Nutr Res Pract.  2017 Oct;11(5):357-364. 10.4162/nrp.2017.11.5.357.

Black soybean anthocyanins attenuate inflammatory responses by suppressing reactive oxygen species production and mitogen activated protein kinases signaling in lipopolysaccharide-stimulated macrophages

• Kim JN
• Han SN
• Ha TJ
• Kim HK

• KMID: 2390127
• DOI: http://doi.org/10.4162/nrp.2017.11.5.357

Abstract

BACKGROUND/OBJECTIVES
Oxidative stress is closely related with inflammation and development of many diseases. Black soybean seed coat contains high amount of anthocyanins, which are well-known for free radical scavenging activities. This study investigated inflammatory response and action mechanism of black soybean anthocyanins with regard to antioxidant activity in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells.
MATERIALS/METHODS
RAW 264.7 cells were treated with anthocyanins extracted from black soybean seed coats in a concentration range of 12.5 to 100 µg/mL. The production of reactive oxygen species (ROS), secretion of pro-inflammatory mediators and cytokines, and the signaling in the mitogen activated protein kinases (MAPKs) pathway were examined.
RESULTS
Black soybean anthocyanins significantly decreased LPS-stimulated production of ROS, inflammatory mediators such as nitric oxide (NO) and prostaglandin E₂, and pro-inflammatory cytokines, including tumor necrosis factor α and interleukin-6, in a dose-dependent manner without cytotoxicity (P < 0.001). Black soybean anthocyanins downregulated the expression of inducible NO synthase and cyclooxygenase-2 in LPS-stimulated RAW 264.7 cells (P < 0.001). Moreover, black soybean anthocyanins inhibited LPS-induced phosphorylation of MAPKs, including extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 (P < 0.001).
CONCLUSION
These results suggest that black soybean anthocyanins exert anti-inflammatory activity by inhibiting ROS generation and subsequent MAPKs signaling, thereby inhibiting inflammatory responses.

Keyword

Anthocyanins; anti-inflammatory agents; macrophage; mitogen-activated protein kinases; reactive oxygen species

MeSH Terms

Anthocyanins*
Anti-Inflammatory Agents
Cyclooxygenase 2
Cytokines
Inflammation
Interleukin-6
JNK Mitogen-Activated Protein Kinases
Macrophages*
Mitogen-Activated Protein Kinases*
Nitric Oxide
Nitric Oxide Synthase
Oxidative Stress
Phosphorylation
Phosphotransferases
RAW 264.7 Cells
Reactive Oxygen Species*
Soybeans*
Tumor Necrosis Factor-alpha
Anthocyanins
Anti-Inflammatory Agents
Cyclooxygenase 2
Cytokines
Interleukin-6
JNK Mitogen-Activated Protein Kinases
Mitogen-Activated Protein Kinases
Nitric Oxide
Nitric Oxide Synthase
Phosphotransferases
Reactive Oxygen Species
Tumor Necrosis Factor-alpha

Figure

• 

  Fig. 1 Effect of black soybean anthocyanins (BSA) on the viability of RAW 264.7 cells. Cells were treated with the indicated concentrations of BSA for 24 h, and the cell viability was measured with a MTS assay. The data are presented as the means ± SD (n = 4). *P < 0.001 was used to indicate a significant difference compared with the control by ANOVA followed by student's t-test.

• 

  Fig. 2 Effect of black soybean anthocyanins (BSA) on LPS-induced ROS production in RAW 264.7 macrophages. The cells were treated with the indicated concentrations of BSA for 1 h and later stimulated with LPS (1 µg/mL) for 24 h. Intracellular peroxide was measured by labeling with DCFH-DA for 30 min, and then analyzing the fluorescent intensity using a flow cytometer. The data are presented as the means ± SD from three independent experiments. Mean values without a common letter are significantly different as determined by a one-way ANOVA followed by Tukey's test (P < 0.001). LPS, lipopolysaccharide; CON, control; ROS, reactive oxygen species; DCFH-DA, 2,7-Dichlorofluorescein diacetate.

• 

  Fig. 3 Effect of black soybean anthocyanins (BSA) on NO/PGE2 production and iNOS/COX-2 expression in RAW 264.7 cells. The cells were treated with the indicated concentrations of BSA for 1 h and later stimulated with LPS (1 µg/mL) for 24 h. The levels of nitrite (A) and PGE2 (B) were measured in the culture media using the Griess reagent and an EIA kit, respectively. Western blot analysis was performed to determine the protein levels of iNOS (C) and COX-2 (D). The data are presented as the means ± SD from three independent experiments. Mean values without a common letter are significantly different as determined by a one-way ANOVA followed by Tukey's test (P < 0.001). LPS, lipopolysaccharide; CON, control; NO, nitric oxide; PGE2, prostaglandin E2; iNOS, inducible nitric oxide synthase; COX-2, cyclooxygenase-2.

• 

  Fig. 4 Effect of black soybean anthocyanins (BSA) on the production of pro-inflammatory cytokines in RAW 264.7 cells. The cells were treated with the indicated concentrations of BSA for 1 h and later stimulated with LPS (1 µg/mL) for 24 h. The protein levels of TNFα (A) and IL-6 (B) were measured in the culture media by EIA. The data are presented as the means ± SD from three independent experiments. Mean values without a common letter are significantly different as determined by a one-way ANOVA followed by Tukey's test (P < 0.001). LPS, lipopolysaccharide; CON, control; TNFα, tumor necrosis factor alpha; IL, interleukin.

• 

  Fig. 5 Effect of black soybean anthocyanins on the activation of MAPKs in RAW 264.7 cells. The cells were treated with the indicated concentrations of black soybean anthocyanins for 1 h and later stimulated with LPS (1 µg/mL) for 30 min. Western blot analysis was performed to determine the protein levels of JNK (A), ERK (B) and p38 (C). The data are presented as the means ± SD from three independent experiments. Mean values without a common letter are significantly different as determined by a one-way ANOVA followed by Tukey's test (P < 0.01). LPS, lipopolysaccharide; CON, control; JNK, c-Jun N-terminal kinase; p-JNK, phospho-c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; p-ERK, phospho-extracellular signal-regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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