To investigate the effect of Chang’an II Decoction (肠安 II 号方))-containing serum on intestinal epithelial barrier dysfunction in rats.
Tumor necrosis factor (TNF)-α-induced injury of Caco-2 monolayers were established as an inflammatory model of human intestinal epithelium. Caco-2 monolayers were treated with blank serum and Chang’an II Decoction-containing serum that obtained from the rats which were treated with distilled water and Chang’an II Decoction intragastrically at doses of 0.49, 0.98, 1.96 g/(kg·d) for 1 week, respectively. After preparation of containing serum, cells were divided into the normal group, the model group, the Chang’an II-H, M, and L groups (treated with 30 ng/mL TNF-α and medium plus 10% high, middle-, and low-doses Chang’an II serum, respectively). Epithelial barrier function was assessed by transepithelial electrical resistance (TER) and permeability of fluorescein isothiocyanate (FITC)-labeled dextran. Transmission electron microscopy was used to observe the ultrastructure of tight junctions (TJs). Immunofluorescence of zonula occludens-1 (ZO-1), claudin-1 and nuclear transcription factor-kappa p65 (NF-κ Bp65) were measured to determine the protein distribution. The mRNA expression of myosin light chain kinase (MLCK) was measured by real-time polymerase chain reaction. The expression levels of MLCK, myosin light chain (MLC) and p-MLC were determined by Western blot.
Chang’an II Decoction-containing serum significantly attenuated the TER and paracellular permeability induced by TNF-α. It alleviated TNF-α-induced morphological alterations in TJ proteins. The increases in MLCK mRNA and MLCK, MLC and p-MLC protein expressions induced by TNF-α were significantly inhibited in the Chang’an II-H group. Additionally, Chang’an II Decoction significantly attenuated translocation of NF-κ Bp65 into the nucleus.
High-dose Chang’an II-containing serum attenuates TNF-α-induced intestinal barrier dysfunction. The underlying mechanism may be involved in inhibiting the MLCK-MLC phosphorylation signaling pathway mediated by NF-κ Bp65.
This is a preview of subscription content, log in to check access.
Buy single article
Instant access to the full article PDF.
Tax calculation will be finalised during checkout.
Lacy BE, Mearin F, Chang L, Chey WD, Lembo AJ, Simren M, et al. Bowel disorders. Gastroenterology 2016;150:1393–1407.
Canavan C, West J, Card T. The epidemiology of irritable bowel syndrome. Clin Epidemiol 2014;6:71–80.
Stasi C, Rosselli M, Bellini M, Laffi G, Milani S. Altered neuro-endocrine-immune pathways in the irritable bowel syndrome: the top-down and the bottom-up model. J Gastroenterol 2012; 47:1177–1185.
Bercik P, Wang L, Verdu EF, Mao YK, Blennerhassett P, Khan WI, et al. Visceral hyperalgesia and Intestinal dysmotility in a mouse model of postinfective gut dysfunction. Gastroenterology 2004;127:179–187.
Ledochowski M, Propst T, Fuchs D. The role of psychological and biological factors in post infective gut dysfunction. Gut 2000;46:140–141.
Kimball ES, Palmer JM, D’Andrea MR, Hornby PJ, Wade PR. Acute colitis induction by oil of mustard results in later development of an IBS-like accelerated upper GI transit in mice. Amer J Physiol Gastrointest Liver Phys 2005;288:G1266–G1273.
Ohman L, Tornblom H, Simren M. Crosstalk at the mucosal border: importance of the gut microenvironment in IBS. Nat Rev Gastroenterol Hepatol 2015;12:36–49.
Peterson LW, Artis D. Intestinal epithelial cells: regulators of barrier function and immune homeostasis. Nature Review Immunol 2014;14:141–153.
Bertiaux-Vandaele N, Youmba SB, BeLmonte L, Lecleire S, Antonietti M, Gourcerol G, et al. The expression and the cellular distribution of the tight junction proteins are altered in Irritable bowel syndrome patients with differences according to the disease subtype. Ame J Gastroenterol 2011;106:2165–2173.
