Inhibitory Effect of Gallic acid on Production of Interleukins in Mouse Macrophage Stimulated by Lipopolysaccharide

Gallic acid가 Lipopolysaccharide로 활성화된 마우스 대식세포의 인터루킨 생성에 미치는 영향

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  • ABSTRACT

    Objectives

    Gallic acid (GA) is the major component of tannin which could be easily founded in various natural materials such as green tea, red tea, grape juice, and Corni Fructus. The purpose of this study is to investigate the effect of Gallic acid (GA) on production of interleukin (IL) in mouse macrophage Raw 264.7 cells stimulated by l ipopolysaccharide (LPS).

    Methods

    Productions of interleukins were measured by High-throughput Multiplex Bead based Assay with Bio-plex Suspension Array System based on xMAP® (multi-analyte profiling beads) technology. Firstly, cell culture supernatant was obtained after treatment with LPS and GA for 24 hour. Then, it was incubated with the antibody-conjugated beads for 30 minutes. And detection antibody was added and incubated for 30 minutes. And Strepavidin-conjugated Phycoerythrin (SAPE) was added. After incubation for 30 minutes, the level of SAPE fluorescence was analyzed on Bio-plex Suspension Array System and concentration of interleukin was determined.

    Results

    The results of the experiment are as follows.

    1. GA significantly inhibited the production of IL-3, IL-10, IL-12p40, and IL-17 in LPS-induced mouse macrophage RAW 264.7 cells at the concentration of 25, 50, 100, 200 uM (p<0.05).

    2. GA significantly inhibited the production of IL-6 in LPS-induced mouse macrophage RAW 264.7 cells at the concentration of 50, 100, 200 uM (p<0.05).

    3. GA diminished the production of some cytokine such as IL-4, IL-5, and IL-13 in LPS-induced mouse macrophage RAW 264.7 cells.

    4. GA did not show the inhibitory effect on the production of IL-1αand IL-9 in LPS-induced mouse macrophage RAW 264.7 cells.

    Conclusions

    These results suggest that GA has anti-inflammatory activity related with its inhibitory effects on the production of interleukins such as IL-3, IL-10, IL-12p40, IL-17, and IL-6 in LPS-induced macrophages.


  • KEYWORD

    Gallic acid , Macrophage , Interleukin , Inflammation , lipopolysaccharide.

