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Oleifolioside A, a New Active Compound, Attenuates LPS-Stimulated iNOS and COX-2 Expression through the Downregulation of NF-κB and MAPK Activities in RAW 264.7 Macrophages.

Yu HY, Kim KS, Lee YC, Moon HI, Lee JH - Evid Based Complement Alternat Med (2012)

Bottom Line: Oleifolioside A, a new triterpenoid compound isolated from Dendropanax morbifera Leveille (D. morbifera), was shown in this study to have potent inhibitory effects on lipopolysaccharide (LPS-)stimulated nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production in RAW 264.7 macrophages.These results were found to be associated with the inhibition of the degradation and phosphorylation of IκB-α and subsequent translocation of the NF-κB p65 subunit to the nucleus.Taken together, our results suggest that oleifolioside A has the potential to be a novel anti-inflammatory agent capable of targeting both the NF-κB and MAPK signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: College of Natural Resources and Life Science, BK21 Center for Silver-Bio Industrialization, Dong-A University, Busan 604-714, Republic of Korea.

ABSTRACT
Oleifolioside A, a new triterpenoid compound isolated from Dendropanax morbifera Leveille (D. morbifera), was shown in this study to have potent inhibitory effects on lipopolysaccharide (LPS-)stimulated nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production in RAW 264.7 macrophages. Consistent with these findings, oleifolioside A was further shown to suppress the expression of LPS-stimulated inducible nitric oxide synthase (iNOS) and cyclooxigenase-2 (COX-2) in a dose-dependent manner at both the protein and mRNA levels and to significantly inhibit the DNA-binding activity and transcriptional activity of NF-κB in response to LPS. These results were found to be associated with the inhibition of the degradation and phosphorylation of IκB-α and subsequent translocation of the NF-κB p65 subunit to the nucleus. Inhibition of NF-κB activation by oleifolioside A was also shown to be mediated through the prevention of p38 MAPK and ERK1/2 phosphorylation. Taken together, our results suggest that oleifolioside A has the potential to be a novel anti-inflammatory agent capable of targeting both the NF-κB and MAPK signaling pathways.

No MeSH data available.


Effects of oleifoliosides A on LPS-stimulated NF-κB activation and phosphorylation of MAPK signaling pathway. (a) Cells were pretreated with the indicated the concentrations of oleifoliosides A for 1 h and then stimulated with LPS (500 ng/mL) for 1 h. Nuclear extracts were prepared and analyzed for NF-κB DNA-binding activity by EMSA. The C.P. represents 100-fold cold probe only. (b) Cells were transiently cotransfected with pNF-κB-Luc reporter plasmid and then were pretreated with the indicated the concentrations of oleifoliosides A for 1 h. LPS (500 ng/mL) was then added and cells were further incubated 24 h. The cells were harvested and then the luciferase activities were determined by using the dual luciferase report assay system. (c) Cells were pretreated with the indicated concentrations of oleifoliosides A for 1 h before LPS (500 ng/mL) treatment for 15 min. and the levels of total/phospho-ERK1/2, JNK, and p38 MAPK were determined by Western blotting. The values are presented as mean±S.E. of three independent experiments. n = 6 per experiment, *P < 0.05 versus LPS and **P < 0.01 versus LPS.
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fig4: Effects of oleifoliosides A on LPS-stimulated NF-κB activation and phosphorylation of MAPK signaling pathway. (a) Cells were pretreated with the indicated the concentrations of oleifoliosides A for 1 h and then stimulated with LPS (500 ng/mL) for 1 h. Nuclear extracts were prepared and analyzed for NF-κB DNA-binding activity by EMSA. The C.P. represents 100-fold cold probe only. (b) Cells were transiently cotransfected with pNF-κB-Luc reporter plasmid and then were pretreated with the indicated the concentrations of oleifoliosides A for 1 h. LPS (500 ng/mL) was then added and cells were further incubated 24 h. The cells were harvested and then the luciferase activities were determined by using the dual luciferase report assay system. (c) Cells were pretreated with the indicated concentrations of oleifoliosides A for 1 h before LPS (500 ng/mL) treatment for 15 min. and the levels of total/phospho-ERK1/2, JNK, and p38 MAPK were determined by Western blotting. The values are presented as mean±S.E. of three independent experiments. n = 6 per experiment, *P < 0.05 versus LPS and **P < 0.01 versus LPS.

Mentions: NF-κB is a key transcription factor involved in general inflammation as well as immune responses. Its activation is critically required for the expression of proteins, including iNOS and COX-2, in macrophages. To evaluate the molecular mechanism through which oleifolioside A inhibits the expression of such proinflammatory mediators, we examined NF-κB DNA-binding activity by EMSA. As shown in Figure 4(a), treatment with LPS alone caused a marked increase in the DNA-binding activity of NF-κB. In contrast, pretreatment with oleifolioside A significantly attenuated LPS-stimulated NF-κB DNA-binding activity in a dose-dependent manner. To confirm the results of EMSA, we also investigated the effects of oleifolioside A on NF-κB-dependent reporter gene expression following LPS treatment by luciferase reporter assay. RAW 264.7 macrophages were transiently cotransfected with a pNF-κB-leu reporter vector generated by inserting four spaced NF-κB-binding sites into the pLuc-promoter vector and then stimulated with 500 ng/mL LPS with or without oleifolioside A. As shown in Figure 4(b), treatment with oleifolioside A significantly reduced the level of NF-κB luciferase activity stimulated by LPS in a dose-dependent manner. Taken together, these findings demonstrate that oleifolioside A suppresses the expression of iNOS and COX-2, at least in part via an NF-κB-dependent mechanism.


