed intestinal microenvironments without overt disruption of the epithelial barrier. Am J Physiol Gastrointest Liver Physiol. 2012;302:G781–G793.CrossRefPubMedGoogle Scholar
  57. 57.
    Muccioli GG, Naslain D, Bäckhed F, et al. The endocannabinoid system links gut microbiota to adipogenesis. Mol Syst Biol. 2010;6:392.CrossRefPubMedPubMedCentralGoogle Scholar
  58. 58.
    Feng JS, Yang Z, Zhu YZ, Liu Z, Guo CC, Zheng XB. Serum IL-17 and IL-6 increased accompany with TGF-β and IL-13 respectively in ulcerative colitis patients. Int J Clin Exp Med. 2014;7:5498–5504.PubMedPubMedCentralGoogle Scholar
  59. 59.
    Hunter CA, Jones SA. IL-6 as a keystone cytokine in health and disease. Nat Immunol. 2015;16:448–457.CrossRefPubMedGoogle Scholar
  60. 60.
    Kanai T, Mikami Y, Sujino T, Hisamatsu T, Hibi T. RORγt-dependent IL-17A-producing cells in the pathogenesis of intestinal inflammation. Mucosal Immunol. 2012;5:240–247.CrossRefPubMedGoogle Scholar
  61. 61.
    Sugihara T, Kobori A, Imaeda H, et al. The increased mucosal mRNA expressions of complement C3 and interleukin-17 in inflammatory bowel disease. Clin Exp Immunol. 2010;160:386–393.CrossRefPubMedPubMedCentralGoogle Scholar
  62. 62.
    D’Andrea N, Vigliarolo R, Sanguinetti CM. Respiratory involvement in inflammatory bowel diseases. Multidiscip Respir Med. 2010;5:173–182.CrossRefPubMedPubMedCentralGoogle Scholar
  63. 63.
    Gironella M, Mollà M, Salas A, et al. The role of P-selectin in experimental colitis as determined by antibody immunoblockade and genetically deficient mice. J Leukoc Biol. 2002;72:56–64.PubMedGoogle Scholar
  64. 64.
    Docena G, Rovedatti L, Kruidenier L, et al. Down-regulation of p38 mitogen-activated protein kinase activation and pro-inflammatory cytokine production by mitogen-activated protein kinase inhibitors in inflammatory bowel disease. Clin Exp Immunol. 2010;162:108–115.CrossRefPubMedPubMedCentralGoogle Scholar
  65. 65.
    Feng YJ, Li YY. The role of p38 mitogen-activated protein kinase in the pathogenesis of inflammatory bowel disease. J Dig Dis. 2011;12:327–332.CrossRefPubMedGoogle Scholar
  66. 66.
    Michler T, Storr M, Kramer J, et al. Activation of cannabinoid receptor 2 reduces inflammation in acute experimental pancreatitis via intra-acinar activation of p38 and MK2-dependent mechanisms. Am J Physiol Gastrointest Liver Physiol. 2013;304:G181–G192.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Sisi Lin
    • 1
  • Yongyu Li
    • 1
    Email author
  • Li Shen
    • 2
  • Ruiqin Zhang
    • 1
  • Lizhi Yang
    • 1
  • Min Li
    • 3
  • Kun Li
    • 1
  • Jakub Fichna
    • 4
  1. 1.Department of Pathophysiology, Institute of Digestive DiseaseTongji University School of MedicineShanghaiChina
  2. 2.Department of Immunology and Pathogenic BiologyTongji University School of MedicineShanghaiChina
  3. 3.Department of PhysiologyZunyi Medical and Pharmaceutical CollegeZunyiChina
  4. 4.Department of Biochemistry, Faculty of MedicineMedical University of LodzLodzPoland

Personalised recommendations