title: Intestinal NADPH oxidase 2 activity increases in a neonatal rat model of necrotizing enterocolitis.
abstract: Necrotizing enterocolitis (NEC) is a complication of prematurity. The etiology is unknown, but is related to enteral feeding, ischemia, infection, and inflammation. Reactive oxygen species production, most notably superoxide, increases in NEC. NADPH oxidase (NOX) generates superoxide, but its activity in NEC remains unknown. We hypothesize that NOX-derived superoxide production increases in NEC. Newborn Sprague-Dawley rats were divided into control, formula-fed, formula/LPS, formula/hypoxia, and NEC (formula, hypoxia, and LPS). Intestinal homogenates were analyzed for NADPH-dependent superoxide production. Changes in superoxide levels on days 0-4 were measured. Inhibitors for nitric oxide synthase (L-NAME) and NOX2 (GP91-ds-tat) were utilized. RT-PCR for eNOS, NOX1, GP91phox expression was performed. Immunofluorescence studies estimated the co-localization of p47phox and GP91phox in control and NEC animals on D1, D2, and D4. NEC pups generated more superoxide than controls on D4, while all other groups were unchanged. NADPH-dependent superoxide production was greater in NEC on days 0, 3, and 4. GP91-ds-tat decreased superoxide production in both groups, with greater inhibition in NEC. L-NAME did not alter superoxide production. Temporally, superoxide production varied minimally in controls. In NEC, superoxide generation was decreased on day 1, but increased on days 3-4. GP91phox expression was higher in NEC on days 2 and 4. NOX1 and eNOS expression were unchanged from controls. GP91phox and p47phox had minimal co-localization in all control samples and NEC samples on D1 and D2, but had increased co-localization on D4. In conclusion, this study proves that experimentally-induced NEC increases small intestinal NOX activity. All components of NEC model are necessary for increased NOX activity. NOX2 is the major source, especially as the disease progresses.
authors.affiliation: Division of Neonatology, Department of Pediatrics, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America; Children's Research Institute, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America; Translational Vascular Biology Program, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America.
authors.forename: Scott R
authors.initials: SR
authors.lastname: Welak
authors.affiliation: Children's Research Institute, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America; Division of Pediatric Surgery, Department of Surgery, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America.
authors.forename: Rebecca M
authors.initials: RM
authors.lastname: Rentea
authors.affiliation: Division of Neonatology, Department of Pediatrics, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America; Children's Research Institute, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America; Translational Vascular Biology Program, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America.
authors.forename: Ru-Jeng
authors.initials: RJ
authors.lastname: Teng
authors.affiliation: Children's Research Institute, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America; Division of Pediatric Surgery, Department of Surgery, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America.
authors.forename: Nathan
authors.initials: N
authors.lastname: Heinzerling
authors.affiliation: Children's Research Institute, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America; Division of Pediatric Surgery, Department of Surgery, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America.
authors.forename: Ben
authors.initials: B
authors.lastname: Biesterveld
authors.affiliation: Division of Neonatology, Department of Pediatrics, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America; Children's Research Institute, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America; Division of Critical Care, Department of Pediatrics, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Liedel
authors.affiliation: Translational Vascular Biology Program, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America; Division of Pediatric Surgery, Department of Surgery, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America.
authors.forename: Kirkwood A
authors.initials: KA
authors.lastname: Pritchard
authors.affiliation: Translational Vascular Biology Program, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America; Division of Pediatric Surgery, Department of Surgery, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America.
authors.forename: Katherine M
authors.initials: KM
authors.lastname: Fredrich
authors.affiliation: Children's Research Institute, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America; Translational Vascular Biology Program, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America; Division of Pediatric Surgery, Department of Surgery, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America.
authors.forename: David M
authors.initials: DM
authors.lastname: Gourlay
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D004195:Disease Models, Animal; D004750:Enteral Nutrition; D020345:Enterocolitis, Necrotizing / Q000201:enzymology* / Q000209:etiology / Q000473:pathology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005455:Fluorescent Antibody Technique; D000860:Hypoxia / Q000150:complications / Q000503:physiopathology; D007422:Intestines / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013481:Superoxides / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D013481:Superoxides; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; C500593:Cybb protein, rat; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D019331:NG-Nitroarginine Methyl Ester