미세아교세포의 염증반응에 미치는 청뇌명신환의 영향

Anti-inflammatory Effects of Cheongnoimyungshin-hwan in Microglia Cells

  • ABSTRACT

    Objectives

    Activated microglia cells play an important role in inflammatory responses in the central nervous system (CNS) which are involved in neurodegenerative diseases. We attempted to determine the anti-inflammatory effects of Cheongnoimyungshin-hwan (CNMSH) in microglia cells.

    Methods

    We examined the effect of CNMSH on the inflammatory responses in BV2 microglia cells induced by lipopolysaccharide (LPS) and explored the mechanism underlying the action of CNMSH.

    Results

    BV2 cells treated with LPS showed an up-regulation of nitric oxide (NO), prostaglandin PGE2 (PGE2) and interleukin 1β (IL-1β) release, whereas CNMSH suppressed this up-regulation. CNMSH inhibited the induction of COX-2, iNOS and IL-1β proteins in LPS-treated BV2 cells and blocked the LPS-induced phosphorylation and nuclear translocation of nuclear factor κB (NF-κB). Furthermore, CNMSH attenuated the LPS-induced phosphorylation of extracellular signal-regulated kinase and p38 mitogen activated protein kinase (MAPK), as well as the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway, but did not inhibit the LPS-induced phosphorylation of c-Jun amino terminal kinase.

    Conclusions

    These results suggest that the inhibitory effect of CNMSH on the LPS-induced production of inflammatory mediators and cytokines in BV2 cells is associated with the suppression of the NF-κB and PI3KAkt signaling pathways.


  • KEYWORD

    Cheongnoimyungshin-hwan (CNMSH) , Microglia cells , Neurodegenerative diseases , NF-κB , PI3KAkt