Oleifolioside A, a New Active Compound, Attenuates LPS-Stimulated iNOS and COX-2 Expression through the Downregulation of NF-κB and MAPK Activities in RAW 264.7 Macrophages.

Yu HY, Kim KS, Lee YC, Moon HI, Lee JH - Evid Based Complement Alternat Med (2012)

Effects of oleifoliosides A on LPS-stimulated NF-κB activation and phosphorylation of MAPK signaling pathway. (a) Cells were pretreated with the indicated the concentrations of oleifoliosides A for 1 h and then stimulated with LPS (500 ng/mL) for 1 h. Nuclear extracts were prepared and analyzed for NF-κB DNA-binding activity by EMSA. The C.P. represents 100-fold cold probe only. (b) Cells were transiently cotransfected with pNF-κB-Luc reporter plasmid and then were pretreated with the indicated the concentrations of oleifoliosides A for 1 h. LPS (500 ng/mL) was then added and cells were further incubated 24 h. The cells were harvested and then the luciferase activities were determined by using the dual luciferase report assay system. (c) Cells were pretreated with the indicated concentrations of oleifoliosides A for 1 h before LPS (500 ng/mL) treatment for 15 min. and the levels of total/phospho-ERK1/2, JNK, and p38 MAPK were determined by Western blotting. The values are presented as mean±S.E. of three independent experiments. n = 6 per experiment, *P < 0.05 versus LPS and **P < 0.01 versus LPS.
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Related In: Results  -  Collection

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fig4: Effects of oleifoliosides A on LPS-stimulated NF-κB activation and phosphorylation of MAPK signaling pathway. (a) Cells were pretreated with the indicated the concentrations of oleifoliosides A for 1 h and then stimulated with LPS (500 ng/mL) for 1 h. Nuclear extracts were prepared and analyzed for NF-κB DNA-binding activity by EMSA. The C.P. represents 100-fold cold probe only. (b) Cells were transiently cotransfected with pNF-κB-Luc reporter plasmid and then were pretreated with the indicated the concentrations of oleifoliosides A for 1 h. LPS (500 ng/mL) was then added and cells were further incubated 24 h. The cells were harvested and then the luciferase activities were determined by using the dual luciferase report assay system. (c) Cells were pretreated with the indicated concentrations of oleifoliosides A for 1 h before LPS (500 ng/mL) treatment for 15 min. and the levels of total/phospho-ERK1/2, JNK, and p38 MAPK were determined by Western blotting. The values are presented as mean±S.E. of three independent experiments. n = 6 per experiment, *P < 0.05 versus LPS and **P < 0.01 versus LPS.
Mentions: NF-κB is a key transcription factor involved in general inflammation as well as immune responses. Its activation is critically required for the expression of proteins, including iNOS and COX-2, in macrophages. To evaluate the molecular mechanism through which oleifolioside A inhibits the expression of such proinflammatory mediators, we examined NF-κB DNA-binding activity by EMSA. As shown in Figure 4(a), treatment with LPS alone caused a marked increase in the DNA-binding activity of NF-κB. In contrast, pretreatment with oleifolioside A significantly attenuated LPS-stimulated NF-κB DNA-binding activity in a dose-dependent manner. To confirm the results of EMSA, we also investigated the effects of oleifolioside A on NF-κB-dependent reporter gene expression following LPS treatment by luciferase reporter assay. RAW 264.7 macrophages were transiently cotransfected with a pNF-κB-leu reporter vector generated by inserting four spaced NF-κB-binding sites into the pLuc-promoter vector and then stimulated with 500 ng/mL LPS with or without oleifolioside A. As shown in Figure 4(b), treatment with oleifolioside A significantly reduced the level of NF-κB luciferase activity stimulated by LPS in a dose-dependent manner. Taken together, these findings demonstrate that oleifolioside A suppresses the expression of iNOS and COX-2, at least in part via an NF-κB-dependent mechanism.

Bottom Line: Oleifolioside A, a new triterpenoid compound isolated from Dendropanax morbifera Leveille (D. morbifera), was shown in this study to have potent inhibitory effects on lipopolysaccharide (LPS-)stimulated nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production in RAW 264.7 macrophages.These results were found to be associated with the inhibition of the degradation and phosphorylation of IκB-α and subsequent translocation of the NF-κB p65 subunit to the nucleus.Taken together, our results suggest that oleifolioside A has the potential to be a novel anti-inflammatory agent capable of targeting both the NF-κB and MAPK signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: College of Natural Resources and Life Science, BK21 Center for Silver-Bio Industrialization, Dong-A University, Busan 604-714, Republic of Korea.

ABSTRACT
Oleifolioside A, a new triterpenoid compound isolated from Dendropanax morbifera Leveille (D. morbifera), was shown in this study to have potent inhibitory effects on lipopolysaccharide (LPS-)stimulated nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production in RAW 264.7 macrophages. Consistent with these findings, oleifolioside A was further shown to suppress the expression of LPS-stimulated inducible nitric oxide synthase (iNOS) and cyclooxigenase-2 (COX-2) in a dose-dependent manner at both the protein and mRNA levels and to significantly inhibit the DNA-binding activity and transcriptional activity of NF-κB in response to LPS. These results were found to be associated with the inhibition of the degradation and phosphorylation of IκB-α and subsequent translocation of the NF-κB p65 subunit to the nucleus. Inhibition of NF-κB activation by oleifolioside A was also shown to be mediated through the prevention of p38 MAPK and ERK1/2 phosphorylation. Taken together, our results suggest that oleifolioside A has the potential to be a novel anti-inflammatory agent capable of targeting both the NF-κB and MAPK signaling pathways.

No MeSH data available.