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...
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-${\alpha}$ 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 ($NF-{\kappa}B$), 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-${\alpha}$, 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 $NF-{\kappa}B$ 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 $NF-{\kappa}B$ 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, $NF-{\kappa}B$, and MAPKs pathways.
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-${\alpha}$ 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 ($NF-{\kappa}B$), 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-${\alpha}$, 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 $NF-{\kappa}B$ 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 $NF-{\kappa}B$ 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, $NF-{\kappa}B$, and MAPKs pathways.
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제안 방법
Despite these encouraging studies, reports on the cellular and molecular mechanisms responsible for the anti-inflammatory effect of red rice extract have not been published. Here, we investigated the anti-inflammatory effect of red rice extracts by measuring their ability to inhibit inflammatory mediators and cytokine production. Next, we determined the molecular mechanism underlying the anti-inflammatory impact of red rice extract in LPS-induced Raw 264.
대상 데이터
All experiments were performed in triplicate. Quantifications are defined as mean ± SD of three independent experiments and expressed as percentage of the control, which was considered 100%.
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and LPS were obtained from Sigma Aldrich (St Louis, MO, USA). Antibodies for detection of ERK1/2, p38, JNK, c-Jun, p65, and iNOS were purchased from Cell Signaling Technology (Danver, MA, USA). Antibody for detection of COX-2 was purchased from Calbiochem (San Diego, CA, USA).
Antibody for detection of COX-2 was purchased from Calbiochem (San Diego, CA, USA). The extraction solvents and the mobile phase of HPLC, including n-butanol, ethanol, and methanol, were purchased from RCI Labscan (Samutsakorn, Thailand).
데이터처리
Quantifications are defined as mean ± SD of three independent experiments and expressed as percentage of the control, which was considered 100%. Throughout this study statistically significant differences were calculated using one-way ANOVA (Prism version 6.0 software). A difference between the experimental groups was considered statistically significant and determined at *P < 0.
이론/모형
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.
성능/효과
In conclusion, this study has shown that RR-P inhibited the production of NO, IL-6, TNF-α, COX-2, and iNOS in LPS-treated Raw 264.7 cells.
Our results demonstrated that RR-P was rich in phenolic compounds and proanthocyanidins, while lower amounts or none at all were detected in RR-NP. The main phenolic component in RR-P was a catechin followed by hydroxybenzoic acid and vanilic acid.
Results of this study indicated that RR-NP did not exhibit an obvious anti-inflammatory activity at the concentration of up to 200 μg/ml.
7 cells. The results showed that the phosphorylation of p38, JNK, and ERK1/2 by LPS was inhibited by RR-P treatment. Thus, it is likely that the inhibition of MAPKs phosphorylation by RR-P may contribute to the RR-P mediated inhibition of NF-κB and AP-1 in LPS-induced macrophages.
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