Nutr Res Pract.  2014 Oct;8(5):516-520. 10.4162/nrp.2014.8.5.516.

Carnosic acid inhibits TLR4-MyD88 signaling pathway in LPS-stimulated 3T3-L1 adipocytes

• Park MY
• Mun ST

• KMID: 2313772
• DOI: http://doi.org/10.4162/nrp.2014.8.5.516

Abstract

BACKGROUND/OBJECTIVES
Carnosic acid (CA), found in rosemary (Rosemarinus officinalis) leaves, is known to exhibit anti-obesity and anti-inflammatory activities. However, whether its anti-inflammatory potency can contribute to the amelioration of obesity has not been elucidated. The aim of the current study was to investigate the effect of CA on Toll-like receptor 4 (TLR4) pathways in the presence of lipopolysaccharide (LPS) in 3T3-L1 adipocytes.
MATERIALS/METHODS
3T3-L1 adipocytes were treated with CA (0-20 microM) for 1 h, followed by treatment with LPS for 30 min; mRNA expression of adipokines and protein expression of TLR4-related molecules were then measured.
RESULTS
LPS-stimulated 3T3-L1 adipocytes showed elevated mRNA expression of tumor necrosis factor (TNF)-alpha, interleukin-6, and monocyte chemoattractant protein-1, and CA significantly inhibited the expression of these adipokine genes. LPS-induced up regulation of TLR4, myeloid differentiation factor 88, TNF receptor-associated factor 6, and nuclear factor-kappaB, as well as phosphorylated extracellular receptor-activated kinase were also suppressed by pre-treatment of 3T3-L1 adipocytes with CA.
CONCLUSIONS
Results of this study suggest that CA directly inhibits TLR4-MyD88-dependent signaling pathways and decreases the inflammatory response in adipocytes.

Keyword

3T3-L1 adipocytes; carnosic acid; inflammation; toll-like receptor 4

MeSH Terms

Adipocytes*
Adipokines
Chemokine CCL2
Inflammation
Interleukin-6
Myeloid Differentiation Factor 88
Obesity
Phosphotransferases
RNA, Messenger
TNF Receptor-Associated Factor 6
Toll-Like Receptor 4
Tumor Necrosis Factor-alpha
Up-Regulation
Adipokines
Chemokine CCL2
Interleukin-6
Myeloid Differentiation Factor 88
Phosphotransferases
RNA, Messenger
TNF Receptor-Associated Factor 6
Toll-Like Receptor 4
Tumor Necrosis Factor-alpha

Figure

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  Fig. 1 (A, B) LPS-induced expression of IL-6 mRNA in differentiated 3T3-L1 cells. (C) LPS-induced phosphorylation of ERK in differentiated 3T3-L1 cells. (D) Inhibition of IL-6 mRNA expression by CA in LPS-stimulated 3T3-L1 cells.

• 

  Fig. 2 Effect of CA on viability of differentiated 3T3-L1 cells. Differentiated 3T3-L1 cells were pretreated with various concentrations of CA for 1 h and then stimulated with or without LPS (100 ng/ml) for 30 min. After incubation, cell viability was assessed using the MTT method. Values are expressed as mean ± SD (n = 4).

• 

  Fig. 3 Effect of CA on LPS-induced TNF-α, IL-6, and MCP-1 mRNA levels in 3T3-L1 cells. Differentiated 3T3-L1 cells were pretreated with various concentrations of CA for 1 h. Cells were further stimulated with LPS (100 ng/ml). After 30 min, LPS-induced TNF-α, IL-6, and MCP-1 mRNA levels were determined. Data are expressed as a fold-induction as compared with vehicle (#P < 0.05 vs. the control; *P < 0.05 vs. the LPS group). Values are expressed as mean ± SD (n = 4).

• 

  Fig. 4 Effect of CA on LPS-induced expression of TLR4 in 3T3-L1 cells. Differentiated 3T3-L1 cells were pretreated with various concentrations of CA for 1 h. Cells were further stimulated with LPS (100 ng/ml). After 30 min, cell lysates were analyzed for TLR4 immunoblotting. Data are expressed as a fold-induction as compared with vehicle (#P < 0.05 vs. the control; *P < 0.05 vs. the LPS group). Values are expressed as mean ± SD (n = 4).

• 

  Fig. 5 Effect of CA on LPS-induced expression of MyD88 and TRAF6 in 3T3-L1 cells. Differentiated 3T3-L1 cells were pretreated with various concentrations of CA for 1 h. Cells were further stimulated with LPS (100 ng/ml). After 30 min, cell lysates were analyzed for MyD88 and TRAF6 immunoblotting. Data are expressed as a fold-induction as compared with vehicle (#P < 0.05 vs. the control; *P < 0.05 vs. the LPS group). Values are expressed as mean ± SD (n = 4).

• 

  Fig. 6 Effect of CA on LPS-induced activation of NF-κB in 3T3-L1 cells. Differentiated 3T3-L1 cells were pretreated with various concentrations of CA for 1 h. Cells were further stimulated with LPS (100 ng/ml). After 30 min, nuclear extracts were analyzed for NF-κB activation. Data are expressed as a % activation as compared with vehicle (#P < 0.05 vs. the control; *P < 0.05 vs. the LPS group). Values are expressed as mean ± SD (n = 4).

• 

  Fig. 7 Effect of CA on LPS-induced expression of phospho-ERK in 3T3-L1 cells. Differentiated 3T3-L1 cells were pretreated with various concentrations of CA for 1 h. Cells were further stimulated with LPS (100 ng/ml). After 30 min, cell lysates were analyzed for phospho-ERK and ERK immunoblotting.

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