Brief hypoxic stress suppresses postbacteremic NF-kappaB activation and TNF-alpha bioactivity in perfused liver.
Reductions in hepatic O(2) delivery are common early after gram-negative bacteremic sepsis owing to cardiopulmonary dysfunction and derangements in sinusoidal perfusion. Although gram-negative endotoxin and cellular hypoxia independently enhance activation of nuclear factor-kappaB (NF-kappaB) via generation of reactive O(2) species (ROS), the combination of these stimuli downregulates hepatic TNF-alpha gene expression. Here we tested the hypothesis that hypoxic suppression of postbacteremic TNF-alpha gene expression is transcriptionally mediated by reduced activation of NF-kappaB. Buffer-perfused rat livers (n = 52) were studied over 180 min after intraportal infection at t = 0 with 10(9) live Escherichia coli (EC), serotype O55:B5, or 0.9% NaCl controls under normoxic conditions, compared with 0.5 h of constant-flow hypoxia (PO(2) approximately 41 +/- 7 Torr) beginning at t = 30 min, followed by 120 min of reoxygenation. In parallel studies, tissue was obtained at peak hypoxia (t = 60 min). To determine the role of xanthine oxidase (XO)-induced ROS in modulating NF-kappaB activity after hypoxia/reoxygenation (H/R), livers were pretreated with the XO inhibitor allopurinol, with results confirmed in organs of tungstate-fed animals. Electrophoretic mobility shift assays were performed on nuclear extracts of whole liver lysates using (32)P-labeled oligonucleotides specific for NF-kappaB. Compared with normoxic EC controls, hypoxia reduced postbacteremic NF-kappaB nuclear translocation and TNF-alpha bioactivity, independent of reoxygenation, tissue levels of reduced glutathione, or posthypoxic O(2) consumption. XO inhibition reversed the hypoxic suppression of NF-kappaB nuclear translocation and ameliorated decreases in cell-associated TNF-alpha. Thus decreases in hepatic O(2) delivery reduce postbacteremic nuclear translocation of NF-kappaB and hepatic TNF-alpha biosynthesis by signaling mechanisms involving low-level generation of XO-mediated ROS.
Loftis
L L
LL
Division of Critical Care Medicine, Department of Pediatrics, Saint Louis University School of Medicine, Saint Louis 63104, Missouri, USA.
Johanns
C A
CA
Lechner
A J
AJ
Matuschak
G M
GM
D000493:Allopurinol / Q000494:pharmacology; D000818:Animals; D016470:Bacteremia / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004927:Escherichia coli Infections / Q000378:metabolism; D015870:Gene Expression; D000860:Hypoxia / Q000378:metabolism*; D066298:In Vitro Techniques; D008099:Liver / Q000378:metabolism*; D008111:Liver Function Tests; D008297:Male; D016328:NF-kappa B / Q000032:analysis / Q000378:metabolism*; D010477:Perfusion; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism; D013312:Stress, Physiological / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000235:genetics; D014969:Xanthine Oxidase / Q000037:antagonists & inhibitors / Q000378:metabolism
D004791:Enzyme Inhibitors; D016328:NF-kappa B; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; D000493:Allopurinol; D014969:Xanthine Oxidase