Immune Netw.  2012 Oct;12(5):181-188. 10.4110/in.2012.12.5.181.

Immunomodulatory Effects of Dioscoreae Rhizome Against Inflammation through Suppressed Production of Cytokines Via Inhibition of the NF-kappaB Pathway

• Kim S
• Shin S
• Hyun B
• Kong H
• Han S
• Lee A
• Lee S
• Kim K

• KMID: 2150747
• DOI: http://doi.org/10.4110/in.2012.12.5.181

Abstract

Dioscoreae Rhizome (DR) has been used in traditional medicine to treat numerous diseases and is reported to have anti-diabetes and anti-tumor activities. To identify a bioactive traditional medicine with anti-inflammatory activity of a water extract of DR (EDR), we determined the mRNA and protein levels of proinflammatory cytokines in macrophages through RT-PCR and western blot analysis and performed a FACS analysis for measuring surface molecules. EDR dose-dependently decreased the production of NO and pro-inflammatory cytokines such as IL-1beta, IL-6, TNF-alpha, and PGE2, as well as mRNA levels of iNOS, COX-2, and pro-inflammatory cytokines, as determined by western blot and RT-PCR analysis, respectively. The expression of co-stimulatory molecules such as B7-1 and B7-2 was also reduced by EDR. Furthermore, activation of the nuclear transcription factor, NF-kappaB, but not that of IL-4 and IL-10, in macrophages was inhibited by EDR. These results show that EDR decreased pro-inflammatory cytokines via inhibition of NF-kappaB-dependent inflammatory protein level, suggesting that EDR could be a useful immunomodulatory agent for treating immunological diseases.

Keyword

Dioscoreae Rhizoma; Anti-inflammatory; Inflammatory cytokines; Immunomodulatory agent

MeSH Terms

Blotting, Western
Cytokines
Dinoprostone
Dioscorea
Immune System Diseases
Inflammation
Interleukin-10
Interleukin-4
Interleukin-6
Macrophages
Medicine, Traditional
NF-kappa B
Rhizome
RNA, Messenger
Transcription Factors
Tumor Necrosis Factor-alpha
Water
Cytokines
Dinoprostone
Interleukin-10
Interleukin-4
Interleukin-6
NF-kappa B
RNA, Messenger
Transcription Factors
Tumor Necrosis Factor-alpha
Water

Figure

• 

  Figure 1 EDR inhibits the production of NO (A), expression of iNOS mRNA (B), and protein (C) in LPS-stimulated RAW 264.7 cells. RAW 264.7 cells were treated with various concentrations (25, 50, 100, 200 µg/ml) of EDR in the absence or presence of LPS (100 ng/ml) overnight. Culture supernatants were then collected and NO concentrations were measured using Griess reagent (A). Cell lysates were extracted, and protein levels of iNOS were then analyzed by Western blotting (B). RAW 264.7 cells were incubated with EDR in the absence or presence of LPS for 24 h. Total RNA was isolated, and levels of iNOS mRNA were then measured by RT-PCR (C). Each value represents the mean±S.D. of three independent experiments. ††p<0.01 vs. cells only based on Student's t-test. **p<0.01 vs. LPS only based on Student's t-test.

• 

  Figure 2 EDR inhibits the production of PGE2 (A), COX-2 mRNA (B) and protein (C) in LPS-stimulated RAW 264.7 cells. RAW 264.7 cells were treated with various concentrations (25, 50, 100, 200 µg/ml) of EDR in the absence or presence of LPS (100 ng/ml) for 48 h. Culture supernatants were then collected and PGE2 concentrations were measured using ELISA kits (A). Cell lysates were extracted, and protein levels of COX-2 were then analyzed by Western blotting (B). RAW 264.7 cells were incubated with EDR in the absence or presence of LPS for 24 h. Total RNA was isolated, and levels of COX-2 mRNA were then measured by RT-PCR (C). Each value represents the mean±S.D. of three independent experiments. †p<0.01 vs. cells only based on Student's t-test.

• 

  Figure 3 EDR inhibits pro-inflammatory cytokine production in LPS-stimulated RAW 264.7 cells. RAW 264.7 cells were treated with various concentrations (25, 50, 100, 200 µg/ml) of EDR in the absence or presence of LPS (100 ng/ml) for 48 h. Culture supernatants were then collected and cytokine concentrations were measured using ELISA kits (A~C). RAW 264.7 cells were incubated with EDR in the absence or presence of LPS for 24 h. Cell lysates were extracted, and protein levels of each cytokine were then analyzed by Western blotting (D). Total RNA was isolated, and mRNA levels of each cytokine were then measured by RT-PCR (E). Each value represents the mean±S.D. of three independent experiments. ††p<0.01 vs. cells only based on Student's t-test. *p<0.05, **p<0.01 vs. LPS only based on Student's t-test.

• 

  Figure 4 Effects of EDR on the anti-inflammatory cytokine production in LPS-stimulated RAW 264.7 cells. RAW 264.7 cells were treated with various concentrations (25, 50, 100, 200 µg/ml) of EDR in the absence or presence of LPS (100 ng/ml) for 24 h. Cell lysates were extracted, and protein levels of each cytokine were then analyzed by Western blotting.

• 

  Figure 5 EDR inhibits the expression of co-stimulatory molecules in LPS-stimulated RAW 264.7 cells. RAW 264.7 cells were cultured and activated with LPS (100 ng/ml) in the absence or presence of various EDR concentrations for 24 hrs. The surface B7-1 (A) and B7-2 (B) were labeled with anti-B7-1/-2 antibodies and the cells were then stained using anti-Vβ8.1+8.2-FITC, anti-Vβ2-PE, or anti-Vβ2-FITC, which served as an isotype control for nonspecific binding.

• 

  Figure 6 EDR inhibits the IκBα phosphorylation in the cytoplasm and the nuclear translocation of NF-κB p65 in LPS-stimulated RAW 264.7 cells. RAW 264.7 cells were treated with various concentrations (25, 50, 100, 200 µg/ml) of EDR in the absence or presence of LPS (100 ng/ml) for 24 hrs. Cell lysates were extracted, and protein levels of each cytokine were then analyzed by Western blotting.

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