Phenylmethimazole inhibits production of proinflammatory mediators and is protective in an experimental model of endotoxic shock*.
BACKGROUND
One form of sepsis, or endotoxic shock, is a hyperactivated systemic response caused by excessive expression of proinflammatory mediators, which results from Gram-negative bacterial lipopolysaccharide-stimulated Toll-like receptor-4 signaling. This lipopolysaccharide signaling is known to consist of a MyD88-dependent nuclear factor-κB-mediated pathway that results in production of proinflammatory mediators (tumor necrosis factor-α, interleukin-6, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, inducible nitric oxide synthase, cyclooxygenase-2) and a MyD88-independent interferon regulatory factor-mediated pathway that regulates production of Type 1 interferon-inducible proteins (interferon γ-induced protein-10, monocyte chemotactic protein-1). In prior studies, phenylmethimazole markedly decreased virally induced Toll-like receptor-3 expression and signaling and significantly suppressed murine colitis in an experimental model wherein lipopolysaccharide is known to play an important role.


OBJECTIVE
In this study, we probed the hypothesis that phenylmethimazole inhibits lipopolysaccharide-mediated Toll-like receptor-4 signaling and is efficacious in attenuating inflammatory changes and improving survival in an in vivo murine model of endotoxic shock.


METHODS
Experimental animal model.


METHODS
University laboratory.


METHODS
Male C57BL/6J mice weighing 18-22 g.


METHODS
Phenylmethimazole (1 mg/kg) was administered intraperitoneally to mice before a lethal lipopolysaccharide challenge (25 mg/kg). RAW264.7 mouse macrophage cells were pretreated with phenylmethimazole followed by lipopolysaccharide stimulation.


RESULTS
: Macroscopic observations revealed that phenylmethimazole was significantly protective in controlling clinical manifestations of endotoxic shock and death under conditions wherein flunixin of meglumine and prednisolone were marginally effective. A combination of enzyme-linked immunosorbent assay, Northern blot, reverse transcriptase-polymerase chain reaction, immunohistochemistry, and Western blot analyses showed that phenylmethimazole attenuated lipopolysaccharide-induced increases in production of proinflammatory cytokines (tumor necrosis factor-α, interleukin-6, interferon-γ), endothelial cell adhesion molecules (intercellular adhesion molecule-1, vascular cell adhesion molecule-1), inducible nitric oxide synthase and cyclooxygenase-2, interferon regulatory factor-1, interferon-inducible proteins (interferon γ-induced protein-10, monocyte chemotactic protein-1), and signal transducer and activator of transcription-1 phosphorylation in multiple tissues in mice. Consistent with these observations, electrophoretic mobility shift assay demonstrated that phenylmethimazole inhibited in vitro lipopolysaccharide-induced nuclear factor-κB and interferon regulatory factor-1 activation in RAW 264.7 mouse macrophages.


CONCLUSIONS
Collectively, these results provide direct evidence that phenylmethimazole diminishes lipopolysaccharide-induced MyD88-dependent as well as MyD88-independent signaling pathways and is protective in an experimental model of endotoxic shock.
Departments of Biomedical Sciences, Ohio University Heritage, College of Osteopathic Medicine, and Edison Biotechnology Institute, Ohio University, Athens, OH, USA.
Uruguaysito
U
Benavides
Mariana
M
Gonzalez-Murguiondo
Norikazu
N
Harii
Christopher J
CJ
Lewis
Anthony L
AL
Schwartz
Cesidio
C
Giuliani
Giorgio
G
Napolitano
Nilesh M
NM
Dagia
Ramiro
R
Malgor
Kelly D
KD
McCall
Leonard D
LD
Kohn
D000818:Animals; D016207:Cytokines / Q000096:biosynthesis* / Q000187:drug effects*; D004195:Disease Models, Animal; D007249:Inflammation / Q000276:immunology; D008297:Male; D008713:Methimazole / Q000031:analogs & derivatives* / Q000627:therapeutic use; D051379:Mice; D008810:Mice, Inbred C57BL; D012772:Shock, Septic / Q000276:immunology* / Q000378:metabolism / Q000517:prevention & control*; D013871:Thiones / Q000627:therapeutic use*
D016207:Cytokines; D013871:Thiones; C489637:phenyl methimazole; D008713:Methimazole