title: Glutamine and alanyl-glutamine increase RhoA expression and reduce Clostridium difficile toxin-a-induced intestinal epithelial cell damage.
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.
authors.affiliation: Department of Morphology, Faculty of Medicine, Federal University of Ceará, Delmiro de Farias, 60416-030 Fortaleza, CE, Brazil.
authors.forename: Ana A Q A
authors.initials: AA
authors.lastname: Santos
authors.forename: Manuel B
authors.initials: MB
authors.lastname: Braga-Neto
authors.forename: Marcelo R
authors.initials: MR
authors.lastname: Oliveira
authors.forename: Rosemeire S
authors.initials: RS
authors.lastname: Freire
authors.forename: Eduardo B
authors.initials: EB
authors.lastname: Barros
authors.forename: Thiago M
authors.initials: TM
authors.lastname: Santiago
authors.forename: Luciana M
authors.initials: LM
authors.lastname: Rebelo
authors.forename: Claudia
authors.initials: C
authors.lastname: Mermelstein
authors.forename: Cirle A
authors.initials: CA
authors.lastname: Warren
authors.forename: Richard L
authors.initials: RL
authors.lastname: Guerrant
authors.forename: Gerly A C
authors.initials: GA
authors.lastname: Brito
mesh_terms: D000818:Animals; D001427:Bacterial Toxins / Q000633:toxicity*; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D048429:Cell Size / Q000187:drug effects; D003092:Colitis / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D003599:Cytoskeleton / Q000378:metabolism / Q000473:pathology; D004151:Dipeptides / Q000494:pharmacology*; D004768:Enterotoxins / Q000633:toxicity*; D004847:Epithelial Cells / Q000201:enzymology* / Q000473:pathology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D005973:Glutamine / Q000494:pharmacology*; D007413:Intestinal Mucosa / Q000201:enzymology* / Q000473:pathology; D018625:Microscopy, Atomic Force; D008855:Microscopy, Electron, Scanning; D051381:Rats; D020742:rhoA GTP-Binding Protein / Q000096:biosynthesis*
chemical_list: D001427:Bacterial Toxins; D004151:Dipeptides; D004768:Enterotoxins; C041348:tcdA protein, Clostridium difficile; D005973:Glutamine; D020742:rhoA GTP-Binding Protein; C054122:alanylglutamine