Anti-Inflammatory Effect of Lupinalbin A Isolated from Apios americana on Lipopolysaccharide-Treated RAW264.7 Cells

Abstract

Apios americana, a leguminous plant, is used as food in some countries. Although the biological activities of Apios extract have been reported, there have been no reports about the anti-inflammatory mechanism of lupinalbin A on the RAW264.7 cells. In this study, we investigated the anti-inflammatory effect of A. americana lupinalbin A on lipopolysaccharide (LPS)-treated RAW264.7 cells. Lupinalbin A significantly inhibited nitric oxide production and inducible nitric oxide synthase expression in LPS-treated RAW264.7 cells. The expression of cytokines, including interleukin-6, tumor necrosis factor-α, and chemokine of monocyte chemoattractant protein, was reduced under lupinalbin A exposure in LPS-treated RAW264.7 cells. In addition, lupinalbin A significantly decreased LPS-induced interferon (IFN)-β production and STAT1 protein levels in RAW264.7 cells. Taken together, these results suggest that A. americana lupinalbin A exerts anti-inflammatory effects via the inhibition of pro-inflammatory cytokines and blocking of IFN-β/STAT1 pathway activation.

Keywords: Apios americana; anti-inflammatory effects; lipopolysaccharide; lupinalbin A.

Figure 1

Chemical structure of lupinalbin A and effect of lupinalbin A on cell viability in RAW264.7 cells. (A) The chemical structure of lupinalbin A; (B) Cell viability was measured by EZ-Cytox cell viability assay. Cells were treated with various concentrations of lupinalbin A (5–200 µM) for 24 h. The data are presented as mean ± SDs (n = 6).

Figure 1

Chemical structure of lupinalbin A and effect of lupinalbin A on cell viability in RAW264.7 cells. (A) The chemical structure of lupinalbin A; (B) Cell viability was measured by EZ-Cytox cell viability assay. Cells were treated with various concentrations of lupinalbin A (5–200 µM) for 24 h. The data are presented as mean ± SDs (n = 6).

Figure 2

Effect of lupinalbin A on NO production and iNOS protein and iNOS mRNA expression in LPS-treated RAW264.7 cells. (A) Cells were treated with various concentrations of lupinalbin A (5–100 µM) for 2 h and then incubated with LPS (1 μg/mL) for 18 h. The data are presented as mean ± SDs (n = 6). ^# p < 0.05 vs. control group, * p < 0.05 vs. LPS-treated group; (B) iNOS protein levels were measured by western blot; (C) Cells were treated with various concentrations of lupinalbin A (5–100 µM) for 2 h and then stimulated with LPS (1 μg/mL) for 18 h. iNOS mRNA expression levels were measured by qRT-PCR. The data are presented as mean ± SDs (n = 3). ^# p < 0.05 vs. control group, * p < 0.05 vs. LPS-treated group.

Figure 2

Effect of lupinalbin A on NO production and iNOS protein and iNOS mRNA expression in LPS-treated RAW264.7 cells. (A) Cells were treated with various concentrations of lupinalbin A (5–100 µM) for 2 h and then incubated with LPS (1 μg/mL) for 18 h. The data are presented as mean ± SDs (n = 6). ^# p < 0.05 vs. control group, * p < 0.05 vs. LPS-treated group; (B) iNOS protein levels were measured by western blot; (C) Cells were treated with various concentrations of lupinalbin A (5–100 µM) for 2 h and then stimulated with LPS (1 μg/mL) for 18 h. iNOS mRNA expression levels were measured by qRT-PCR. The data are presented as mean ± SDs (n = 3). ^# p < 0.05 vs. control group, * p < 0.05 vs. LPS-treated group.

Figure 3

Effect of lupinalbin A on (A) IL-6; (B) TNF-α; and (C) MCP-1 production in LPS-treated RAW264.7 cells. The cells were treated with various concentrations of lupinalbin A (5–100 µM) for 2 h and then stimulated with LPS (1 μg/mL) for 18 h. IL-6, TNF-α, and MCP-1 production was determined by ELISA. The data are presented as mean ± SDs (n = 3). ^# p < 0.05 vs. control group, * p < 0.05 vs. LPS-treated group.

Figure 3

Effect of lupinalbin A on (A) IL-6; (B) TNF-α; and (C) MCP-1 production in LPS-treated RAW264.7 cells. The cells were treated with various concentrations of lupinalbin A (5–100 µM) for 2 h and then stimulated with LPS (1 μg/mL) for 18 h. IL-6, TNF-α, and MCP-1 production was determined by ELISA. The data are presented as mean ± SDs (n = 3). ^# p < 0.05 vs. control group, * p < 0.05 vs. LPS-treated group.

Figure 4

Effect of lupinalbin A on (A) IL-6; (B) TNF-α; and (C) MCP-1 mRNA expression in LPS-treated RAW264.7 cells. Cells were treated with various concentrations of lupinalbin A (5–100 µM) for 2 h and then stimulated with LPS (1 μg/mL) for 18 h. IL-6, TNF-α, and MCP-1 mRNA expression levels were determined by qRT-PCR. The data are presented as mean ± SDs (n = 3). ^# p < 0.05 vs. control group, * p < 0.05 vs. LPS-treated group.

Figure 5

Effect of lupinalbin A on IFN-β production and STAT1 protein in LPS-treated RAW264.7 cells. (A) Cells were treated with various concentrations of lupinalbin A (5–100 µM) for 2 h and then stimulated with LPS (1 μg/mL) for 18 h. IFN-β production was determined by ELISA. The data are presented as mean ± SDs (n = 3). ^# p < 0.05 vs. control group, * p < 0.05 vs. LPS-treated group; (B) STAT1 protein levels were measured by western blot analysis.