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Fisetin Suppresses Macrophage-Mediated Inflammatory Responses by Blockade of Src and Syk.

Kim JH, Kim MY, Kim JH, Cho JY - Biomol Ther (Seoul) (2015)

Bottom Line: In agreement, the upstream phosphorylation events for NF-κB activation, composed of Src, Syk, and IκBα, were also reduced by fisetin.The phospho-Src level, triggered by overexpression of wild-type Src, was also inhibited by fisetin.Therefore, these results strongly suggest that fisetin can be considered a bioactive immunomodulatory compound with anti-inflammatory properties through suppression of Src and Syk activities.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetic Engineering, Sungkyunkwan University, Suwon 440-746.

ABSTRACT
Flavonoids, such as fisetin (3,7,3',4'-tetrahydroxyflavone), are plant secondary metabolites. It has been reported that fisetin is able to perform numerous pharmacological roles including anti-inflammatory, anti-microbial, and anti-cancer activities; however, the exact anti-inflammatory mechanism of fisetin is not understood. In this study, the pharmacological action modes of fisetin in lipopolysaccharide (LPS)-stimulated macrophage-like cells were elucidated by using immunoblotting analysis, kinase assays, and an overexpression strategy. Fisetin diminished the release of nitric oxide (NO) and reduced the mRNA levels of inducible NO synthase (iNOS), tumor necrosis factor (TNF)-α, and cyclooxygenase (COX)-2 in LPS-stimulated RAW264.7 cells without displaying cytotoxicity. This compound also blocked the nuclear translocation of p65/nuclear factor (NF)-κB. In agreement, the upstream phosphorylation events for NF-κB activation, composed of Src, Syk, and IκBα, were also reduced by fisetin. The phospho-Src level, triggered by overexpression of wild-type Src, was also inhibited by fisetin. Therefore, these results strongly suggest that fisetin can be considered a bioactive immunomodulatory compound with anti-inflammatory properties through suppression of Src and Syk activities.

No MeSH data available.


Related in: MedlinePlus

The effect of fisetin on the activity of Src and Syk. (A) Inhibitory activity of fisetin (30 μM) on the kinase activity of purified Src or Syk was examined by kinase assay. (B) Inhibitory activity of fisetin (20 and 30 μM) on the phosphorylation of overexpressed Src was determined by immunoblotting analysis. (C) RAW264.7 cells (1×106 cells/mL) were treated with LPS (1 μg/mL) in the presence or absence of PP2 (10 and 20 μM) for 24 h. The supernatants were then collected, and the NO concentration in the supernatants was determined using the Griess assay. Data (A and C) are expressed as the mean ± SD of experiments, which were performed with six samples. *p<0.05 and **p<0.01 compared to normal or control groups.
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f5-bt-23-414: The effect of fisetin on the activity of Src and Syk. (A) Inhibitory activity of fisetin (30 μM) on the kinase activity of purified Src or Syk was examined by kinase assay. (B) Inhibitory activity of fisetin (20 and 30 μM) on the phosphorylation of overexpressed Src was determined by immunoblotting analysis. (C) RAW264.7 cells (1×106 cells/mL) were treated with LPS (1 μg/mL) in the presence or absence of PP2 (10 and 20 μM) for 24 h. The supernatants were then collected, and the NO concentration in the supernatants was determined using the Griess assay. Data (A and C) are expressed as the mean ± SD of experiments, which were performed with six samples. *p<0.05 and **p<0.01 compared to normal or control groups.

Mentions: In order to confirm inhibitory activity of Src/Syk phosphorylation, we checked whether these enzymes can directly block the kinase activity of Src and Syk. As expected, the kinase activities of purified Src and Syk were completely inhibited by fisetin (30 μM). This result indicates that fisetin can act as a direct inhibitor of these enzymes (Fig. 5A). To further validate fisetin-mediated suppression of Src activity, we employed an overexpression strategy using HA-Src. We found that over-expressed Src increased the phospho-Src level and fisetin suppressed the phosphorylation of Src in a dose-dependent manner (Fig. 5B).


