Inhibitory effect of Buthus martensi Karsch extracts on interleukin-1beta-induced expression of nitric oxide (NO) synthase and production of NO in human chondrocytes and LPS-induced NO and prostaglandin E2 production in mouse peritoneal macrophages.
We examined the effect of Buthus martensi Karsch (BMK) extract on IL-1beta-induced production of nitrogen oxide (NO) in primary human osteoarthritis (OA) chondrocytes. The cells were treated with BMK (10 microg/ml) and IL-1beta (2 ng/ml) for different periods, and inducible nitric oxide synthase (iNOS) mRNA and protein expression were determined by real-time quantitative reverse transcriptase-polymerase chain reaction and Western blotting, respectively. The cytotoxicity of BMK on human OA chondrocytes was very low (IC50 > 250 microg/ml) as measured by the XTT assay method. Production of NO was determined as nitrite in culture supernatant. Human chondrocytes cotreated with BMK produced significantly less NO compared with chondrocytes stimulated with IL-1beta alone. Activation and translocation of and NF-(kappa)B DNA binding activity were determined by Western blotting and specific enzyme-linked immunosorbent assay. The inhibition of NO production correlated with the suppression of induction and expression of nuclear factor-kappaB (NF-(kappa)B) and activation protein-1 (AP-1)-dependent gene. BMK inhibited the activation and translocation of NF-(kappa)B to the nucleus, indicating that BMK inhibits the IL-1beta-induced production of NO in human chondrocytes by interfering with the activation of NF-(kappa)B through a novel mechanism. In addition, BMK reduced prostaglandin E2 (PGE2) production in mouse peritoneal macrophages stimulated with lipopolysaccharide, whereas no influence on the activity of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2) or cyclooxygenase-1 (COX-1) was observed. Our data, therefore, suggest that BMK may be a therapeutically effective inhibitor of IL-1beta-induced inflammatory effects that are dependent on NF-(kappa)B activation in human OA chondrocytes. The results indicate that BMK exerts anti-inflammatory effects related to the inhibition of neutrophil functions and of NO and PGE2 production, which could be due to a decreased expression of iNOS and COX-2 through the transcription factors NF-(kappa)B and AP-1.
Kim
Kap-Sung
KS
Department of Biochemistry, Molecular Biology and Acupuncture, College of Oriental Medicine, Dongguk University, Kyungju, Republic of Korea.
Cho
Hyun-Seok
HS
Lee
Seung-Deok
SD
Kim
Kyung-Ho
KH
Cho
Jae-Yong
JY
Chung
Kang-Hyun
KH
Lee
Young-Choon
YC
Moon
Sung-Kwon
SK
Kim
Cheorl-Ho
CH
D000818:Animals; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism; D015153:Blotting, Western; D002470:Cell Survival / Q000187:drug effects; D019902:Chondrocytes / Q000187:drug effects / Q000378:metabolism*; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D015232:Dinoprostone / Q000096:biosynthesis*; D005260:Female; D006801:Humans; D007375:Interleukin-1 / Q000037:antagonists & inhibitors* / Q000494:pharmacology*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D017737:Macrophages, Peritoneal / Q000187:drug effects / Q000378:metabolism*; D008565:Membrane Proteins; D051379:Mice; D016328:NF-kappa B / Q000187:drug effects; D009504:Neutrophils / Q000187:drug effects; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000096:biosynthesis* / Q000235:genetics; D052247:Nitric Oxide Synthase Type II; D010003:Osteoarthritis / Q000378:metabolism; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012604:Scorpion Venoms / Q000633:toxicity*; D018808:Transcription Factor AP-1 / Q000378:metabolism
D007375:Interleukin-1; D008070:Lipopolysaccharides; D008565:Membrane Proteins; D016328:NF-kappa B; D012333:RNA, Messenger; D012604:Scorpion Venoms; D018808:Transcription Factor AP-1; D009569:Nitric Oxide; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; C496511:PTGS1 protein, human; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; C496512:Ptgs1 protein, mouse; D015232:Dinoprostone