Nutr Res Pract.  2016 Jun;10(3):251-258. 10.4162/nrp.2016.10.3.251.

Anti-inflammatory effects of proanthocyanidin-rich red rice extract via suppression of MAPK, AP-1 and NF-κB pathways in Raw 264.7 macrophages

• Limtrakul
• Yodkeeree
• Pitchakarn P
• Punfa W

• KMID: 2342126
• DOI: http://doi.org/10.4162/nrp.2016.10.3.251

Abstract

BACKGROUND/OBJECTIVES
Several pharmacological properties of red rice extract have been reported including anti-oxidant, anti-tumor, and reduced cancer cell invasion. This study was conducted to evaluate the anti-inflammatory effects of red rice extract on the production of inflammatory mediators in lipopolysaccharide (LPS)-induced Raw 264.7 macrophages.
MATERIALS/METHODS
Pro-inflammatory cytokines including tumor necrosis factor-α and interleukin-6 were determined by ELISA and cyclooxygenase-2 and inducible nitric oxide synthase expression was evaluated using western blot analysis. In addition, the signaling pathway controlling the inflammatory cascade such as nuclear factor kappa B (NF-κB), activator proteins-1 (AP-1), and mitogen-activated protein kinase (MAPK) was determined.
RESULTS
Our results showed that red rice polar extract fraction (RR-P), but not non-polar extract fraction, inhibited interleukin-6, tumor necrosis factor-α, and nitric oxide production in LPS-induced Raw 264.7 cells. RR-P also reduced the expression of inflammatory enzymes, inducible nitric oxide synthase, and cyclooxygenase-2. In addition, activation of AP-1 and NF-κB transcription factor in the nucleus was abrogated by RR-P. RR-P inhibited the phosphorylation of extracellular signaling-regulated kinase 1/2, c-Jun NH2-terminal kinase, and p38 MAPK signaling responsible for the expression of inflammatory mediators in LPS-stimulated Raw 264.7 cells. Based on chemical analysis, high amounts of proanthocyanidin and catechins were detected in the RR-P fraction. However, only proanthocyanidin reduced NF-κB and AP-1 activation in LPS-activated Raw 264.7 cells.
CONCLUSION
These observations suggest that the anti-inflammatory properties of RR-P may stem from the inhibition of pro-inflammatory mediators via suppression of the AP-1, NF-κB, and MAPKs pathways.

Keyword

Cytokines; inflammation; lipopolysaccharide; phenolic; proanthocyanidin

MeSH Terms

Blotting, Western
Catechin
Cyclooxygenase 2
Cytokines
Enzyme-Linked Immunosorbent Assay
Inflammation
Interleukin-6
Macrophages*
Necrosis
NF-kappa B
Nitric Oxide
Nitric Oxide Synthase Type II
p38 Mitogen-Activated Protein Kinases
Phosphorylation
Phosphotransferases
Protein Kinases
RAW 264.7 Cells
Transcription Factor AP-1*
Transcription Factors
Catechin
Cyclooxygenase 2
Cytokines
Interleukin-6
NF-kappa B
Nitric Oxide
Nitric Oxide Synthase Type II
Phosphotransferases
Protein Kinases
Transcription Factor AP-1
Transcription Factors
p38 Mitogen-Activated Protein Kinases

Figure

• 

  Fig. 1 Effect of red rice extract fractions on the viability of Raw 264.7 cells. The cells were seeded into 96-well plates and treated with RR-P or RR-NP for 48 h. The cell viability was determined by MTT assay. The results are expressed as percentages of cell viability relative to the untreated cells and presented as the mean ± SD of three independent experiments.

• 

  Fig. 2 Effect of red rice extract fraction on the level of IL-6, TNF-α, and NO production in LPS-induced Raw 264.7 cells. The cells were pre-treated with 200 µg/ml of RR-P or RR-NP for 4h, followed by treatment with LPS for 24h. The levels of IL-6 and TNF-α were determined with ELISA and NO production was assayed using Griess reagent (A). The effects of various concentrations of RR-P (0-200 µg/ml) on the level of IL-6, TNF-α, and NO in LPS-induced Raw macrophages are presented (B). Data were obtained from three independent experiments and expressed as mean ± SD. Significant differences from the LPS induction group are depicted by * P < 0.05, ** P < 0.01, or **** P < 0.0001.

• 

  Fig. 3 RR-P reduced the expression of iNOS and COX-2 in LPS-induced Raw 264.7 cells. The cells were pretreated with RR-P for 4 h before stimulation with or without LPS for 24 h. The expression levels of iNOS (A) and COX-2 (B) in the cells were determined by western blot analysis. Relative densities were calculated as the ratio of iNOS and COX-2 expression to β-actin levels. Data are represented as the mean ± SD of three experiments. * P < 0.05, *** P < 0.001 or **** P < 0.0001 compared to LPS alone.

• 

  Fig. 4 Effect of RR-P on the level of NF-κB and AP-1 in the nucleus of LPS-induced Raw macrophages. The cells were pretreated with RR-P for 4 h, and stimulated with LPS for 30 min. The cell nucleus fraction was prepared and the level of NF-κB and AP-1 (c-Jun) was determined by western blot analysis. Relative densities were determined as the ratio of NF-κB expression to PARP level and c-Jun expression to PARP level. Data are represented as the mean ± SD of three experiments. * P < 0.05 or *** P < 0.001 compared to LPS alone.

• 

  Fig. 5 Effect of RR-P on LPS induced phosphorylation of MAPK in Raw 264.7 cells. The cells were pretreated with RR-P for 4 h, and stimulated with LPS for 20 min. The whole cells lysate were prepared for measurement of phosphorylated and non-phosphorylated forms of ERK1/2, p-38, and JNK by western blot analysis (A). Relative protein levels were quantified by scanning densitometry and normalized to control protein levels (B). Data are presented as the mean ± SD of three experiments. ** P < 0.01, *** P < 0.001, or **** P < 0.0001 compared to LPS alone.

• 

  Fig. 6 Effect of proanthocyanidin and catechin on LPS induced NF-κB and AP-1activities in Raw 264.7 cells. The cells were pretreated with 50 µg/ml of proanthocyanidin or catechin for 4 h and stimulated with LPS for 30 min. The nucleus-extracted fraction was prepared and used for determining NF-κB and AP-1 (c-Jun) levels by western blot analysis. Relative densities were determined as the ratio of NF-κB expression to PARP level and c-Jun expression to PARP level. Data are presented as the mean ± SD of three experiments. * P < 0.05 or ** P < 0.01 compared to LPS alone.

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