Anat Cell Biol.  2011 Sep;44(3):194-203. 10.5115/acb.2011.44.3.194.

Resveratrol activates AMPK and suppresses LPS-induced NF-kappaB-dependent COX-2 activation in RAW 264.7 macrophage cells

• Yi CO
• Jeon BT
• Shin HJ
• Jeong EA
• Chang KC
• Lee JE
• Lee DH
• Kim HJ
• Kang SS
• Cho GJ
• Choi WS
• Roh GS

• KMID: 1447431
• DOI: http://doi.org/10.5115/acb.2011.44.3.194

Abstract

AMP-activated protein kinase (AMPK), an enzyme involved in energy homeostasis, regulates inflammatory responses, but its precise mechanisms are not fully understood. Recent evidence has shown that resveratrol (RES), an AMPK activator, reduces prostaglandin E2 production in lipopolysaccharide (LPS)-treated microglia. Here, we examined the effect of RES on nuclear factor kappa B (NF-kappaB) dependent cyclooxygenase (COX)-2 activation in LPS-treated RWA 264.7 macrophages. We found that treatment with RES increased AMPK activation. AMPK and acetyl CoA carboxylase phosphorylation were attenuated in cells treated with LPS+RES, compared to cells treated with LPS alone. RES inhibited tumor necrosis factor (TNF)-alpha and TNF receptor 1 in LPS-treated cells. Finally, RES inhibited LPS-induced NF-kappaB translocation into the nucleus and COX-2 expression. Moreover, the effects of 5-aminoimidazole-4-carboxamide ribose and compound C were consistent with the effects of RES in LPS-treated cells. Taken together, these results suggest that the anti-inflammatory action of RES in RAW 264.7 macrophages is dependent on AMPK activation and is associated with inhibition of the LPS-stimulated NF-kappaB-dependent COX-2 signaling pathway.

Keyword

Resveratrol; AMP-activated protein kinases; Macrophages

MeSH Terms

Acetyl-CoA Carboxylase
AMP-Activated Protein Kinases
Dinoprostone
Homeostasis
Macrophages
Microglia
NF-kappa B
Phosphorylation
Prostaglandin-Endoperoxide Synthases
Receptors, Tumor Necrosis Factor
Ribose
Stilbenes
Tumor Necrosis Factor-alpha
AMP-Activated Protein Kinases
Acetyl-CoA Carboxylase
Dinoprostone
NF-kappa B
Prostaglandin-Endoperoxide Synthases
Receptors, Tumor Necrosis Factor
Ribose
Stilbenes
Tumor Necrosis Factor-alpha

Figure

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  Fig. 1 Effect of resveratrol (RES) treatment on AMP-activated protein kinase (AMPK) activation in RAW 264.7 cells. (A) Concentration-dependent effects of RES on RAW 264.7 cell viability. Cells were incubated with different concentrations (0.1 or 10 µM) of RES for 3-h. (B) Western blot analysis for AMPK activation in different concentrations of RES. p-AMPK was normalized to β-actin and represented as arbitrary units. Data are expressed as mean±SEM of the ratio of p-AMPK protein expression for three independent experiments. P<0.05 by ANOVA. *P<0.005,†P<0.001 vs. control.

• 

  Fig. 2 Effect of resveratrol (RES) treatment on AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) phosphorylation in lipopolysaccharide (LPS)-treated RAW 264.7 cells. Cells were incubated with 0.5 µM RES for 3-h, 1 mM 5-aminoimidazole-4-carboxamide ribose (AICAR) for 1-h, and 10 µM compound C (CC) for 1-h. Cells were then treated with 50 ng/ml LPS for 3-h. Cell lysates were subjected to Western blot analysis with p-AMPK and total AMPK (A) and p-ACC and total ACC (B) antibodies. p-AMPK or p-ACC was normalized to total AMPK or ACC and represented as arbitrary units, respectively. Data are expressed as mean±SEM for three independent experiments. P<0.05 by ANOVA. *P<0.005, †P<0.001 vs. control (CTL) or LPS-treated cells, respectively.

• 

  Fig. 3 Effect of resveratrol (RES) treatment on tumor necrosis factor (TNF)-α and TNF receptor (TNFR) expression in lipopolysaccharide (LPS)-treated RAW 264.7 cells. Cells were incubated with 0.5 µM RES for 3-h, 1 mM 5-aminoimidazole-4-carboxamide ribose (AICAR) for 1-h, and 10 µM compound C (CC) for 1-h. Cells were then treated with 50 ng/ml LPS for 3-h. (A) Cell supernatant TNF-α concentrations measured by enzyme-linked immunosorbent assay. Cell lysates were subjected to Western blot analyses with TNF-α (B) and TNFR (C) antibodies, respectively. TNF-α and TNFR1 were normalized to β-actin and represented as arbitrary units. Data are expressed as mean±SEM for three independent experiments. P<0.05 by ANOVA. *P<0.005, †P<0.001 vs. the respective control (CTL) or LPS-treated cells, respectively.

• 

  Fig. 4 Effect of resveratrol (RES) treatment on nuclear factor κB (NF-κB) translocation in lipopolysaccharide (LPS)-treated RAW 264.7 cells. (A) Representative photomicrographs of immunolabeling for NF-κB (red) and nuclear counterstaining with DAPI (blue) in control (CTL), LPS, LPS+RES, LPS+5-aminoimidazole-4-carboxamide ribose (AICAR), and LPS+compound C (CC)-treated RAW 264.7 cells. LPS treatment induced increased colocalization of NF-κB and DAPI (purple) in LPS-treated cells. However, RES treatment reduced NF-κB translocation, as shown by decreased colocalization with DAPI. Arrows indicate translocation from the cytoplasm to the nucleus. Scale bar=100 µm. (B) Nuclear translocation of cytoplasmic NF-κB from each group was assessed by Western blotting. (C) Quantitative analysis of NF-κB levels calculated from Western blots. Cytoplasmic (cyto) or nuclear (nu) NF-κB was normalized to β-actin or lamin A and represented as arbitrary units, respectively. Data are expressed as mean±SEM for three independent experiments. P<0.05 by ANOVA. *P<0.05, †P<0.001 vs. the respective control (CTL) or LPS-treated cells, respectively.

• 

  Fig. 5 Effect of resveratrol (RES) treatment on cyclooxygenase (COX)-2 expression in lipopolysaccharide (LPS)-treated RAW 264.7 cells. (A) Cells were incubated with 0.5 µM RES for 3-h, 1 mM 5-aminoimidazole-4-carboxamide ribose (AICAR) for 1-h, and 10 µM compound C (CC) for 1-h. Cells were then treated with 50 ng/ml LPS for 3-h. Cell lysates were subjected to Western blot analysis with a COX-2 antibody. (B) Quantitative analysis of COX-2 levels calculated from Western blots. COX-2 was normalized to β-actin and represented as arbitrary units. Data are expressed as mean±SEM for three independent experiments. P<0.05 by ANOVA. *P<0.001 vs. the respective control (CTL) or LPS-treated cells, respectively.

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