Nuclear factor-kappaB as a target of cyclosporin in acute hypovolemic hemorrhagic shock.
BACKGROUND
Cyclosporin is an immunosuppressive drug that blocks Nuclear Factor kappaB (NF-kappaB) activation. We investigated the role of NF-kappaB in acute hypovolemic hemorrhagic (Hem) shock and the effects of cyclosporin in this model of experimental shock.


METHODS
Hem shock was induced in male anesthetized rats by intermittently withdrawing blood from an iliac catheter over a period of 20 min (bleeding period) until mean arterial blood pressure (MAP) fell and stabilized within the range of 20-30 mmHg. Two minutes after bleeding cessation, animals received intravenously cyclosporin (1 mg kg(-1)) or its vehicle. Survival rate and survival time were evaluated for 120 min after bleeding was discontinued. Plasma TNF-alpha levels were investigated at different time points after bleeding cessation. Moreover we investigated levels of TNF-alpha mRNA in the liver, vascular reactivity, liver NF-kappaB binding activity and levels of the inhibitory protein IkappaBalpha in the cytoplasm.


RESULTS
Hemorrhagic shocked rats died in 27+/-6 min following the cessation of bleeding, experienced a marked hypotension (mean arterial blood pressure=20-30 mmHg) and had enhanced plasma levels of Tumor Necrosis Factor-alpha (208+/-22 pg ml(-1), 20 min after the end of bleeding). Furthermore, aortas taken 20 min after bleeding from hemorrhagic shocked rats showed a marked hypo-reactivity to phenylephrine (PE: 1 nM-10 microM) compared with aortas harvested from sham shocked rats. Hem shocked rats also had increased levels of TNF-alpha mRNA in the liver (15-20 min after the end of bleeding). Electrophoretic mobility shift assay showed that liver NF-kappaB binding activity increased in the nucleus 10 min after the end of hemorrhage and remained elevated until the death of animals. Western blot analysis suggested that the levels of inhibitory protein IkappaBalpha in the cytoplasm decreased at 5 min after the end of bleeding. Cyclosporin inhibited the loss of IkappaBalpha protein from the cytoplasm and prevented NF-kappaB binding activity in the nucleus. Furthermore, cyclosporin increased survival time (118+/-7 min; P<0.01) and survival rate (vehicle=0% and cyclosporin=80%, at 120 min after the end of bleeding), reverted the marked hypotension, decreased liver mRNA for TNF-alpha, reduced plasma TNF-alpha (28+/-7 pg ml(-1)), and restored to control values the hypo-reactivity to PE.


CONCLUSIONS
Our results suggest that acute blood loss (50% of the estimated total blood volume over a period of 20 min) causes early activation of NF-kappaB which triggers an inflammatory cascade leading to a fatal outcome. Cyclosporin blocks NF-kappaB activation and protects against hypovolemic hemorrhagic shock.
Altavilla
D
D
Department of Experimental and Clinical Medicine and Pharmacology, Section of Pharmacology, School of Medicine, University of Messina, Messina, Italy.
Saitta
A
A
Guarini
S
S
Galeano
M
M
Squadrito
G
G
Santamaria
L B
LB
Venuti
F S
FS
Bazzani
C
C
Bertolini
A
A
Squadrito
F
F
D000818:Animals; D001011:Aorta / Q000187:drug effects; D015153:Blotting, Western / Q000379:methods; D002478:Cells, Cultured; D016572:Cyclosporine / Q000627:therapeutic use*; D003593:Cytoplasm / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000032:analysis; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D006861:Hydrogen Peroxide / Q000494:pharmacology; D020672:I-kappa B Proteins; D007166:Immunosuppressive Agents / Q000627:therapeutic use*; D066298:In Vitro Techniques; D008099:Liver / Q000378:metabolism; D008262:Macrophage Activation; D017737:Macrophages, Peritoneal / Q000187:drug effects / Q000378:metabolism; D008297:Male; D023421:Models, Animal; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism*; D010656:Phenylephrine / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012769:Shock / Q000097:blood / Q000378:metabolism / Q000517:prevention & control*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis / Q000235:genetics; D014662:Vasoconstrictor Agents / Q000494:pharmacology
D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D007166:Immunosuppressive Agents; D016328:NF-kappa B; C000606206:Nfkbia protein, rat; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D014662:Vasoconstrictor Agents; D000072000:NF-KappaB Inhibitor alpha; D010656:Phenylephrine; D016572:Cyclosporine; D006861:Hydrogen Peroxide; D013755:Tetradecanoylphorbol Acetate