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  • [Fig. 1] Effect of GA on producton of IL-3 in RAW 264.7 cells using Bioplex cytokine assay. Cells were incubated with LPS (1 ug/mL) and GA (0, 25, 50, 100, 200 uM) for 24 h. Results are represented as mean ± SEM. Nor : Normal group treated with culture media only. Con : Control group treated with LPS only. * represents p < 0.05 compared to the control.
    Effect of GA on producton of IL-3 in RAW 264.7 cells using Bioplex cytokine assay. Cells were incubated with LPS (1 ug/mL) and GA (0, 25, 50, 100, 200 uM) for 24 h. Results are represented as mean ± SEM. Nor : Normal group treated with culture media only. Con : Control group treated with LPS only. * represents p < 0.05 compared to the control.
  • [Fig. 2] Effect of GA on producton of IL-10 in RAW 264.7 cells using Bioplex cytokine assay. Cells were incubated with LPS (1 ug/mL) and GA (0, 25, 50, 100, 200 uM) for 24 h. Results are represented as mean ± SEM. Nor : Normal group treated with culture media only. Con : Control group treated with LPS only. * represents p < 0.05 compared to the control.
    Effect of GA on producton of IL-10 in RAW 264.7 cells using Bioplex cytokine assay. Cells were incubated with LPS (1 ug/mL) and GA (0, 25, 50, 100, 200 uM) for 24 h. Results are represented as mean ± SEM. Nor : Normal group treated with culture media only. Con : Control group treated with LPS only. * represents p < 0.05 compared to the control.
  • [Fig. 3] Effect of GA on producton of IL-12p40 in RAW 264.7 cells using Bioplex cytokine assay. Cells were incubated with LPS (1 ug/mL) and GA (0, 25, 50, 100, 200 uM) for 24 h. Results are represented as mean ± SEM. Nor : Normal group treated with culture media only. Con : Control group treated with LPS only. * represents p < 0.05 compared to the control.
    Effect of GA on producton of IL-12p40 in RAW 264.7 cells using Bioplex cytokine assay. Cells were incubated with LPS (1 ug/mL) and GA (0, 25, 50, 100, 200 uM) for 24 h. Results are represented as mean ± SEM. Nor : Normal group treated with culture media only. Con : Control group treated with LPS only. * represents p < 0.05 compared to the control.
  • [Fig. 4] Effect of GA on producton of IL-17 in RAW 264.7 cells using Bioplex cytokine assay. Cells were incubated with LPS (1 ug/mL) and GA (0, 25, 50, 100, 200 uM) for 24 h. Results are represented as mean ± SEM. Nor : Normal group treated with culture media only. Con : Control group treated with LPS only. * represents p < 0.05 compared to the control.
    Effect of GA on producton of IL-17 in RAW 264.7 cells using Bioplex cytokine assay. Cells were incubated with LPS (1 ug/mL) and GA (0, 25, 50, 100, 200 uM) for 24 h. Results are represented as mean ± SEM. Nor : Normal group treated with culture media only. Con : Control group treated with LPS only. * represents p < 0.05 compared to the control.
  • [Fig. 5] Effect of GA on producton of IL-6 in RAW 264.7 cells using Bioplex cytokine assay. Cells were incubated with LPS (1 ug/mL) and GA (0, 25, 50, 100, 200 uM) for 24 h. Results are represented as mean ± SEM. Nor : Normal group treated with culture media only. Con : Control group treated with LPS only. * represents p < 0.05 compared to the control.
    Effect of GA on producton of IL-6 in RAW 264.7 cells using Bioplex cytokine assay. Cells were incubated with LPS (1 ug/mL) and GA (0, 25, 50, 100, 200 uM) for 24 h. Results are represented as mean ± SEM. Nor : Normal group treated with culture media only. Con : Control group treated with LPS only. * represents p < 0.05 compared to the control.
  • [Fig. 6] Effect of GA on producton of IL-4 in RAW 264.7 cells using Bioplex cytokine assay. Cells were incubated with LPS (1 ug/mL) and GA (0, 25, 50, 100, 200 uM) for 24 h. Results are represented as mean ± SEM. Nor : Normal group treated with culture media only. Con : Control group treated with LPS only. * represents p < 0.05 compared to the control.
    Effect of GA on producton of IL-4 in RAW 264.7 cells using Bioplex cytokine assay. Cells were incubated with LPS (1 ug/mL) and GA (0, 25, 50, 100, 200 uM) for 24 h. Results are represented as mean ± SEM. Nor : Normal group treated with culture media only. Con : Control group treated with LPS only. * represents p < 0.05 compared to the control.
  • [Fig. 7] Effect of GA on producton of IL-5 in RAW 264.7 cells using Bioplex cytokine assay. Cells were incubated with LPS (1 ug/mL) and GA (0, 25, 50, 100, 200 uM) for 24 h. Results are represented as mean ± SEM. Nor : Normal group treated with culture media only. Con : Control group treated with LPS only. * represents p < 0.05 compared to the control.
    Effect of GA on producton of IL-5 in RAW 264.7 cells using Bioplex cytokine assay. Cells were incubated with LPS (1 ug/mL) and GA (0, 25, 50, 100, 200 uM) for 24 h. Results are represented as mean ± SEM. Nor : Normal group treated with culture media only. Con : Control group treated with LPS only. * represents p < 0.05 compared to the control.
  • [Fig. 8] Effect of GA on producton of IL-13 in RAW 264.7 cells using Bioplex cytokine assay. Cells were incubated with LPS (1 ug/mL) and GA (0, 25, 50, 100, 200 uM) for 24 h. Results are represented as mean ± SEM. Nor : Normal group treated with culture media only. Con : Control group treated with LPS only. * represents p < 0.05 compared to the control.
    Effect of GA on producton of IL-13 in RAW 264.7 cells using Bioplex cytokine assay. Cells were incubated with LPS (1 ug/mL) and GA (0, 25, 50, 100, 200 uM) for 24 h. Results are represented as mean ± SEM. Nor : Normal group treated with culture media only. Con : Control group treated with LPS only. * represents p < 0.05 compared to the control.
  • [Fig. 9.] Effect of GA on producton of IL-1αin RAW 264.7cells using Bioplex cytokine assay. Cells wereincubated with LPS (1 ug/mL) and GA (0, 25, 50,100, 200 uM) for 24 h. Results are represented asmean ± SEM. Nor : Normal group treated withculture media only. Con : Control group treatedwith LPS only. * represents p < 0.05 compared tothe control.
    Effect of GA on producton of IL-1αin RAW 264.7cells using Bioplex cytokine assay. Cells wereincubated with LPS (1 ug/mL) and GA (0, 25, 50,100, 200 uM) for 24 h. Results are represented asmean ± SEM. Nor : Normal group treated withculture media only. Con : Control group treatedwith LPS only. * represents p < 0.05 compared tothe control.
  • [Fig. 10.] Effect of GA on producton of IL-9 in RAW 264.7cells using Bioplex cytokine assay. Cells wereincubated with LPS (1 ug/mL) and GA (0, 25, 50,100, 200 uM) for 24 h. Results are represented asmean ± SEM. Nor : Normal group treated withculture media only. Con : Control group treatedwith LPS only. * represents p < 0.05 compared tothe control.
    Effect of GA on producton of IL-9 in RAW 264.7cells using Bioplex cytokine assay. Cells wereincubated with LPS (1 ug/mL) and GA (0, 25, 50,100, 200 uM) for 24 h. Results are represented asmean ± SEM. Nor : Normal group treated withculture media only. Con : Control group treatedwith LPS only. * represents p < 0.05 compared tothe control.