Korean J Physiol Pharmacol.  2015 Sep;19(5):441-449. 10.4196/kjpp.2015.19.5.441.

Scutellarein Reduces Inflammatory Responses by Inhibiting Src Kinase Activity

• Sung NY
• Kim MY
• Cho JY

• KMID: 2285589
• DOI: http://doi.org/10.4196/kjpp.2015.19.5.441

Abstract

Flavonoids are plant pigments that have been demonstrated to exert various pharmacological effects including anti-cancer, anti-diabetic, anti-atherosclerotic, anti-bacterial, and anti-inflammatory activities. However, the molecular mechanisms in terms of exact target proteins of flavonoids are not fully elucidated yet. In this study, we aimed to evaluate the anti-inflammatory mechanism of scutellarein (SCT), a flavonoid isolated from Erigeron breviscapus, Clerodendrum phlomidis and Oroxylum indicum Vent that have been traditionally used to treat various inflammatory diseases in China and Brazil. For this purpose, a nitric oxide (NO) assay, polymerase chain reaction (PCR), nuclear fractionation, immunoblot analysis, a kinase assay, and an overexpression strategy were employed. Scutellarein significantly inhibited NO production in a dose-dependent manner and reduced the mRNA expression levels of inducible NO synthase (iNOS) and tumor necrosis factor (TNF)-alpha in lipopolysaccharide (LPS)-activated RAW264.7 cells. In addition, SCT also dampened nuclear factor (NF)-kappaB-driven expression of a luciferase reporter gene upon transfection of a TIR-domain-containing adapter-inducing interferon-beta (TRIF) construct into Human embryonic kidney 293 (HEK 293) cells; similarly, NF-kappa B nuclear translocation was inhibited by SCT. Moreover, the phosphorylation levels of various upstream signaling enzymes involved in NF-kappaB activation were decreased by SCT treatment in LPS-treated RAW264.7 cells. Finally, SCT strongly inhibited Src kinase activity and also inhibited the autophosphorylation of overexpressed Src. Therefore, our data suggest that SCT can block the inflammatory response by directly inhibiting Src kinase activity linked to NF-kappaB activation.

Keyword

Anti-inflammatory effect; Flavonoid; Macrophages; NF-kappaB; Scutellarein; Src

MeSH Terms

Brazil
China
Clerodendrum
Erigeron
Flavonoids
Genes, Reporter
Humans
Interferon-beta
Kidney
Luciferases
Macrophages
NF-kappa B
Nitric Oxide
Nitric Oxide Synthase
Phosphorylation
Phosphotransferases*
Plants
Polymerase Chain Reaction
RNA, Messenger
Transfection
Tumor Necrosis Factor-alpha
Flavonoids
Interferon-beta
Luciferases
NF-kappa B
Nitric Oxide
Nitric Oxide Synthase
Phosphotransferases
RNA, Messenger
Tumor Necrosis Factor-alpha

Figure

• 

  Fig. 1 Chemical structure and anti-inflammatory activity of SCT. (A) Molecular structure of SCT. (B) RAW-264.7 cells (1×106 cells/ml) were pretreated with SCT (left panel) or L-NAME (right panel) for 1 h and incubated with LPS (1 µg/ml) for 24 h. The nitric oxide levels were measured by the Griess assay. (C) Viability of RAW264.7 cells after treatment with SCT for 24 h. Viability was determined by a conventional MTT assay. *p<0.05 and **p<0.01 compared with the control group.

• 

  Fig. 2 Effect of SCT on inflammatory gene expression in LPS-treated RAW264.7 cells. (A) RAW264.7 cells (1×106 cells/ml) were incubated with LPS (1 µg/ml) for 6 h after treatment with SCT. The mRNA expression levels of iNOS and TNF-α were determined by RT-PCR. (B and C) iNOS and TNF-α mRNA expression levels were measured by real-time PCR. **p<0.01 compared with the control group.

• 

  Fig. 3 Effect of SCT on the transcriptional activation of inflammatory gene expression. (A, left panel) HEK 293 cells (5×105 cells/ml) were incubated with SCT for 24 h. Cell viability was measured by the MTT assay. (A, right panel) HEK 293 cells were treated with SCT for 24 h after cotransfection with NF-κB-Luc, TRIF, or pcDNA for 24 h. Luciferase activity was determined using a luminometer. (B left panel) Nuclear translocation of NF-κB subunits was detected by immunoblot analysis of nuclear fractions. Relative intensity (B right panel) was calculated using total levels by the DNR Bio-Imaging system. *p<0.05 and **p<0.01 compared with the control group.

• 

  Fig. 4 Effect of SCT on signaling events upstream of NF-κB activation. (Left panels of A and B) RAW264.7 cells (1×106 cells/ml) were incubated with LPS (1 µg/ml) for the indicated times after treatment with SCT (200 µM). The levels of phosphorylated IκBα, IKKα/β, AKT, p85/PI3K, and Src were detected by immunoblot analysis. Relative intensity (Right panels of A and B) was calculated using total levels by the DNR Bio-Imaging system. *p<0.05 and **p<0.01 compared with the control group.

• 

  Fig. 5 Effect of SCT on the regulation of Src kinase. (A) The inhibitory effect of SCT on Src kinase activity was determined using an established kinase assay with purified Src enzyme. (B and C) HEK 293 cells (5×105 cells/ml) were transfected with Flag-hMyD88 or HA-Src plasmids and then incubated for 24 h. The transfected cells were then treated with SCT for 12 h. The level of phosphorylated Src was measured by immunoblotting. Relative intensity (Bottom panels of B and C) was calculated using β-actin levels by the DNR Bio-Imaging system. **p<0.01 compared with the control group.

• 

  Fig. 6 Schematic diagram of the inhibitory action of SCT on LPS-induced inflammatory responses in macrophage cells.

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