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. 2008 May 29;5:7.
doi: 10.1186/1476-9255-5-7.

JNK Pathway Is Involved in the Inhibition of Inflammatory Target Gene Expression and NF-kappaB Activation by Melittin

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Free PMC article

JNK Pathway Is Involved in the Inhibition of Inflammatory Target Gene Expression and NF-kappaB Activation by Melittin

Hye Ji Park et al. J Inflamm (Lond). .
Free PMC article

Abstract

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.

Conclusion: 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.

Figures

Figure 1
Figure 1
Effect of melittin and bee venom on LPS and SNP-induced phosphorylation of MAPKs. A, Raw 264.7 macrophages were treated with 5 or 10 μg/ml melittin or 0.5–5 μg/ml bee venom in the presence of LPS (1 μg/ml) or SNP (200 μM) at 37°C for 24 hr. B, Synoviocytes were treated with 5 or 10 μg/ml melittin or 0.5–5 μg/ml bee venom in the presence of 1 μg/ml LPS or 200 μM SNP at 37°C for 24 hr. Equal amounts of total proteins (80 μg/lane) were subjected to 10% SDS ± PAGE, and the expression of p-ERK/ERK, p-JNK/JNK, or p-p38/p38 were detected by western blotting using specific antibodies. Each panel representative of three independent experiments. Quantification of band intensities from three independent experimental results was determined by a densitometry (Imaging System). Data was described as means ± S.E. from three experiments performed in triplicate for p-ERK/ERK, p-JNK/JNK, or p-p38/p38. *p < 0.05 indicate statistically significant differences from the LPS or SNP-treated group.
Figure 2
Figure 2
JNK inhibitor suppressed the inhibitory effect of melittin and bee venom on the NF-κB DNA binding activity induced by LPS or SNP. Raw 264.7 macrophages (A) and synoviocytes (B) were pretreated with 10, 20, and 50 μM SP600125 1 h prior to the treatment with melittin or bee venom with or without LPS or SNP for 2 h. The DNA binding activation of NF-κB was investigated using EMSA. Nuclear extracts from Raw 264.7 cells or synoviocytes treated for 1 hr were incubated with 32P-end-labeled oligonucleotide containing the κB sequence. Each panel is representative of three similar experiments with duplicates.
Figure 3
Figure 3
JNK inhibitor suppressed the inhibitory effect of melittin and bee venom on the nuclear translocation of the p50 subunit and the release of IκB induced by LPS or SNP. Raw 264.7 cells (A) or synoviocytes (B) were pretreated with 10 and 20 μM SP600125 1 h prior to the treatment with melittin or bee venom with or without LPS (1 μg/ml) or SNP (200 μM) at 37°C for 24 hr. 80 μg of nuclear (p50 and p65), cytosolic IκB or total protein extracted after treatment were used to determine of p50, p65, p-IκBα, IκBα, or IκBβ; β-actin protein was used as an internal control. Each panel is representative of three similar experiments. C, Raw 264.7 cells were treated with LPS, BV and SP for 24 hr, and then the intracellular location of p50 was determined by immunofluorescence confocal scanning microscope (magnification, 630×). Double staining (Merge, pink) with p50 (red) and DAPI (blue) staining demonstrates the localization of p50 in the nucleus.
Figure 4
Figure 4
JNK inhibitor suppressed the inhibitory effect of melittin and bee venom on the on NF-κB-dependent luciferase induced by LPS or SNP. Raw 264.7 cells (A) and THP-1 cells (B) were transfected with pNF-κB-Luc plasmid (5× NF-κB), Raw 264.7 cells or THP-1 cells were pretreated with 10 and 20 μM SP600125 1 hr prior to the treatment with melittin or bee venom with or without LPS (1 μg/ml) or SNP (200 μM) at 37°C for 2 hr, and then luciferase activities were determined. All values represent means ± S.E. of three independent experiments performed in triplicate.
Figure 5
Figure 5
JNK inhibitor suppressed inhibitory effect of melittin and bee venom on the inflammatory gene expression induced by LPS or SNP. Raw 264.7 cells (A) or synoviocytes (B) were pretreated with 10 and 20 μM SP600125 1 hr prior to the treatment with melittin or bee venom with or without LPS (1 μg/ml) or SNP (200 μM) at 37°C for 24 hr. Equal amounts of total proteins (80 μg/lane) were subjected to 10% SDS ± PAGE, and the expression of iNOS, COX-2 and β-actin were detected by western blotting using specific antibodies. Each panel representative of three independent experiments.
Figure 6
Figure 6
JNK inhibitor suppressed the inhibitory effect of melittin and bee venom on the generation of NO induced by LPS or SNP. Raw 264.7 cells (A, C) or synoviocytes (B, D) were pretreated with 10 and 20 μM SP600125 1 hr prior to the treatment with melittin or bee venom with or without LPS (1 μg/ml) or SNP (200 μM) at 37°C for 24 hr. The amounts of NO in the medium of cultured Raw264.7 cells (A, C) or synoviocytes (B, D) were determined by the methods described in the methods. Results are expressed as means ± SE of three independent experiments performed in triplicate. * indicates significantly different from the LPS or SNP treated groups (p < 0.05). # and ##, indicates significantly different from LPS or SNP + melittin or BV treated group (p < 0.05).
Figure 7
Figure 7
JNK inhibitor suppressed the inhibitory effect of melittin and bee venom on the generation of PGE2 induced by LPS or SNP. Raw 264.7 cells (A, C) or synoviocytes (B, D) were pretreated with 10 and 20 μM SP600125 1 hr prior to the treatment with melittin or bee venom with or without LPS (1 μg/ml) or SNP (200 μM) at 37°C for 24 hr. The amounts of PGE2 in the medium of cultured Raw264.7 cells (A, C) or synoviocytes (B, D) were determined by the methods described in the methods. Results are expressed as means ± SE of three independent experiments performed in triplicate. * indicates significantly different from the LPS or SNP treated groups (p < 0.05). # and ##, indicates significantly different from LPS or SNP + melittin or BV treated group (p < 0.05).

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