IRFI 042, a novel dual vitamin E-like antioxidant, inhibits activation of nuclear factor-kappaB and reduces the inflammatory response in myocardial ischemia-reperfusion injury.
BACKGROUND
Nuclear factor-kappaB (NF-kappaB) is a ubiquitous rapid response transcription factor involved in inflammatory reactions which exerts its effect by expressing cytokines, chemokines, and cell adhesion molecules. Oxidative stress causes NF-kappaB activation. IRFI 042 is a novel dual vitamin E-like antioxidant and we, therefore, investigated its ability to protect the heart from oxidative stress and to halt the inflammatory response in a model of myocardial ischaemia-reperfusion injury.


METHODS
Anaesthetized rats were subjected to total occlusion (45 min) of the left main coronary artery followed by 5-h reperfusion (MI/R). Sham myocardial ischaemia rats (sham-operated rats) were used as controls. Myocardial necrosis, cardiac output, cardiac and plasma vitamin E levels, myocardial malondialdehyde (MAL), cardiac SOD activity and elastase content (an index of leukocyte infiltration), hydroxyl radical (OH&z.ccirf;) formation, cardiac amount of mRNA codifying for ICAM-1 (evaluated by the means of reverse transcriptase polymerase chain reaction) and ICAM-1 immunostaining in the at-risk myocardium were investigated. NF-kappaB activation and the inhibitory protein of NF-kappaB, I-kappaBalpha, were also studied in at-risk myocardium by using electrophoretic mobility shift assay (EMSA) and Western blot analysis, respectively.


RESULTS
The ischaemia-reperfusion model produced wide heart necrosis (area at risk-necrotic area=52+/-5%; necrotic area-left ventricle=28+/-3%), increased cardiac MAL, an index of lipid peroxidation (area at risk=62.5+/-3.9 nmol/g tissue; necrotic area=80.3+/-5.1 nmol/g tissue), induced tissue neutrophil infiltration, and caused a marked decrease in endogenous antioxidants. Furthermore, myocardial ischaemia plus reperfusion caused in the area at risk peak message for ICAM-1 at 3 h of reperfusion and increased cardiac ICAM-1 immunostaining at 5 h of reperfusion. NF-kappaB activation was also evident at 0.5 h of reperfusion and reached its maximum at 2 h of reperfusion. I-kappaBalpha was markedly decreased at 45 min of occlusion; peak reduction was observed at 1 h of reperfusion and thereafter it was gradually restored. Intraperitoneal administration of IRFI 042 (5, 10, 20 mg/kg, 5 min after reperfusion) reduced myocardial necrosis expressed as a percentage either of the area at risk (18+/-4%) or the total left ventricle (11+/-2%), and improved cardiac output. This treatment also limited membrane lipid peroxidation in the area at risk (MAL=14.3+/-2.5 nmol/g tissue) and in the necrotic area (MAL=26.5+/-3.7 nmol/g tissue), restored the endogenous antioxidants vitamin E and superoxide dismutase, and inhibited detrimental hydroxyl radical formation. Finally, IRFI 042 blocked the activation of NF-kappaB, reduced cardiac ICAM-1 expression, and blunted tissue elastase content, an index of leukocytes accumulation at the site of injury.


CONCLUSIONS
Our data suggest that IRFI 042 is cardioprotective during myocardial infarction by limiting reperfusion-induced oxidative stress and by halting the inflammatory response.
Altavilla
D
D
Institute of Pharmacology, School of Medicine, University of Messina, Policlinico Universitario, Via Consolare Valeria, Policlinico Gazzi Torre Biologica 5 degrees Piano, 98125, Messina, Italy.
Deodato
B
B
Campo
G M
GM
Arlotta
M
M
Miano
M
M
Squadrito
G
G
Saitta
A
A
Cucinotta
D
D
Ceccarelli
S
S
Ferlito
M
M
Tringali
M
M
Minutoli
L
L
Caputi
A P
AP
Squadrito
F
F
D000704:Analysis of Variance; D000818:Animals; D000975:Antioxidants / Q000627:therapeutic use*; D001572:Benzofurans / Q000737:chemistry* / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D017665:Hydroxyl Radical / Q000032:analysis; D020672:I-kappa B Proteins / Q000378:metabolism; D007150:Immunohistochemistry; D007249:Inflammation; D018799:Intercellular Adhesion Molecule-1 / Q000032:analysis / Q000235:genetics; D015227:Lipid Peroxidation / Q000187:drug effects; D008297:Male; D015428:Myocardial Reperfusion Injury / Q000188:drug therapy* / Q000276:immunology / Q000378:metabolism; D009206:Myocardium / Q000737:chemistry / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects; D010196:Pancreatic Elastase / Q000032:analysis; D012313:RNA / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013482:Superoxide Dismutase / Q000032:analysis; D014810:Vitamin E / Q000032:analysis / Q000097:blood
D000975:Antioxidants; D001572:Benzofurans; D020672:I-kappa B Proteins; C413683:IRFI 042; D016328:NF-kappa B; D018799:Intercellular Adhesion Molecule-1; D014810:Vitamin E; D017665:Hydroxyl Radical; D012313:RNA; D013482:Superoxide Dismutase; D010196:Pancreatic Elastase