Brief hypoxic stress suppresses postbacteremic NF-κB activation and TNF-α bioactivity in perfused liver

Reductions in hepatic O2 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-κB (NF-κB) via generation of reactive O2 species (ROS), the combination of these stimuli downregulates hepatic TNF-α gene expression. Here we tested the hypothesis that hypoxic suppression of postbacteremic TNF-α gene expression is transcriptionally mediated by reduced activation of NF-κB. Buffer-perfused rat livers ( n = 52) were studied over 180 min after intraportal infection at t = 0 with 109 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 ∼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-κB 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 using32P-labeled oligonucleotides specific for NF-κB. Compared with normoxic EC controls, hypoxia reduced postbacteremic NF-κB nuclear translocation and TNF-α bioactivity, independent of reoxygenation, tissue levels of reduced glutathione, or posthypoxic O2 consumption. XO inhibition reversed the hypoxic suppression of NF-κB nuclear translocation and ameliorated decreases in cell-associated TNF-α. Thus decreases in hepatic O2delivery reduce postbacteremic nuclear translocation of NF-κB and hepatic TNF-α biosynthesis by signaling mechanisms involving low-level generation of XO-mediated ROS.