Effects of shokyo (Zingiberis Rhizoma) and kankyo (Zingiberis Processum Rhizoma) on prostaglandin E2 production in lipopolysaccharide-treated mouse macrophage RAW264.7 cells

Abstract

We previously reported that shokyo and kankyo, which are water-extracted fractions of ginger, reduced LPS-induced PGE₂ production in human gingival fibroblasts. In this study, we examined the effects of these herbs on LPS-treated mouse macrophage RAW264.7 cells. Both shokyo and kankyo reduced LPS-induced PGE₂ production in a concentration-dependent manner. Shokyo and kankyo did not inhibit cyclooxygenase (COX) activity, nor did they alter the expression of molecules in the arachidonic acid cascade. In addition, these herbs did not alter NF-κB p65 translocation into nucleus, or phosphorylation of p65 or ERK. These results suggest that shokyo and kankyo inhibit cPLA₂ activity. Although 6-shogaol produced similar results to those of shokyo and kankyo, the concentration of 6-shogaol required for the reduction of PGE₂ production were higher than those of 6-shogaol in shokyo and kankyo. Therefore, several gingerols and shogaols other than 6-shogaol may play a role in the reduction of LPS-induced PGE₂ production. Thus, 6-shogaol, and other gingerols and shogaols inhibit cPLA₂ activity and reduce LPS-induced PGE₂ production via a different mechanism from traditional anti-inflammatory drugs. Moreover, kampo medicines that contain shokyo or kankyo are considered to be effective for inflammatory diseases.

Keywords: 6-shogaol; Antiinflammatory effect; Arachidonic acid cascade; Herb; Kampo medicine; Kankyo; Macrophage; Prostaglandin E2; Shokyo.

Figure 1. Cytotoxicity of shokyo (A) and kankyo (B).

RAW264.7 cells were treated with each herb (0, 100, 300, or 1,000 µg/ml) for 24 h. Then, the numbers of viable cells were measured by WST-8. P-values vs. without each herb were calculated by Dunnett’s method. *P < 0.05, ***P < 0.001.

Figure 2. Effects of shokyo and kankyo on PGE₂ production.

(A) Time schedule of treatment with LPS and/or each herb. Simultaneous treatment: Cells were treated with combinations of LPS (0 or 100 ng/ml) and medium or each herb (100 µg/ml) for 24 h. Sequential treatment: Cells were treated with medium or LPS (100 ng/ml) for 30 min, washed, and further treated with medium or each herb (100 µg/ml) for 24 h. (B, C) Concentrations of PGE₂ were measured by ELISA, adjusted by cell number, and expressed as 100% at LPS alone (mean ± SD, n = 3) in simultaneous (B) and sequential (C) treatment experiments. med, medium; s, shokyo; k, kankyo. (D, E) Concentration-dependent effects of shokyo (D) and kankyo (E) on LPS-induced PGE₂ production. Cells were treated with combinations of LPS (100 ng/ml) and each herb (0, 1, 10, or 100 µg/ml) for 24 h. P-values vs. with LPS alone were calculated by Dunnett’s method. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3. Effects of shokyo and kankyo on the arachidonic acid cascade.

(A) Time schedule of treatment with LPS and/or each herb. Simultaneous treatment: RAW264.7 cells were treated with LPS (100 ng/ml) and medium or each herb (100 µg/ml) for 6 h, washed, and then treated with 10 µM arachidonic acid for 30 min. Sequential treatment: RAW264.7 cells were treated with LPS (100 ng/ml) for 6 h, and further treated with medium or each herb (100 µg/ml) for 1 h. Then, the cells were washed and treated with 10 µM arachidonic acid for 30 min. (B, C) Effects of shokyo and kankyo on COX activity. Concentrations of PGE₂ were measured by ELISA, adjusted by cell number, and expressed as per 10,000 cells (mean ± SD, n = 4) in simultaneous (B) and sequential (C) treatment experiments. P-values vs. with LPS alone by Dunnett’s test are indicated. (D) Effects of herbs on cPLA₂, annexin 1, and COX-2 expression. RAW264.7 cells were treated with a combination of LPS (0 or 100 ng/ml) and medium, each herb (100 µg/ml), or dexamethasone (100 nM) for 8 h, and protein levels were examined by Western blotting. med, medium; s, shokyo; k, kankyo; and Dex, dexamethasone. The band densities were normalized against LPS alone and actin. The values were indicated below each band.

