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1Department of Genetic Engineering, Sungkyunkwan University, Suwon 440-746
2School of Systems Biological Science, Soongsil University, Seoul 156-743
3Department of Veterinary Physiology, College of Veterinary Medicine, Biosafety Research Institute, Chonbuk National University, Jeonju 561-756, Republic of Korea
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.
Inflammation is one of the common biological reactions that protect our body from infection of bacteria, virus, and fungi. When inflammation occurs, the body responds by activating many different types of immune cells to clear out pathogens. Macrophages, the most common inflammatory cells, play a critical role in removing infectious materials and activate other immune cells. For these processes, activated macrophages produce pro-inflammatory cytokines and inflammatory mediators such as nitric oxide (NO) (Yang
Flavonoids are plant pigments that possess various physiological effects including antioxidant, antiviral, anti-cancer, anti-bacterial and anti-inflammatory activities (Rice-Evans
Fisetin (purity: >98%), polyethylenimine (PEI), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (a tetrazole) (MTT), and LPS (
Wild-type Src (Src-WT) was used as reported previously (Yang
RAW264.7 cells were cultured in RPMI1640 with 10% heat-inactivated FBS and 1% penicillin/streptomycin at 37°C in 5% CO2. HEK293 cells were maintained in DMEM media supplemented with 5% heat-inactivated FBS and 1% penicillin/streptomycin at 37°C in 5% CO2. The stock solutions of fisetin for the
After pre-incubation of RAW264.7 cells (1×106 cells/mL) for 18 h, fisetin was added to the cells for 30 min. After that, the cells were treated with LPS (1 μg/mL) for 24 h. The effect of fisetin (0 to 30 μM) on the production of NO was determined by analyzing NO levels using Griess reagents (Kim
The cytotoxic effects of fisetin (0 to 30 μM) were then evaluated using a conventional MTT assay as previously reported (Pauwels
In order to determine cytokine mRNA expression levels, total RNA was isolated from LPS-treated RAW264.7 cells using TRIzol Reagent, according to the manufacturer’s instructions. Total RNA was stored at −70°C until use. Semi-quantitative RT reactions were conducted as previously reported (Lee
RAW264.7 or HEK293 cells (5×106 cells/mL) were washed three times in cold phosphate buffered saline (PBS) and lysed in lysis buffer as reported previously (Kim
In order to evaluate the inhibition of the kinase activities of Src or Syk using purified enzymes, the kinase profiler service from Millipore (Billerica, MA, USA) was used. Purified Src or Syk (human) (1–5 mU) were incubated with the reaction buffer in a final reaction volume of 25 μl. The reaction was initiated by the addition of Mg-ATP. After incubation for 40 min at room temperature, the reaction was stopped by the addition of 5 mL of a 3% phosphoric acid solution. Ten microliters of the reaction were then spotted onto a P30 Filtermat that was washed three times for 5 min in 75 mM phosphoric acid and once in methanol prior to drying and scintillation counting.
All of the data presented in this paper are expressed as means ± SD. For statistical comparisons, results were analyzed using either ANOVA/Scheffe’s
First, to confirm the function of fisetin as an inflammatory-response suppressor in macrophage cells, we tested its capability to suppress NO production in LPS-treated RAW264.7 cells. As expected, production of NO was dose-dependently decreased by fisetin in RAW264.7 cells stimulated by LPS (Fig. 2A). It was found that there was no cytotoxic activity of fisetin at its effective anti-inflammatory concentrations (Fig. 2B).
To check whether the anti-inflammatory effect of fisetin occurs at the transcriptional level, we determined the mRNA expression levels of inflammatory genes such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and tumor necrosis factor-α (TNF-α). As expected, the expression of inflammatory mediator genes was suppressed in cells treated with either 20 or 30 μM of fisetin according to both semi-quantitative (Fig. 3A) and real-time RT-PCR (Fig. 3B) analyses. We also examined transcription factor levels in nuclear extracts by immunoblotting analysis and found that fisetin treatment was capable of inhibiting the nuclear translocation of p65/NF-κB at 60 min but not of p50/NF-κB (Fig. 3C).
Many studies have demonstrated that the NF-κB pathway is regulated by intracellular signaling cascades (Byeon
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).
It was reported that fisetin exhibits anti-inflammatory, anti-oxidative, and anti-proliferative activities (Pal
Previously, we determined that Src and Syk are major NF-κB regulatory protein tyrosine kinases (Byeon
Our data, demonstrating fisetin’s inhibitory potency and its anti-inflammatory mechanisms, strongly suggest that it can be developed as an anti-inflammatory drug. However, development of a single compound from natural plants as an anti-inflammatory drug is difficult due to high production cost and low purification yield. Although the amount of this compound is very low, it has been demonstrated that fisetin is one of the major anti-inflammatory components in various plants, such as
In summary, we have demonstrated that fisetin can suppress IκBα phosphorylation, p65 translocation, inflammatory gene expression, and NO production by direct blockade of Src and Syk kinase activities as summarized in Fig. 6. Because fisetin shows potent anti-inflammatory activity, we suggest that fisetin-rich plants from edible sources could be applied to the development of therapeutic remedies to cure various inflammatory symptoms. Therefore, relevant works will be further proved in the future projects.
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