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  • [Table 1.] Antibodies Used in the Present Study
    Antibodies Used in the Present Study
  • [Fig. 1.] The CNMSH and/or in combination with lipopolysaccharide (LPS) on BV2 microglia viability. The cells were incubated for 24 h at the indicated concentrations of CNMSH with or without LPS (500 ng/ml). In combination treatment, CNMSH were pretreated 1 h before LPS treatment. Cell viability was determined by the MTT assay. Data are expressed as mean±SE of three independent experiments.
    The CNMSH and/or in combination with lipopolysaccharide (LPS) on BV2 microglia viability. The cells were incubated for 24 h at the indicated concentrations of CNMSH with or without LPS (500 ng/ml). In combination treatment, CNMSH were pretreated 1 h before LPS treatment. Cell viability was determined by the MTT assay. Data are expressed as mean±SE of three independent experiments.
  • [Fig. 2.] The effects of CNMSH on NO production and iNOS expression in LPS-stimulated BV2 microglia. Cells were pre-treated with 125, 250 and 500 μg/mL of CNMSH 1 h before LPS treatment for 24 h. (A) Following 24 h treatment, and the amounts of NO production were measured with the supernatants. Data are expressed as mean±SD of three independent experiments (*p<0.05 as compared with control group, #p<0.05 as compared with LPS treated group). (B) Following 24 h treatment, the expression of iNOS were assessed by Western blot analysis. Actin was used as the internal control.
    The effects of CNMSH on NO production and iNOS expression in LPS-stimulated BV2 microglia. Cells were pre-treated with 125, 250 and 500 μg/mL of CNMSH 1 h before LPS treatment for 24 h. (A) Following 24 h treatment, and the amounts of NO production were measured with the supernatants. Data are expressed as mean±SD of three independent experiments (*p<0.05 as compared with control group, #p<0.05 as compared with LPS treated group). (B) Following 24 h treatment, the expression of iNOS were assessed by Western blot analysis. Actin was used as the internal control.
  • [Fig. 3.] The effects of CNMSH on PGE2 production and COX-2 expression in LPS-stimulated BV2 microglia. Cells were pre-treated with 125, 250 and 500 μg/mL of CNMSH 1 h before LPS treatment for 24 h. (A) Following 24 h treatment, and the amounts of PGE2 production were measured with the supernatants. Data are expressed as mean±SD of three independent experiments (*p<0.05 as compared with control group, #p<0.05 as compared with LPS treated group). (B) Following 24 h treatment, the expression of COX-2 were assessed by Western blot analysis. Actin was used as the internal control.
    The effects of CNMSH on PGE2 production and COX-2 expression in LPS-stimulated BV2 microglia. Cells were pre-treated with 125, 250 and 500 μg/mL of CNMSH 1 h before LPS treatment for 24 h. (A) Following 24 h treatment, and the amounts of PGE2 production were measured with the supernatants. Data are expressed as mean±SD of three independent experiments (*p<0.05 as compared with control group, #p<0.05 as compared with LPS treated group). (B) Following 24 h treatment, the expression of COX-2 were assessed by Western blot analysis. Actin was used as the internal control.
  • [Fig. 4.] The effects of CNMSH on IL-1β production and protein expression in LPS-stimulated BV2 microglia. Cells were pre-treated with 125, 250 and 500 μg/mL of CNMSH 1 h before LPS treatment for 24 h. (A) Following 24 h treatment, and the amounts of IL-1β production were measured with the supernatants. Data are expressed as mean±SD of three independent experiments. (*p<0.05 as compared with control group, #p<0.05 as compared with LPS treated group). (B) Following 24 h treatment, the expression of IL-1β were assessed by Western blot analysis. Actin was used as the internal control.
    The effects of CNMSH on IL-1β production and protein expression in LPS-stimulated BV2 microglia. Cells were pre-treated with 125, 250 and 500 μg/mL of CNMSH 1 h before LPS treatment for 24 h. (A) Following 24 h treatment, and the amounts of IL-1β production were measured with the supernatants. Data are expressed as mean±SD of three independent experiments. (*p<0.05 as compared with control group, #p<0.05 as compared with LPS treated group). (B) Following 24 h treatment, the expression of IL-1β were assessed by Western blot analysis. Actin was used as the internal control.
  • [Fig. 5.] The effects of CNMSH on TNF-α production and protein expression in LPS-stimulated BV2 microglia. Cells were pre-treated with 125, 250 and 500 μg/mL of CNMSH 1 h before LPS treatment for 24 h. (A) Following 24 h treatment, and the amounts of TNF-α production were measured with the supernatants. Data are expressed as mean±SD of three independent experiments (*p<0.05 as compared with control group). (B) Following 24 h treatment, the expression of IL-1α were assessed by Western blot analysis. Actin was used as the internal control.