Figure 4. Effects of shokyo and kankyo on the intracellular signal transduction.

(A) Effects of herbs on NF-κB p65 translocation into nucleus. RAW264.7 cells were treated with each herb (100 µg/ml), or BAY 11-7082 (10 µM) for 2 h, and further treated with LPS (100 ng/ml), and 6-shogaol or BAY 11-7082 for 30 min. The cellular localization of p65 was determined by immunofluorescence analysis. Nuclei of the corresponding cells were visualized with DAPI, and observed at 400× magnification. The bar represents 20 µm. (B, C) Effects of herbs on NF-κB p65 and ERK phosphorylation. RAW264.7 cells were treated with each herb (100 µg/ml), BAY 11-7082 (10 µM), or PD98059 (20 µM) for 1 h, and further treated with LPS (100 ng/ml) and each herb, BAY 11-7082, or PD98059 for 15 min. p65 and phosphorylated p65 levels (B), and ERK and phosphorylated ERK (pERK) levels (C) were examined by Western blotting. The upper band indicates ERK1 (p44 MAPK) and the lower band indicates ERK2 (p42 MAPK) in (C). med, medium; s, shokyo; k, kankyo; BAY, BAY 11-7082, and PD, PD98059. The band densities were normalized against LPS alone, and p65 or ERK. The values were indicated below each band.

Figure 5. Effects of 6-shogaol on LPS-induced PGE₂ production, and the arachidonic acid cascade.

(A) Effects of 6-shogaol on LPS-induced PGE₂. RAW264.7 cells were treated with LPS (100 ng/ml) and 6-shogaol (0 to 1,000 nM) for 24 h. Concentrations of PGE₂ were measured by ELISA, adjusted by cell number, and expressed as per 10,000 cells (mean ± SD, n = 3). P-values vs. with LPS alone were calculated by Dunnett’s method. *P < 0.05, **P < 0.01, ***P < 0.001. Red and blue lines represent the effects of shokyo and kankyo in Figs. 2D and 2E, respectively. The concentrations of 6-shogaol in shokyo and kankyo were calculated using the results shown in Table 1. (B) Effects of 6-shogaol on cPLA₂, annexin 1, and COX-2 expression. RAW264.7 cells were treated with a combination of LPS (0 or 100 ng/ml) and 6-shogaol (0, 0.1, 1, or 10 µM), or dexamethasone (100 nM) for 8 h, and protein levels were examined by Western blotting. The band densities were normalized against LPS alone and actin. The values were indicated below each band.

Figure 6. Effects of 6-shogaol on the intracellular signal transduction.

(A) Effects of 6-shogaol on NF-κB p65 translocation into nucleus. RAW264.7 cells were treated with 6-shogaol (0, 0.1, 1, or 10 µM), or BAY 11-7082 (10 µM) for 2 h, and further treated with LPS (100 ng/ml), and 6-shogaol or BAY 11-7082 for 30 min. The cellular localization of p65 was determined by immunofluorescence analysis. Nuclei of the corresponding cells were visualized with DAPI, and observed at 400× magnification. The bar represents 20 µm. (B, C) Effects of 6-shogaol on NF-κB p65 and ERK phosphorylation. RAW264.7 cells were treated with 6-shogaol (0, 0.1, 1, or 10 µM), BAY 11-7082 (10 µM), or PD98059 (20 µM) for 1 h and further treated with LPS (100 ng/ml), and 6-shogaol, BAY 11-7082, or PD98059 for 15 min. p65 and phosphorylated p65 levels (B), and ERK and phosphorylated ERK (pERK) levels (C) were examined by Western blotting. BAY: BAY 11-7082, and PD: PD98059. The band densities were normalized against LPS alone, and p65 or ERK. The values were indicated below each band.