Chonnam Med J.  2011 Aug;47(2):104-110. 10.4068/cmj.2011.47.2.104.

Theaflavin Inhibits LPS-Induced IL-6, MCP-1, and ICAM-1 Expression in Bone Marrow-Derived Macrophages Through the Blockade of NF-kappaB and MAPK Signaling Pathways

• Kim S
• Joo YE

• KMID: 2048793
• DOI: http://doi.org/10.4068/cmj.2011.47.2.104

Abstract

Theaflavin, the main polyphenol in black tea, has anti-inflammatory, antioxidative, anti-mutagenic, and anti-carcinogenic properties. The aim of this study was to evaluate the effects of theaflavin on lipopolysaccharide (LPS)-induced innate signaling and expression of pro-inflammatory mediators in bone marrow-derived macrophages isolated from ICR mice. The effects of theaflavin on the expression of proinflammatory mediators, LPS-induced nuclear factor-kappa B (NF-kappaB), and mitogen-activated protein kinase (MAPK) signaling pathways were examined by reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting, and immunofluorescence. LPS-induced interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and intercellular adhesion molecule-1 (ICAM-1) expression was inhibited by theaflavin. LPS-induced inhibitor kappa B alpha (IkappaBalpha) degradation and nuclear translocation of RelA were blocked by theaflavin. LPS-induced phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2), c-Jun-N-terminal kinase (JNK), and p38 MAPK was inhibited by theaflavin. The inhibitory effect of theaflavin on IL-6, MCP-1, and ICAM-1 expression was completely inhibited by Bay11-7082 (NF-kappaB inhibitor). The inhibitory effect of theaflavin on IL-6 and ICAM-1 expression was inhibited by SB203580 (p38 MAPK inhibitor). The inhibitory effect of theaflavin on MCP-1 expression was inhibited by SP600125 (JNK inhibitor). These results indicate that theaflavin prevents LPS-induced IL-6, MCP-1, and ICAM-1 expression through blockade of NF-kappaB and MAPK signaling pathways in bone marrow-derived macrophages.

Keyword

Theaflavin; NF-kappa B; MAP kinase; Macrophage

MeSH Terms

Animals
Anthracenes
Biflavonoids
Blotting, Western
Catechin
Chemokine CCL2
Fluorescent Antibody Technique
Imidazoles
Intercellular Adhesion Molecule-1
Interleukin-6
Macrophages
Mice
Mice, Inbred ICR
NF-kappa B
Nitriles
p38 Mitogen-Activated Protein Kinases
Phosphorylation
Phosphotransferases
Protein Kinases
Pyridines
Sulfones
Tea
Anthracenes
Biflavonoids
Catechin
Chemokine CCL2
Imidazoles
Intercellular Adhesion Molecule-1
Interleukin-6
NF-kappa B
Nitriles
Phosphotransferases
Protein Kinases
Pyridines
Sulfones
Tea
p38 Mitogen-Activated Protein Kinases

Figure

• 

  FIG. 1 Effect of theaflavin or LPS on cell viability. Cells were preincubated for 24 h with the indicated concentrations of theaflavin or LPS. No significant cytotoxicity of theaflavin or LPS was observed under the experimental conditions. Statistical significance was p>0.05.

• 

  FIG. 2 Effect of theaflavin on LPS-induced expression of pro-inflammatory mediators. Cells were pretreated with 50 µg/ml theaflavin for 1 h before stimulation for 1 or 4 h with 1 µg/ml LPS. RNA was isolated by the TRIzol procedure, and 1 µg of total RNA was reverse transcribed and amplified by using primers specific for IL-6, MCP-1, ICAM-1, and GAPDH. Theaflavin was found to significantly reduce the LPS-induced mRNA accumulation of IL-6, MCP-1, and ICAM-1.

• 

  FIG. 3 Effect of theaflavin on LPS-induced NF-κB activation.Cells were pretreated with theaflavin (50 µg/ml) and then stimulated with LPS (1 µg/ml) for the times indicated. (A) IκBα phosphorylation/degradation was evaluated by Western blotting. The LPS-induced IκBα phosphorylation/degradation was dramatically inhibited by co-incubation of LPS plus theaflavin. (B) Nuclear translocation of NF-κB/p65 was visualized by immunofluorescence. Co-incubation with LPS plus theaflavin inhibited the nuclear translocation of NF-κB/p65.

• 

  FIG. 4 Effect of theaflavin on LPS-induced phosphorylation of MAPK signal proteins. Cells were pretreated with 50 µg/ml theaflavin for 1 h and stimulated with LPS (1 µg/ml) for various times. Total protein was extracted, and 20 µg of protein was subjected to SDS-PAGE followed by phospho-ERK1/2, total ERK1/2, phospho-JNK, total JNK, phospho-p38, total p38, and β-tubulin immunoblotting using the ECL technique. Results are representative of three independent experiments. Theaflavin pretreatment resulted in a decrease in the level of phosphorylated ERK1/2, JNK, and p38 MAPK in BMMs.

• 

  FIG. 5 Effect of MAPK and NF-κB inhibitors on LPS-induced expression of pro-inflammatory mediators. Cells were pretreated with a specified concentration of theaflavin (50 µg/ml), PD98059 (ERK inhibitor; 20 µM), SP600125 (JNK inhibitor; 20 µM), SB203580 (p38 inhibitor; 20 µM), or Bay11-7082 (NF-κB inhibitor; 5 µM) and then stimulated with LPS (1 µg/ml) for the times indicated. Pretreatment with Bay11-7082 (A) markedly inhibited the LPS-induced expression of pro-inflammatory mediators, whereas pretreatment with either SB203580 or SP600125 had an inhibitory effect on the LPS-induced expression of IL-6, ICAM-1, or MCP-1, respectively (B).

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