Fisetin Suppresses Macrophage-Mediated Inflammatory Responses by Blockade of Src and Syk.

Kim JH, Kim MY, Kim JH, Cho JY - Biomol Ther (Seoul) (2015)

The effect of fisetin on the activity of Src and Syk. (A) Inhibitory activity of fisetin (30 μM) on the kinase activity of purified Src or Syk was examined by kinase assay. (B) Inhibitory activity of fisetin (20 and 30 μM) on the phosphorylation of overexpressed Src was determined by immunoblotting analysis. (C) RAW264.7 cells (1×106 cells/mL) were treated with LPS (1 μg/mL) in the presence or absence of PP2 (10 and 20 μM) for 24 h. The supernatants were then collected, and the NO concentration in the supernatants was determined using the Griess assay. Data (A and C) are expressed as the mean ± SD of experiments, which were performed with six samples. *p<0.05 and **p<0.01 compared to normal or control groups.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556200&req=5

f5-bt-23-414: The effect of fisetin on the activity of Src and Syk. (A) Inhibitory activity of fisetin (30 μM) on the kinase activity of purified Src or Syk was examined by kinase assay. (B) Inhibitory activity of fisetin (20 and 30 μM) on the phosphorylation of overexpressed Src was determined by immunoblotting analysis. (C) RAW264.7 cells (1×106 cells/mL) were treated with LPS (1 μg/mL) in the presence or absence of PP2 (10 and 20 μM) for 24 h. The supernatants were then collected, and the NO concentration in the supernatants was determined using the Griess assay. Data (A and C) are expressed as the mean ± SD of experiments, which were performed with six samples. *p<0.05 and **p<0.01 compared to normal or control groups.
Mentions: In order to confirm inhibitory activity of Src/Syk phosphorylation, we checked whether these enzymes can directly block the kinase activity of Src and Syk. As expected, the kinase activities of purified Src and Syk were completely inhibited by fisetin (30 μM). This result indicates that fisetin can act as a direct inhibitor of these enzymes (Fig. 5A). To further validate fisetin-mediated suppression of Src activity, we employed an overexpression strategy using HA-Src. We found that over-expressed Src increased the phospho-Src level and fisetin suppressed the phosphorylation of Src in a dose-dependent manner (Fig. 5B).

Bottom Line: In agreement, the upstream phosphorylation events for NF-κB activation, composed of Src, Syk, and IκBα, were also reduced by fisetin.The phospho-Src level, triggered by overexpression of wild-type Src, was also inhibited by fisetin.Therefore, these results strongly suggest that fisetin can be considered a bioactive immunomodulatory compound with anti-inflammatory properties through suppression of Src and Syk activities.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetic Engineering, Sungkyunkwan University, Suwon 440-746.

ABSTRACT
Flavonoids, such as fisetin (3,7,3',4'-tetrahydroxyflavone), are plant secondary metabolites. It has been reported that fisetin is able to perform numerous pharmacological roles including anti-inflammatory, anti-microbial, and anti-cancer activities; however, the exact anti-inflammatory mechanism of fisetin is not understood. In this study, the pharmacological action modes of fisetin in lipopolysaccharide (LPS)-stimulated macrophage-like cells were elucidated by using immunoblotting analysis, kinase assays, and an overexpression strategy. Fisetin diminished the release of nitric oxide (NO) and reduced the mRNA levels of inducible NO synthase (iNOS), tumor necrosis factor (TNF)-α, and cyclooxygenase (COX)-2 in LPS-stimulated RAW264.7 cells without displaying cytotoxicity. This compound also blocked the nuclear translocation of p65/nuclear factor (NF)-κB. In agreement, the upstream phosphorylation events for NF-κB activation, composed of Src, Syk, and IκBα, were also reduced by fisetin. The phospho-Src level, triggered by overexpression of wild-type Src, was also inhibited by fisetin. Therefore, these results strongly suggest that fisetin can be considered a bioactive immunomodulatory compound with anti-inflammatory properties through suppression of Src and Syk activities.

No MeSH data available.


Related in: MedlinePlus