Fig 1.
Experimental design and basal conditions.
A. The concept of experimental designs included leptin supplement, Ad-CM replacement, and co-culture system. B. RAW264.7 cells were treated with a serial dose of lunasin for 24 h and cell proliferation was tested by MTT assay. C. RAW264.7 cells were treated with a serial dose of LPS for 24 h and cell proliferation was tested by MTT assay. D. RAW264.7 cells were treated with various concentrations of LPS for 24 h, and MCP-1 production in culture supernatant was determined by ELISA. Data are presented as mean ± SEM, from three independent experiments. Statistical analysis was based on one-way ANOVA and LSD post hoc test; # p < 0.001 or & p < 0.005 vs. control group. Ad-CM, adipocyte-conditioned medium; MCP-1, monocyte chemoattractant protein-1; TNF-α, tumor necrosis factor-α; IL, interleukin.
Fig 2.
Lunasin inhibited inflammatory cytokine production in leptin-conditioned RAW264.7 cells.
A. Cell proliferation of RAW264.7 cells was determined by MTT assay. Cells were treated with serial doses of leptin or leptin plus 100 ng/mL LPS for 24 h. B. MCP-1 secretion by RAW264.7 cells was analyzed by ELISA. Cells were treated with various concentrations of leptin in the presence or absence of 100 ng/mL LPS for 24 h. C. MCP-1 production after 24 h incubation was analyzed in the culture supernatant by ELISA. Cells were treated with various concentrations of lunasin and were activated by 200 ng/mL leptin, 100 ng/mL LPS, or a combination of both, for 24 h. D. IL-6 production was analyzed by ELISA. E. TNF-α production was analyzed by ELISA. F. IL-1β production was analyzed by ELISA. Data are presented as mean ± SEM, from three independent experiments. Statistical analysis was based on one-way ANOVA and LSD post hoc test; * p < 0.05, # p < 0.001, or & p < 0.005 vs. control group. LPS, lipopolysaccharide; MCP-1, monocyte chemoattractant protein-1; lun, lunasin; IL, interleukin; TNF-α, tumor necrosis factor-α.
Fig 3.
Lunasin inhibited inflammatory cytokine production in Ad-CM-conditioned RAW264.7 cells.
A. Cell proliferation of RAW264.7 cells was determined by MTT assay. Cells were cultured in the presence of increasing concentration of Ad-CM for 24 h. B. MCP-1 production by RAW264.7 cells was analyzed by ELISA. Cells were treated with multiple doses of Ad-CM in the presence or absence of 100 ng/mL LPS for 24 h. C. MCP-1 production after 24 h culture was analyzed in the supernatant by ELISA. Cells were treated with multiple doses of lunasin and were activated by 25% Ad-CM, 100 ng/mL LPS, or a combination of both for 24 h. D. IL-6 production was analyzed by ELISA. E. TNF-α production was analyzed by ELISA. F. IL-1β production was analyzed by ELISA. Data are presented as mean ± SEM from three independent experiments. Statistical analysis was based on one-way ANOVA and LSD post hoc test; * p < 0.05, # p < 0.001, or & p < 0.005 vs. control group. Ad-CM, adipocyte-conditioned medium; LPS, lipopolysaccharide; MCP-1, monocyte chemoattractant protein-1; lun, lunasin; IL, interleukin; TNF-α, tumor necrosis factor-α.
Fig 4.
Lunasin abated inflammatory cytokine production in the RAW264.7 cell and mature 3T3-L1 adipocyte co-culture.
A. RAW264.7 cells were co-cultured with mature adipocytes using a transwell system for 24 h. B. MCP-1 level in the supernatant obtained from 3T3-L1 cell culture, RAW264.7 cell culture, and co-culture of both cells in the presence or absence of 100 ng/mL LPS was measured by ELISA. C. MCP-1, IL-6, TNF-α, and IL1-β secretions in the supernatant obtained from the 24 h co-culture were measured by ELISA. Data are presented as mean ± SEM, from three independent experiments. Statistical analysis was based on one-way ANOVA and LSD post hoc test; * p < 0.05 vs. control group. DMEM, Dulbecco’s modified Eagle’s medium; BS, calf serum; FBS, fetal bovine serum; MCP-1, monocyte chemoattractant protein-1; LPS, lipopolysaccharide; lun, lunasin; IL, interleukin; TNF-α, tumor necrosis factor-α.
Fig 5.
Lunasin inhibited pro-inflammatory adipokine production in 3T3-L1 adipocytes.
3T3-L1 adipocytes were treated with various doses of lunasin and were stimulated with 10 ng/mL TNF-α, 50% RAW-CM, or 1 μg/mL LPS to induce inflammatory conditions for 24 h. A. MCP-1 production was analyzed in the supernatant by ELISA. B. PAI-1 production was analyzed by ELISA. C. Leptin production was analyzed by ELISA. D. Adiponectin production was analyzed by ELISA. Data are presented as mean ± SEM from three independent experiments. Statistical analysis was based on one-way ANOVA and LSD post hoc test; * p < 0.05, or # p < 0.001 vs. control group. LPS, lipopolysaccharide; MCP-1, monocyte chemoattractant protein-1; lun, lunasin; IL, interleukin; PAI-1, plasminogen activator inhibitor-1; RAW-CM, RAW264.7 cell-conditioned medium; TNF-α, tumor necrosis factor-α.
Fig 6.
A schema of the possible influential insights on anti-inflammatory property of lunasin.
In RAW264.7 macrophages, lunasin decreased inflammatory cytokine MCP-1, IL-6, TNF-α, and IL-1β productions. In 3T3-L1 adipocytes, lunasin inhibited the secretion of inflammatory adipokines MCP-1, PAI-1, and leptin. In addition, treatment with lunasin reduced the levels of these pro-inflammatory mediators, thereby disrupting the crosstalk between the two cells in a co-culture. Lunasin apparently exerted anti-inflammatory ability, possibly diminishing the obesity-induced inflammatory diseases.