JNK pathway is involved in the inhibition of inflammatory target gene expression and NF-kappaB activation by melittin.
BACKGROUND
Bee venom therapy has been used to treat inflammatory diseases including rheumatoid arthritis in humans and in experimental animals. We previously found that bee venom and melittin (a major component of bee venom) have anti-inflammatory effect by reacting with the sulfhydryl group of p50 of nuclear factor-kappa B (NF-kappaB) and IkappaB kinases (IKKs). Since mitogen activated protein (MAP) kinase family is implicated in the NF-kappaB activation and inflammatory reaction, we further investigated whether activation of MAP kinase may be also involved in the anti-inflammatory effect of melittin and bee venom.


METHODS
The anti-inflammatory effects of melittin and bee venom were investigated in cultured Raw 264.7 cells, THP-1 human monocytic cells and Synoviocytes. The activation of NF-kappaB was investigated by electrophoretic mobility shift assay. Nitric oxide (NO) and prostaglandin E2 (PGE2) were determined either by Enzyme Linked Immuno Sorbent Assay or by biochemical assay. Expression of IkappaB, p50, p65, inducible nitric oxide synthetase (iNOS), cyclooxygenase-2 (COX-2) as well as phosphorylation of MAP kinase family was determined by Western blot.


RESULTS
Melittin (0.5-5 mug/ml) and bee venom (5 and 10 mug/ml) inhibited lipopolysaccharide (LPS, 1 mug/ml) and sodium nitroprusside (SNP, 200 muM)-induced activation of c-Jun NH2-terminal kinase (JNK) in RAW 264.7 cells in a dose dependent manner. However, JNK inhibitor, anthra [1,9-cd]pyrazole-6 (2H)-one (SP600215, 10-50 muM) dose dependently suppressed the inhibitory effects of melittin and bee venom on NF-kappaB dependent luciferase and DNA binding activity via suppression of the inhibitory effect of melittin and bee venom on the LPS and SNP-induced translocation of p65 and p50 into nucleus as well as cytosolic release of IkappaB. Moreover, JNK inhibitor suppressed the inhibitory effects of melittin and bee venom on iNOS and COX-2 expression, and on NO and PGE2 generation.


CONCLUSIONS
These data show that melittin and bee venom prevent LPS and SNP-induced NO and PGE2 production via JNK pathway dependent inactivation of NF-kappaB, and suggest that inactivation of JNK pathways may also contribute to the anti-inflammatory and anti-arthritis effects of melittin and bee venom.
Park
Hye Ji
HJ
College of Pharmacy, Chungbuk National University, 12 Gaesin-dong, Heungduk-gu, Cheongju, Chungbuk 361-763, Korea. jinthong@chungbuk.ac.kr.
Lee
Hwa Jeong
HJ
Choi
Myung Sook
MS
Son
Dong Ju
DJ
Song
Ho Sueb
HS
Song
Min Jong
MJ
Lee
Jeong Min
JM
Han
Sang Bae
SB
Kim
Youngsoo
Y
Hong
Jin Tae
JT