Piche T, Barbara G, Aubert P, Bruley des Varannes S, Dainese R, Nano JL, et al. Impaired Intestinal barrier integrity in the colon of patients with irritable bowel syndrome: involvement of soluble mediators. Gut 2009;58:196–201.
Bashashati M, Rezaei N, Andrews CN, Chen CQ, Daryani NE, Sharkey KA, et al. Cytokines and irritable bowel syndrome: where do we stand? Cytokine 2012;57:201–209.
Darkoh C, Comer L, Zewdie G, HaroLd S, Snyder N, DuPont HL. Chemotactic chemokines are important in the pathogenesis of irritable bowel syndrome. PLoS One 2014;9:1–8.
Clark IA. How TNF was recognized as a key mechanism of disease. Cytok Growth Factor Rev 2007;18:335–343.
Ma TY, Boivin MA, Ye D, Pedram A, Said HM. Mechanism of TNF-α modulation of Caco-2 intestinal epithelial tight junction barrier: role of myosin light-chain kinase protein expression. Am J Physiol Gastrointest Liver Physiol 2005;288:G422–G430.
Hecht G, Pestic L, Nikcevic G, Koutsouris A, Tripuraneni J, Lorimer DD, et al. Expression of the catalytic domain of myosin light chain kinase increases paracellular permeability. Am J Physiol 1996;271:C1678–C1684.
Ivanov AI, McCall IC, Parkos CA, Nusrat A. Role for actin filament turnover and a myosin II motor in cytoskeleton-driven disassembly of the epithelial apical junctional complex. Mol Biol Cell 2004;15:2639–2651.
Ma TY, Tran D, Hoa N, Nguyen D, Merryfield M, Tarnawski A. Mechanism of extracellular calcium regulation of Intestinal epithelial tight junction permeability: role of cytoskeletal involvement. Microsc Res Tech 2000;51:156–168.
Shen L, Black ED, Witkowski ED, Lencer WI, Guerriero V, Schneeberger EE, et al. Myosin light chain phosphorylation regulates barrier function by remodeling tight junction structure. J Cell Sci 2006;119:2095–2106.
Cao M, Wang P, Sun CH, He W, Wang FJ. Amelioration of IFN-γ and TNF-α-induced intestinal epithelial barrier dysfunction by berberine via suppression of MLCK-MLC phosphorylation signaling pathway. PLoS One 2013;8:1–9.
Liu H, Wang P, Cao M, Li M, Wang F. Protective role of oligomycin against intestinal epithelial barrier dysfunction caused by IFN-γ and TNF-α. Cell Physiol Biochem 2012;29:799–808.
Bian Z, Wu T, Liu L, Miao J, Wong H, Song L, et al. Effectiveness of the Chinese herbal formula TongXieYaoFang for irritable bowel syndrome: a systematic review. J Altern Complement Med 2006;12:401–407.
Wang CW, Shi J, Ke F. Meta-analysis of the effectiveness of Tong Xie Yao Fang in treating irritable bowel syndrome. World Chin J Digest (Chin) 2007;15:1934–1939.
Wang FY, Su M, Zheng YQ, Wang XG, Kang N, Chen T, et al. Herbal prescription Chang’an II repairs intestinal mucosal barrier in rats with post-inflammation irritable bowel syndrome. Acta Pharmacol Sin 2015;36:708–715.
Turner JR. Molecular basis of epithelial barrier regulation: from basic mechanisms to clinical application. Am J Pathol 2006;169:1901–1909.
Zhang K, Hornef MW, Dupont A. The intestinal epithelium as guardian of gut barrier integrity. Cell Microbiol 2015;17:1561–1569.
Watson AJ, Hughes KR. TNF-α -induced intestinal epithelial cell shedding: implications for intestinal barrier function. Ann N Y Acad Sci 2012;1258:1–8.
Noth R, Stüber E, Häsler R. Anti-TNF-α antibodies improve intestinal barrier function in Crohn’s disease. J Crohns Colitis 2012;6:464–469.
