Evidence for a role of nuclear factor-kappaB in acute hypovolemic hemorrhagic shock.
BACKGROUND
In acute hypovolemic shock, a rapid systemic release of the inflammatory cytokine tumor necrosis factor (TNF-alpha) contributes to vascular failure. Nuclear factor kappaB (NF-kappaB) is an ubiquitous rapid-response transcription factor involved in inflammatory reactions and exerts its effect by expressing cytokines, chemokines, and cell adhesion molecules. The purpose of this study was to evaluate the role of NF-kappaB in acute hypovolemic hemorrhagic shock.


METHODS
Hemorrhagic shock was induced in anesthetized male rats by intermittently withdrawing blood from an iliac catheter for 20 minutes (bleeding period) until mean arterial blood pressure (MAP) decreased and stabilized within the range of 20 to 30 mm Hg. Two minutes after bleeding was discontinued the rats received tacrolimus (100 microg/kg), an inhibitor of NF-kappaB activation, or its vehicle. We then evaluated survival rate and survival time, liver NF-kappaB activation by means of electrophoretic mobility shift assay, liver IkappaBalpha protein in the cytoplasm, hepatic TNF-alpha messenger RNA expression, plasma TNF-alpha, arterial blood pressure, and the contractile response of aortic rings to phenylephrine.


RESULTS
Rats that underwent hemorrhagic shock died 28+/-2 minutes after bleeding was discontinued, experienced marked hypotension (MAP, 20-30 mm Hg), and had enhanced plasma levels of TNF-alpha (218 +/- 28 pg/mL 20 minutes after bleeding was discontinued). Aortas taken 20 minutes after bleeding was discontinued in rats that underwent hemorrhagic shock showed marked hyporeactivity to phenylephrine (1 nmol/L-10 micromol/L) compared with aortas harvested from sham shocked rats. Rats that underwent hemorrhagic shock also had increased levels of TNF-alpha messenger RNA in the liver. Furthermore, electrophoretic mobility shift assay showed that liver NF-kappaB binding activity increased in the nucleus, and Western blot analysis suggested that the levels of inhibitory IkappaBalpha protein in the cytoplasm decreased. Tacrolimus (100 microg/kg, administered 2 minutes after bleeding was discontinued) inhibited the loss of IkappaBalpha protein from the cytoplasm and prevented NF-kappaB binding activity in the nucleus. Moreover, tacrolimus increased survival time (118 +/- 7 minutes; P <.01) and survival rate (vehicle = 0 and tacrolimus = 90% 240 minutes after bleeding was discontinued), reverted the marked hypotension, decreased liver messenger RNA for TNF-alpha reduced plasma TNF-alpha (35 +/- 6 pg/mL), and restored the hyporeactivity to phenylephrine to control values.


CONCLUSIONS
Our results suggest that acute blood loss (50% of the estimated total blood volume during a 20-minute period) causes activation of NF-kappaB and that tacrolimus, by inhibiting this transcription factor, protects against acute hypovolemic shock.
Altavilla
Domenica
D
University of Messina, Azienda Ospedaliera Universitaria "G. Martino," Messina, Italy.
Saitta
Antonino
A
Squadrito
Giovanni
G
Galeano
Mariarosaria
M
Venuti
Saverio F
SF
Guarini
Salvatore
S
Bazzani
Carla
C
Bertolini
Alfio
A
Caputi
Achille P
AP
Squadrito
Francesco
F
D000208:Acute Disease; D000818:Animals; D001794:Blood Pressure; D008297:Male; D016328:NF-kappa B / Q000502:physiology*; D009569:Nitric Oxide / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D012769:Shock / Q000209:etiology* / Q000378:metabolism / Q000401:mortality; D012771:Shock, Hemorrhagic / Q000209:etiology* / Q000378:metabolism / Q000401:mortality; D015996:Survival Rate; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics
D016328:NF-kappa B; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide