Immunomodulating activity of Sargassum horneri extracts in RAW264.7 macrophages

doi:10.4163/jnh.2018.51.6.507

Journal List > J Nutr Health > v.51(6) > 1111483


J Nutr Health. 2018 Dec;51(6):507-514. Korean. Published online Dec 31, 2018. https://doi.org/10.4163/jnh.2018.51.6.507
© 2018 The Korean Nutrition Society


Immunomodulating activity of Sargassum horneri extracts in RAW264.7 macrophages
Dong-Sub Kim,¹ Nak-Yun Sung,¹ Sang-Yun Park,¹ Geon Kim,¹ Ji Eom,¹ Jin-Gon Yoo,¹ In-Ra Seo,¹ In-Jun Han,¹ Young-Baik Cho,¹ and Kyung-Ah Kim²
¹Division of Natural Product Research, Korea Prime Pharmacy CO., LTD., Jeonnam 58144, Korea.
²Department of Food and Nutrition, Chungnam National University, Daejeon 34134, Korea.
To whom correspondence should be addressed. tel: +82-42-821-6832, Email: kakim@cnu.ac.kr

Received Oct 23, 2018; Revised Nov 21, 2018; Accepted Dec 10, 2018.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.


Keywords: Sargassum horneri; macrophages; nitric oxide; cytokines; MAPKs

Figures


	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
	Click for larger image


	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
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	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
	Click for larger image


	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
	Click for larger image

Notes

This work was supported by grants from the Ministry of Trade, Industry & Energy (MOTIE), Korea Institute for Advancement of Technology (KIAT) through the Encouragement Program for The Industries of Economic Cooperation Region (P0000611).

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