KE-298 and its active metabolite KE-758 suppress nitric oxide production by murine macrophage cells and peritoneal cells from rats with adjuvant induced arthritis.
OBJECTIVE
To analyze the effects of KE-298 and KE-758 on lipopolysaccharide (LPS) induced nitric oxide (NO) production by the RAW264.7 murine macrophage cell line, and the effect of KE-758 on spontaneous NO production by peritoneal cells from rats with adjuvant induced arthritis.


METHODS
The amount of NO was determined using Griess reagents. The proteins for inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) were detected by Western blot, then mRNA for interferon-beta (IFN)-beta, IFN regulatory factor-1 (IRF-1), and iNOS were detected by RT-PCR. Degradation of iNOS mRNA was analyzed using Northern blot. Nuclear factor-kappa B (NF-kappa B) in nuclear extracts was determined by EMSA. Adjuvant arthritis in rats was induced by inoculating heat killed Mycobacterium butyricum s.c. in the tail.


RESULTS
KE-298 and KE-758 suppressed NO production by LPS activated RAW264.7 cells by inhibiting iNOS gene expression. Neither LPS induced NF-kappa B activation nor degradation of iNOS mRNA was affected by KE-758 treatment. LPS induced IFN-beta and IRF-1 gene expression were markedly suppressed by KE-758. In rats with adjuvant induced arthritis, enhanced NO and iNOS production by cultured peritoneal cells and the development of arthritis were suppressed by KE-758.


CONCLUSIONS
KE-758 suppressed LPS induced iNOS gene expression by murine macrophage cells by inhibiting IFN-beta/IRF-1 expression. The potential of KE-758 to inhibit iNOS production might partly explain its efficacy on adjuvant induced arthritis in rats.
Inoue
T
T
Department of Pharmacology, Pharmaceutical Research Laboratories, Taisho Pharmaceutical Co., Ltd., 1-403, Yoshino-cho, Ohmiya, Saitama 330-8530, Japan. s15376@ccm.taisho.co.jp
Hamada
Y
Y
Takeshita
K
K
Fukushima
K
K
Higaki
M
M
D000818:Animals; D018501:Antirheumatic Agents / Q000737:chemistry / Q000494:pharmacology*; D001169:Arthritis, Experimental / Q000188:drug therapy* / Q000276:immunology / Q000378:metabolism; D002461:Cell Line, Transformed; D051546:Cyclooxygenase 2; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000235:genetics; D004487:Edema / Q000188:drug therapy; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D015870:Gene Expression / Q000187:drug effects / Q000276:immunology; D007275:Injections, Intravenous; D050856:Interferon Regulatory Factor-1; D016899:Interferon-beta / Q000235:genetics; D007527:Isoenzymes / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D017737:Macrophages, Peritoneal / Q000166:cytology / Q000201:enzymology*; D016328:NF-kappa B / Q000378:metabolism; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D010654:Phenylbutyrates / Q000494:pharmacology*; D010666:Phenylpropionates / Q000737:chemistry / Q000494:pharmacology*; D010750:Phosphoproteins / Q000235:genetics; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
D018501:Antirheumatic Agents; D017931:DNA Primers; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D050856:Interferon Regulatory Factor-1; C494259:Irf1 protein, rat; D007527:Isoenzymes; C102509:KE 758; D008070:Lipopolysaccharides; D016328:NF-kappa B; D010654:Phenylbutyrates; D010666:Phenylpropionates; D010750:Phosphoproteins; D012333:RNA, Messenger; D009569:Nitric Oxide; D016899:Interferon-beta; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; C078547:esonarimod; D019331:NG-Nitroarginine Methyl Ester