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BioMed Research International
Volume 2013 (2013), Article ID 152052, 13 pages
http://dx.doi.org/10.1155/2013/152052
Research Article

Glutamine and Alanyl-Glutamine Increase RhoA Expression and Reduce Clostridium difficile Toxin-A-Induced Intestinal Epithelial Cell Damage

1Department of Morphology, Faculty of Medicine, Federal University of Ceará, Delmiro de Farias, 60416-030 Fortaleza, CE, Brazil
2Department of Physiology and Pharmacology, Faculty of Medicine, Federal University of Ceará, 1127 Coronel Nunes de Melo, 60430-270 Fortaleza, CE, Brazil
3Department of Physics, Faculty of Physics, Federal University of Ceará, 922 Campus do Pici, 60455-760 Fortaleza, CE, Brazil
4Biomedical Sciences Institute, Federal University of Rio de Janeiro, 373 Avenue Carlos Chagas, 21941-902 Rio de Janeiro, RJ, Brazil
5Division of Infectious Diseases and International Health, Center for Global Health, University of Virginia, 345 Crispell Drive, Room 2709, Charlottesville, VA 22903, USA

Received 22 August 2012; Accepted 12 November 2012

Academic Editor: Reinaldo B. Oriá

Copyright © 2013 Ana A. Q. A. Santos et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Clostridium difficile is a major cause of antibiotic-associated colitis and is associated with significant morbidity and mortality. Glutamine (Gln) is a major fuel for the intestinal cell population. Alanyl-glutamine (Ala-Gln) is a dipeptide that is highly soluble and well tolerated. IEC-6 cells were used in the in vitro experiments. Cell morphology was evaluated by atomic force microscopy (AFM) and scanning electron microscopy (SEM). Cell proliferation was assessed by WST-1 and Ki-67 and apoptosis was assessed by TUNEL. Cytoskeleton was evaluated by immunofluorescence for RhoA and F-actin. RhoA was quantified by immunoblotting. TcdA induced cell shrinkage as observed by AFM, SEM, and fluorescent microscopy. Additionally, collapse of the F-actin cytoskeleton was demonstrated by immunofluorescence. TcdA decreased cell volume and area and increased cell height by 79%, 66.2%, and 58.9%, respectively. Following TcdA treatment, Ala-Gln and Gln supplementation, significantly increased RhoA by 65.5% and 89.7%, respectively at 24 h. Ala-Gln supplementation increased cell proliferation by 137.5% at 24 h and decreased cell apoptosis by 61.4% at 24 h following TcdA treatment. In conclusion, TcdA altered intestinal cell morphology and cytoskeleton organization, decreased cell proliferation, and increased cell apoptosis. Ala-Gln and Gln supplementation reduced intestinal epithelial cell damage and increased RhoA expression.