Immune Netw.  2010 Oct;10(5):145-152. 10.4110/in.2010.10.5.145.

Flowers of Inula japonica Attenuate Inflammatory Responses

• Choi JH
• Park YN
• Li Y
• Jin MH
• Lee J
• Lee Y
• Son JK
• Chang HW
• Lee E

• KMID: 1456218
• DOI: http://doi.org/10.4110/in.2010.10.5.145

Abstract

BACKGROUND
The flowers of Inula japonica (Inulae Flos) have long been used in traditional medicine for the treatment of inflammatory diseases. In the present study, we investigated the anti-inflammatory properties of Inulae Flos Extract (IFE).
METHODS
The anti-inflammatory effects of IFE against nitric oxide (NO), PGE2, TNF-alpha, and IL-6 release, as well as NF-kappa B and MAP kinase activation were evaluated in RAW 264.7 cells.
RESULTS
IFE inhibited the production of NO and the expression of inducible nitric oxide synthase (iNOS) in LPS-stimulated RAW264.7 cells. In addition, IFE reduced the release of pro-inflammatory cytokines, such as TNF-alpha and IL-6. Furthermore, IFE inhibited the NF-kappa B activation induced by LPS, which was associated with the abrogation of I kappa B-alpha degradation and subsequent decreases in nuclear p65 and p50 levels. Moreover, the phosphorylation of ERK, JNK, and p38 MAP kinases in LPS-stimulated RAW 264.7 cells was suppressed by IFE in a dose-dependent manner.
CONCLUSION
These results suggest that the anti-inflammation activities of IFE might be attributed to the inhibition of NO, iNOS and cytokine expression through the down-regulation of NF-kappa B activation via suppression of I kappa B alpha and MAP kinase phosphorylation in macrophages.

Keyword

Inula japonica; Inulae Flos Extract (IFE); Nitric oxide (NO); iNOS; Cytokine; NF-kappa B; MAP kinase

MeSH Terms

Cytokines
Dinoprostone
Down-Regulation
Flowers
I-kappa B Proteins
Interleukin-6
Inula
Macrophages
Medicine, Traditional
NF-kappa B
Nitric Oxide
Nitric Oxide Synthase Type II
Phosphorylation
Phosphotransferases
Tumor Necrosis Factor-alpha
Cytokines
Dinoprostone
I-kappa B Proteins
Interleukin-6
NF-kappa B
Nitric Oxide
Nitric Oxide Synthase Type II
Phosphotransferases
Tumor Necrosis Factor-alpha

Figure

• 

  Figure 1 Effect of IFE on LPS-induced NO production and iNOS expression in RAW 264.7 cells. Cells were pretreated with different concentrations of IFE for 1 h and then stimulated with LPS (200 ng/ml) for 24 h. The levels of nitrite were measured in the culture media by Griess reagents (A). Cells were harvested and the cell lysates were prepared as described in the experimental procedure. The protein levels of iNOS were measured by Western blot analysis using antibody against iNOS. Quantification of band intensities from three independent results was determined by densitometric analysis. The values were expressed as a percentage of maximal band intensity in the LPS-treated cells, which was set to 100% (B). Values are expressed as means±S.D. of three different samples. *p<0.01, †p<0.001, compared with the control value.

• 

  Figure 2 Effect of IFE on PGE2 generation and COX-2 expression in LPS-treated RAW 264.7 cells. Cells were pretreated with different concentrations of IFE for 1 h and then stimulated with LPS (200 ng/ml) for 24 h. After incubation, the PGE2 levels in the cultured media were measured using an ELISA kit (A). Cells were harvested and equal amounts of cell extracts were subjected to Western blot analysis using antibody against COX-2. Quantification of band intensities from three independent results was determined by densitometric analysis. The values were expressed as a percentage of maximal band intensity in the LPS-treated cells, which was set to 100% (B). Values are expressed as the means±S.D. of three different samples. *p<0.01, †p<0.001, when compared with the control value.

• 

  Figure 3 Effects of IFE on cytokine release in LPS-treated RAW 264.7 cells. Cells were pretreated with different concentrations of IFE for 1 h and then treated with LPS (200 ng/ml) for 24 h. The cytokine levels in the culture media were measured using an ELISA kit as described in the experimental procedures. Values are expressed as the means±S.D. of three independent experiments. *p<0.05, †p<0.001, when compared with the control value.

• 

  Figure 4 Effect of IFE on NF-κB activation in RAW 264.7 cells. Cells were pretreated with different concentrations of IFE for 1 h and then stimulated with LPS (200 ng/ml) for 30 min. Total cellular proteins and nuclear extracts were prepared for Western blot analysis of NF-κB p65 and IκBα proteins. Quantification of band intensities from three independent results was determined by densitometric analysis. The values were expressed as a percentage of maximal band intensity in the LPS-treated cells, which was set to 100% (A, B). Cells were transiently transfected with the pNF-κB-Luc plasmid using a lipofectamine method. Cells were pretreated with different concentrations of IFE for 1 h and then further stimulated with LPS (200 ng/ml) for 6 h. The cells were then harvested and the luciferase activities were determined using a Promega luciferase assay system and a luminometer (C). Data represent the mean±S.D. of three different samples. *p<0.001, when compared with the LPS-treated group.

• 

  Figure 5 Effect of IFE on LPS-stimulated MAPK activation in RAW 264.7 cells. Cells were pretreated with different concentrations of IFE for 1 h and then were incubated with LPS (200 ng/ml) for 30 min. Whole cell lysates were analyzed for ERK (A), JNK (B) and p38 (C) phosphorylation by Western blot analysis. Quantification of band intensities from three independent results was determined by densitometric analysis. The values were expressed as a percentage of maximal band intensity in the LPS-treated cells, which was set to 100%. Data represent the mean±S.D. of three different samples. *p<0.05, †p<0.01, ‡p<0.001, when compared with the LPS-treated group.

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