J Nutr Health.  2018 Dec;51(6):507-514. 10.4163/jnh.2018.51.6.507.

Immunomodulating activity of Sargassum horneri extracts in RAW264.7 macrophages

• Kim DS
• Sung NY
• Park SY
• Kim G
• Eom J
• Yoo JG
• Seo IR
• Han IJ
• Cho YB
• Kim KA

• KMID: 2455839
• DOI: http://doi.org/10.4163/jnh.2018.51.6.507

Abstract

PURPOSE
Sargassum horneri (S. horneri) is a species of brown macroalgae that is common along the coast of Japan and Korea. The present study investigated the immuno-modulatory effects of different types of S. horneri extracts in RAW264.7 macrophages.
METHODS
S. horneri was extracted by three different methods, hot water extraction, 50% ethanol extraction, and supercritical fluid extraction. Cell viability was then measured by MTT assay, while the production levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and nitric oxide (NO) were measured by enzyme-linked immunosorbent assay and Griess assay, respectively. The expression and activation levels of inducible NO synthase (iNOS), mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) were examined by western blot analysis.
RESULTS
The three different S. horneri extracts were nontoxic against RAW 264.7 cells up to 50 µg/mL, among which treatment with hot water extract (HWE) of S. horneri significantly enhanced the production of TNF-α, IL-6, and NO in a dose-dependent manner. Hot water extract of S. horneri also increased the expression level of iNOS, suggesting that up-regulation of iNOS expression by HWE of S. horneri was responsible for the induction of NO production. In addition, treatment of RAW 264.7 macrophages with HWE of S. horneri increased the phosphorylation levels of ERK, p38 and JNK. Furthermore, the activation and subsequent nuclear translocation of NF-κB was enhanced upon treatment with HWE of S. horneri, indicating that HWE of S. horneri activates macrophages to secrete TNF-α, IL-6 and NO and induces iNOS expression via activation of the NF-κB and MAPKs signaling pathways.
CONCLUSION
Taken together, these findings suggest that HWE of S. horneri possesses potential as a functional food with immunomodulatory activity.

Keyword

Sargassum horneri; macrophages; nitric oxide; cytokines; MAPKs

MeSH Terms

Blotting, Western
Cell Survival
Chromatography, Supercritical Fluid
Cytokines
Enzyme-Linked Immunosorbent Assay
Ethanol
Functional Food
Interleukin-6
Japan
Korea
Macrophages*
Necrosis
Nitric Oxide
Nitric Oxide Synthase
Phosphorylation
Protein Kinases
RAW 264.7 Cells
Sargassum*
Seaweed
Up-Regulation
Water
Cytokines
Ethanol
Interleukin-6
Nitric Oxide
Nitric Oxide Synthase
Protein Kinases
Water

Figure

• 

  Fig. 1. Effects of different types of Sargassum horneri extracts on the cell viability of Raw264.7 macrophages. HWE, EE, and SFE were treated at the concentration of 12.5, 25, and 50 μg/mL and LPS (1 μg/mL) used as a positive control. After 24 h, cell proliferation was determined by MTT assay. Results are expressed as the mean ± SD (n = 3). Statistical analysis was performed using Student's two tails t-test with a significant level of ∗ p < 0.05, and ∗∗ p < 0.01 compared to control (CON) group. HWE, hot water extract; EE, ethanol extract; SFE, supercritical fluid extract

• 

  Fig. 2. Effects of different types of Sargassum horneri extracts on the production of TNF-α (A) and IL-6 (B) in Raw264.7 macrophages. HWE, EE, and SFE were treated at the concentration of 3.125, 6.25, 12.5, 25, and 50 μg/mL in RAW 264.7 cells. LPS (1 μg/mL) used as a positive control. After 24hr, cytokine productions in culture supernatant were investigated by using ELISA kits. Results are expressed as the mean ± SD (n = 3). Statistical analysis was performed using Student's two tails t-test with a significant level of ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001, compared to control (CON) group. HWE, hot water extract; EE, ethanol extract; SFE, supercritical fluid extract

• 

  Fig. 3. Effects of different types of Sargassum horneri extracts on the production of NO (A) and iNOS expression level (B) in Raw264.7 macrophages. (A) Dose-dependent effect of HWE from Sargassum horneri (3.125, 6.25, 12.5, 25, and 50 μg/mL) on NO production was determined by Griess reagent assay. LPS (1 μg/mL) used as a positive control. (B) Effect of HWE (12.5, 25, and 50 μg/mL) on iNOS expression in cell lysate was investigated by western blotting. Results are expressed as the mean ± SD (n = 3). Statistical analysis was performed using Student's two tails t-test with a significant level of ∗∗ p < 0.01, and ∗∗∗ p < 0.001, compared to control (CON) group. HWE, hot water extract; EE, ethanol extract; SFE, supercritical fluid extract

• 

  Fig. 4. Effects of hot water extract of Sargassum horneri on MAPKs phosphorylation (A), cytosolic IκB and nuclear NF-κB expression (B) in RAW 264.7 macrophages. HWE of Sargassum horneri was treated at the concentration of 12.5, 25, and 50 μg/mL for 30 min. LPS (0.2 μg/mL) used as a positive control. MAPKs phosphorylation, IκB and NF-κB expression were evaluated by western bolt analysis using specific antibodies to p38, phospho-p38, ERK1/2, phospho-ERK1/2, JNK, phospho-JNK, IκB, NF-κB (p65). HWE, hot water extract

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