Differential activation pattern of redox-sensitive transcription factors and stress-inducible dilator systems heme oxygenase-1 and inducible nitric oxide synthase in hemorrhagic and endotoxic shock.

OBJECTIVE: To investigate the role of redox-sensitive transcription factors nuclear factor kappa-B (NF-kappaB) or activator protein-1 (AP-1) for hepatic gene expression of heme oxygenase (HO)-1 and inducible nitric oxide synthase (iNOS) in models of hemorrhagic or endotoxic shock. DESIGN: Prospective controlled laboratory study. SETTING: Animal research laboratory at a university hospital. SUBJECTS: Male Sprague-Dawley rats (250-350 g). INTERVENTIONS: After anesthesia, animals were assigned to hemorrhagic shock (mean arterial pressure 35-40 mm Hg for 60 mins), sham operation, or endotoxemia (1 mg/kg intraperitoneally). To assess the role of reactive oxygen species for activation of NF-kappaB or AP-1, animals were treated with the antioxidant trolox (6 mg/kg body weight). In additional experiments, animals were pretreated with dexamethasone (10 mg/kg body weight), an inhibitor of the transactivating function of DNA-bound AP-1 or with actinomycin-D (2 mg/kg body weight), an inhibitor of DNA-directed RNA synthesis. Activation of NF-kappaB or AP-1 was assessed by electrophoretic mobility shift assay. HO-1 and iNOS gene expression were assessed by Northern and Western blot. MEASUREMENTS AND MAIN RESULTS: Hemorrhage and resuscitation induced hepatic HO-1 transcripts 12-fold. Induction was abolished by actinomycin-D and was attenuated by dexamethasone and the antioxidant trolox. Activation of AP-1 was observed after hemorrhagic but not after endotoxic shock. AP-1 activation was inhibitable by trolox and correlated with accumulation of HO-1 transcripts. In contrast, a weak activation of NF-kappaB was observed after hemorrhage that was not affected by trolox. A profound activation of NF-kappaB after endotoxic shock correlated with induction of iNOS but failed to induce HO-1 transcripts. CONCLUSIONS: These data suggest that AP-1 but not NF-kappaB activation is dependent on reactive oxygen intermediates in vivo and contributes to HO-1 gene expression. Thus, AP-1-dependent HO-1 induction under oxidative stress conditions may subserve a similar function as a stress-inducible vasodilator system as does NF-kappaB-dependent iNOS expression in liver inflammation.

Differential expression pattern of heme oxygenase-1/heat shock protein 32 and nitric oxide synthase-II and their impact on liver injury in a rat model of hemorrhage and resuscitation.

OBJECTIVE: To investigate the role of the vasodilator systems heme oxygenase-1/heat shock protein 32 (HO-1/HSP32) and nitric oxide synthase-II (NOS-II), generating carbon monoxide and nitric oxide respectively, as modulators of liver injury in an experimental model of reversible hemorrhagic shock. DESIGN: Prospective controlled laboratory study. SETTING: University research laboratory. SUBJECTS: Male Sprague-Dawley rats weighing 250-350 g. INTERVENTIONS: Animals were anesthetized and assigned to a hemorrhagic shock (mean arterial pressure, 35-40 mmHg for 60 mins) or a sham protocol. On the basis of the time course of gene expression, HO-1/HSP32 or NOS-II was blocked 5 hrs after onset of resuscitation. To assess the role of the antioxidative properties of the heme oxygenase (HO) pathway in additional experiments, Trolox, a potent antioxidant, was administered at the time of blockade of HO. Liver injury was assessed morphometrically and by plasma alpha-glutathione-S-transferase (alpha-GST) release 11 hours after onset of resuscitation. MEASUREMENTS AND MAIN RESULTS: Hemorrhage and resuscitation increased HO-1/HSP32 messenger RNA and protein primarily in parenchymal cells, and a faint induction of NOS-II, restricted to nonparenchymal cells, was observed. Inhibition of the HO pathway with tin protoporphyrin-IX (SnPP-IX) increased the incidence of pericentral necrosis (intact acini: shock/vehicle 68.8%; shock/SnPP-IX 42.6%) and alpha-GST levels (sham 94+/-24 microg/L; shock/vehicle 377+/-139 microg/L; shock/SnPP-IX 1708+/-833 microg/L), whereas blockade of NOS-II with S-methylisothiourea did not affect liver injury. Coadministration of Trolox failed to attenuate the aggravation of necrosis associated with blockade of HO, whereas alpha-GST levels were reduced (intact acini: shock/vehicle/Trolox 82.1%, shock/SnPP-IX/Trolox 42.7%; alpha-GST: shock/vehicle/Trolox 202+/-55 microg/L; shock/SnPP-IX/Trolox 236+/-61 microg/L). CONCLUSIONS: These data suggest that HO-1/HSP32, but not the alternative cyclic guanosine monophosphate-generating enzyme NOS-II, is induced after hemorrhage and resuscitation and protects against hepatocellular injury. Both metabolites generated by the heme oxygenase pathway, e.g., carbon monoxide (a vasodilator) and biliverdin (an antioxidant) seem to contribute to the salutary effects of induction of HO-1/HSP32.