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Inhibition of nuclear factor-kappaB activation by IRFI 042, protects against endotoxin-induced shock
Domenica Altavilla1, Giovanni Squadrito, Letteria Minutoli
1Department of Clinical and Experimental Medicine and Pharmacology, Section of Pharmacology School of Medicine, University of Messina, Torre Biologica, Azienda Ospedaliera Universitaria G Martino, Via C. Valeria, Gazzi, Messina, Italy.
Background:
The aim of our study was to investigate the effect of IRFI 042, a novel dual vitamin E-like antioxidant, on nuclear factor-kappaB (NF-kappaB) activation, TNF-alpha gene priming and on the release of the mature protein during endotoxin shock.
Methods:
Endotoxin shock was produced in male rats by a single intravenous (i.v.) injection of 20 mg kg(-1) of Salmonella enteritidis lipopolysaccharide (LPS). Survival rate, mean arterial blood pressure, serum TNF-alpha and plasma malondialdehyde (MAL) levels were investigated. We then evaluated in the liver TNF-alpha mRNA levels, NF-kappaB binding activity and the inhibitory protein IkappaBalpha. Moreover we studied in LPS stimulated (50 microg ml(-1)) peritoneal macrophages (Mphi), NF-kappaB activation, cytoplasmic IkappaB-alpha degradation, the message for TNF-alpha, and TNF-alpha and MAL levels.
Results:
LPS administration reduced survival rate (0%, 72 h after LPS administration), decreased mean arterial blood pressure, augmented serum TNF-alpha (60+/-11 ng ml(-1)) and enhanced plasma malondialdehyde (MAL) levels (55+/-7.1 nmol l(-1)). LPS shocked rats also had increased TNF-alpha mRNA levels, augmented liver NF-kappaB binding activity in the nucleus and decreased levels of the inhibitory protein IkappaBalpha. In addition, in vitro LPS stimulation (50 microg ml(-1)) significantly induced NF-kappaB activation and cytoplasmic IkappaBalpha degradation in Mphi, enhanced TNF-alpha mRNA levels and increased Mphi TNF-alpha and MAL. Treatment with IRFI 042 (20 mg kg(-1), i.v., 5 min after endotoxin challenge) protected against LPS-induced lethality (90% survival rate 24 h and 80% survival rate 72 h after LPS injection, respectively), reduced hypotension, blunted plasma MAL (9.0+/-0.9 nmol l(-1)) and decreased serum TNF-alpha (15+/-3 ng ml(-1)). The antioxidant also inhibited the loss of IkappaBalpha protein from the hepatic cytoplasm, blunted the increased NF-kappaB binding activity in the liver and decreased hepatic liver mRNA for TNF-alpha. Furthermore 'in vitro' IRFI 042 (50 microM) significantly inhibited activation of NF-kappaB through inhibition of IkappaBalpha degradation, reduced the amount of TNF-alpha mRNA, decreased LPS-induced TNF-alpha release and blunted lipid peroxidation (MAL) in LPS stimulated Mphi.
Conclusions:
These data suggest that IRFI 042 blocks the activation of NF-kappaB, reduces TNF-alpha mRNA levels, and finally reverses endotoxic shock.