    The effects of CNMSH on TNF-α production and protein expression in LPS-stimulated BV2 microglia. Cells were pre-treated with 125, 250 and 500 μg/mL of CNMSH 1 h before LPS treatment for 24 h. (A) Following 24 h treatment, and the amounts of TNF-α production were measured with the supernatants. Data are expressed as mean±SD of three independent experiments (*p<0.05 as compared with control group). (B) Following 24 h treatment, the expression of IL-1α were assessed by Western blot analysis. Actin was used as the internal control.
  • [Fig. 6.] The effects of CNMSH on NF-κB activity in LPS-treated BV2 microglia. Cells were pre-treated with CNMSH 1 h before LPS treatment for an hour. (A) Cytosol (30 μg) or nuclear protein (30 μg) was subjected to 10% SDS-PAGE, followed by Western blotting. The NF-κB p65 activity was assessed by Western blot analysis. Actin and lamin B were used as internal controls for cytoplasm and nucleus, respectively. (B) Localization of NF-κB p65 was visualized with fluorescence microscopy after immunofluorescence staining with NF-κB p65 antibody (green). Cells were stained with DAPI to visualize nuclei (blue). These results are representatives of at least two independent experiments that showed similar patterns.
    The effects of CNMSH on NF-κB activity in LPS-treated BV2 microglia. Cells were pre-treated with CNMSH 1 h before LPS treatment for an hour. (A) Cytosol (30 μg) or nuclear protein (30 μg) was subjected to 10% SDS-PAGE, followed by Western blotting. The NF-κB p65 activity was assessed by Western blot analysis. Actin and lamin B were used as internal controls for cytoplasm and nucleus, respectively. (B) Localization of NF-κB p65 was visualized with fluorescence microscopy after immunofluorescence staining with NF-κB p65 antibody (green). Cells were stained with DAPI to visualize nuclei (blue). These results are representatives of at least two independent experiments that showed similar patterns.
  • [Fig. 7.] The effects of CNMSH on phosphorylation of NF-κB in LPS-treated BV2 microglia. Cells were treated with 500 ug/ml CNMSH 1 h before LPS treatment for the indicated times. Total proteins (50 μg) were separated on 10% SDS-polyacrylamide gels, followed by Western blotting using the indicated antibodies. Results are representative of those obtained from three independent experiments.
    The effects of CNMSH on phosphorylation of NF-κB in LPS-treated BV2 microglia. Cells were treated with 500 ug/ml CNMSH 1 h before LPS treatment for the indicated times. Total proteins (50 μg) were separated on 10% SDS-polyacrylamide gels, followed by Western blotting using the indicated antibodies. Results are representative of those obtained from three independent experiments.
  • [Fig. 8.] The effects of CNMSH on MAPK activation in LPS-treated BV2 microglia. Cells were treated with 500 ug/ml CNMSH 1 h before LPS treatment for the indicated times. Total proteins (50 μg) were separated on 10% SDS-polyacrylamide gels, followed by Western blotting using the indicated antibodies. Results are representative of those obtained from three independent experiments.
    The effects of CNMSH on MAPK activation in LPS-treated BV2 microglia. Cells were treated with 500 ug/ml CNMSH 1 h before LPS treatment for the indicated times. Total proteins (50 μg) were separated on 10% SDS-polyacrylamide gels, followed by Western blotting using the indicated antibodies. Results are representative of those obtained from three independent experiments.
  • [Fig. 9.] The effects of CNMSH on phosphorylation of Akt in LPS-treated BV2 microglia. Cells were treated with 500 ug/ml CNMSH 1 h before LPS treatment for the indicated times. Total proteins (50 μg) were separated on 10% SDS-polyacrylamide gels, followed by Western blotting using the indicated antibodies. Results are representative of those obtained from three independent experiments.
    The effects of CNMSH on phosphorylation of Akt in LPS-treated BV2 microglia. Cells were treated with 500 ug/ml CNMSH 1 h before LPS treatment for the indicated times. Total proteins (50 μg) were separated on 10% SDS-polyacrylamide gels, followed by Western blotting using the indicated antibodies. Results are representative of those obtained from three independent experiments.
  • [Fig. 10.] The effects of CNMSH on Akt activation in LPS-treated BV2 microglia. BV2 cells were treated with the indicated does of CNMSH alone and in combination with LY294002 for 1 h before LPS treatment for the indicated times. Following 24 h treatment, the supernatants were taken, and amounts of NO (A), PGE2 (B), IL-1β (C) were measured with the supernatants. The data are shown as means±SD of three independent experiments. (*p<0.05 as compared with control group, #p<0.05 as compared with LPS treated group).
    The effects of CNMSH on Akt activation in LPS-treated BV2 microglia. BV2 cells were treated with the indicated does of CNMSH alone and in combination with LY294002 for 1 h before LPS treatment for the indicated times. Following 24 h treatment, the supernatants were taken, and amounts of NO (A), PGE2 (B), IL-1β (C) were measured with the supernatants. The data are shown as means±SD of three independent experiments. (*p<0.05 as compared with control group, #p<0.05 as compared with LPS treated group).