Forster C. Tight junctions and the modulation of barrier function in disease. Histochem Cell Biol 2008;130:55–70.
Matter K, Balda MS. Signaling to and from tight junctions. Nat Rev Mol Cell Biol 2003;4:225–236.
Steed E, Balda MS, Matter K. Dynamics and functions of tight junctions. Trends Cell Biol 2010;20:142–149.
Suzuki T. Regulation of intestinal epithelial permeability by tight junctions. Cell Mol Life Sci 2013;70:631–659.
Gu L, Li N, Li Q, Zhang Q, Wang C, Zhu W, et al. The effect of berberine in vitro on tight junctions in human Caco-2 intestinal epithelial cells. Fitoterapia 2009;80:241–248.
Grozdanovic MM, Čavić M, Nešić A, Andjelković U, Akbari P, Smit JJ, et al. Kiwifruit cysteine protease actinidin compromises the intestinal barrier by disrupting tight junctions. Biochim Biophys Acta 2016;1860:516–526.
Jeong CH, Seok JS, Petriello MC, Han SG. Arsenic downregulates tight junction claudin proteins through p38 and NF-κ B in intestinal epithelial cell line, HT-29. Toxicology 2017;379:31–39.
Vila L, García-Rodríguez A, Cortés C, Marcos R, Hernández A. Assessing the effects of silver nanoparticles on monolayers of differentiated Caco-2 cells, as a model of intestinal barrier. Food Chem Toxicol 2018;116:1–10.
Wang F, Graham WV, Wang Y, Witkowski ED, Schwarz BT, Turner JR. Interferon-gamma and tumor necrosis factor-alpha synergize to induce intestinal epithelial barrier dysfunction by up-regulating myosin light chain kinase expression. Am J Pathol 2005;166:409–419.
Ma TY, Iwamoto GK, Hoa NT, Akotia V, Pedram A, Boivin MA. TNF-alpha-induced increase in intestinal epithelial tight junction permeability requires NF-kappa B activation. Am J Physiol Gastrointest Liver Physiol 2004;286:G367–G376.
Cunningham KE, Turner JR. Myosin light chain kinase: pulling the strings of epithelial tight junction function. Ann N Y Acad Sci 2012;1258:34–42.
Ye D, Ma I, Ma TY. Molecular mechanism of tumor necrosis factor-alpha modulation of intestinal epithelial tight junction barrier. Am J Physiol Gastrointest Liver Physiol 2006;290:G496–G504.
Widera D, Mikenberg I, Elvers M, Kaltschmidt C, Kaltschmidt B. Tumor necrosis factor alpha triggers proliferation of adult neural stem cells via IKK/NF-kappa B signaling. BMC Neurosci 2006;7:64–82.
Al-Sadi R, Guo S, Ye D, Rawat M, Ma TY. TNF-α modulation of intestinal tight junction permeability is mediated by NIK/IKK-α axis activation of the canonical NF-κB pathway. Am J Pathol 2016;186:1151–1165.
The authors declare no financial or commercial conflict of interest.
Supported by the National Nature Science Foundation of China (No. 81373580, 81173209, 81704070), Visiting Scientist Program of China Academy of Chinese Medical Sciences (No. ZZ070801) and “Ten Diseases and Ten Drugs” Program of Beijing Municipal Science and Technology Commission (No. Z161100000116046)
Electronic Supplementary Material
About this article
Cite this article
Chen, T., Yin, Xl., Kang, N. et al. Chang’an II Decoction (肠安 II 号方)-Containing Serum Ameliorates Tumor Necrosis Factor-α-Induced Intestinal Epithelial Barrier Dysfunction via MLCK-MLC Signaling Pathway in Rats. Chin. J. Integr. Med. 26, 745–753 (2020). https://doi.org/10.1007/s11655-019-3034-6
- myosin light chain kinase-myosin light chain
- signaling pathway
- intestinal epithelial cells
- tight junction
- tumor necrosis factor-α
- Chang’an II Decoction
- drug-containing serum