The role of the glutathione antioxidant system in gut barrier failure in a rodent model of experimental necrotizing enterocolitis.
BACKGROUND
Evidence suggests that intestinal barrier failure in necrotizing enterocolitis results in part from overproduction of nitric oxide and other toxic oxidant species that result in enterocyte death and intestinal barrier failure. We hypothesize that the glutathione detoxifying system is important in maintaining intestinal barrier integrity by protecting against nitrosative stress.


METHODS
Newborn rats were subjected to hypoxia (5% O2, tid) and fed formula by gavage (NEC), or were breast-fed without hypoxia (BF). Rats were killed and the distal ilea were harvested for RNA, protein, and morphologic studies. RNA underwent cDNA microarray analysis. To assess glutathione in protecting against nitrosative stress, IEC-6 cells were exposed to SIN-1 and/or L-buthionine-(S,R)-sulfoximine (BSO), a glutathione inhibitor. Cells were analyzed for glutathione-S-transferase activity, apoptosis and mitochondrial function.


RESULTS
BF controls developed normal intestinal architecture, whereas NEC animals sustained damage to the intestinal epithelium. Microarray analysis demonstrated that 93 genes were overexpressed in NEC compared with controls. In the array, glutathione-S-transferase pi and alpha 2, GSH-dependent detoxifying enzymes, RNA were upregulated compared with BF controls. IEC-6 cells exposed to SIN-1/BSO produced an increase in apoptosis. Poly ADP-ribosylpolymerase cleavage and apoptosis-inducing factor (AIF) nuclear localization, markers of apoptosis, were seen in IEC-6 cells exposed to SIN-1/BSO as opposed to media controls.


CONCLUSIONS
These data support the hypothesis that GSH antioxidant system plays a crucial role in gut barrier protection by attenuating enterocyte death.
Kelly
Natasha
N
Department of Surgery, Children's Hospital of Pittsburgh, PA, USA.
Friend
Kerri
K
Boyle
Patricia
P
Zhang
Xiao Ru
XR
Wong
Catarina
C
Hackam
David J
DJ
Zamora
Ruben
R
Ford
Henri R
HR
Upperman
Jeffrey S
JS
D000818:Animals; D000831:Animals, Newborn; D000975:Antioxidants / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D002463:Cell Membrane Permeability / Q000187:drug effects*; D020345:Enterocolitis, Necrotizing / Q000235:genetics / Q000503:physiopathology / Q000517:prevention & control*; D004847:Epithelial Cells / Q000187:drug effects; D015870:Gene Expression; D005978:Glutathione / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D007413:Intestinal Mucosa / Q000187:drug effects*; D023421:Models, Animal; D009569:Nitric Oxide / Q000378:metabolism; D016877:Oxidants / Q000494:pharmacology; D030421:Peroxynitrous Acid / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley
D000975:Antioxidants; D016877:Oxidants; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; D005978:Glutathione