Skip to main content
Browse Subject Areas

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Immune-enhancing effects of anionic macromolecules extracted from Codium fragile coupled with arachidonic acid in RAW264.7 cells

  • Chaiwat Monmai,

    Roles Data curation, Formal analysis, Investigation, Methodology, Software, Writing – original draft, Writing – review & editing

    Affiliation Department of Marine Food Science and Technology, Gangneung-Wonju National University, Gangneung, Gangwon, Republic of Korea

  • Weerawan Rod-in,

    Roles Methodology

    Affiliation Department of Marine Food Science and Technology, Gangneung-Wonju National University, Gangneung, Gangwon, Republic of Korea

  • A-yeong Jang,

    Roles Methodology

    Affiliation Department of Wellness-Bio Industry, Gangneung-Wonju National University, Gangneung, Gangwon, Republic of Korea

  • Sang-min Lee,

    Roles Writing – review & editing

    Affiliation Department of Marine Biotechnology, Gangneung-Wonju National University, Gangneung, Gangwon, Republic of Korea

  • Seok-Kyu Jung,

    Roles Writing – review & editing

    Affiliation Department of Horticulture, Daegu Catholic University, Gyeongsan, Gyeongbuk, Republic of Korea

  • SangGuan You,

    Roles Writing – review & editing

    Affiliation Department of Marine Food Science and Technology, Gangneung-Wonju National University, Gangneung, Gangwon, Republic of Korea

  • Woo Jung Park

    Roles Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing

    Affiliation Department of Marine Food Science and Technology, Gangneung-Wonju National University, Gangneung, Gangwon, Republic of Korea


Arachidonic acid (ARA) is an integral constituent of the biological cell membrane, conferring it with fluidity and flexibility, which are necessary for the function of all cells, especially nervous system, skeletal muscle, and immune system. Codium species biosynthesize sulfated polysaccharides with very distinct structural features. Some of them have different biological activities with great potential in pharmaceutical applications. In this study, anionic macromolecules extracted from Codium fragile were investigated for their cooperative immune-enhancing activities with ARA. The cooperation between ARA and Codium resulted in increased, dose-dependent nitric oxide production and iNOS gene expression. In addition, co-treatment of ARA and Codium effectively increased pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α), compared with Codium alone. We also demonstrated that the expression of COX-2 mRNA was also increased, which is responsible for the production of inflammatory mediator prostaglandins and their metabolites. Compared to the Codium group, the co-treatment of Codium with ARA enhanced the phosphorylation of nuclear factor-κB p-65, p38, and extracellular signal-related kinase 1/2, indicating that this combination stimulated immune response through nuclear factor-κB and mitogen-activated protein kinase pathways. These results indicated that the coordination of arachidonic acid with polysaccharide extracted from seaweed may be a potential source of immunomodulatory molecules.

1. Introduction

The human immune system was developed to protect the host from infection by foreign materials and pathogens [1]. It has a highly complex structure to eliminate invaders, as well as to help repair infected or damaged sites to restore homeostasis [2]. Macrophages play a role in one of the important parts of the immune system [3], and perform several biological activities such as host defense, inflammation control, and remodeling of tissue [4]. Macrophages exhibit different phenotypes at the different stages of the inflammatory response [5]. Macrophages have at least two different polarizations, the classical (M1) and alternative (M2) [6, 7]. M1 and M2 macrophages can provide their biological activities by secreting different cytokines and effector molecules [8]. The activation of M1 macrophages is associated with regulation of nitric oxide synthase (iNOS) via production of nitric oxide (NO), which is important for removal of infection and cytokine secretion for antigen defense, including anti-bacterial, anti-viral, and anti-tumor functions [913]. The activation of macrophages is thought to help resist infection [14]. The activation of M2 macrophage relate to the natural inflammation resolution. Therefore, M2 macrophages are usually mentioned as having repairing or anti-inflammatory functions [15].

Fatty acids are classified as saturated, monounsaturated, or polyunsaturated fatty acids (PUFA) by their degree of saturation. PUFAs such as dihomo-gamma-linolenic acid (DGLA), arachidonic acid (ARA, 20:4n-6) and eicosapentaenoic acid (EPA, 20:5n-3) are the precursors of eicosanoids which include prostaglandins (PG), thromboxanes (TX), lipoxins (LX), and leukotrienes (LT) [16, 17]. These compounds are associated with the host defense system against infections including resistance to stress stimuli, the immune response, and inflammatory processes [17]. Normally, ARA plays an important role in inflammatory processes and stimulates the production of pro-inflammatory cytokines [18], whereas EPA plays a key role in the anti-inflammatory process [1921]. ARA is generally the preferred substrate of lipoxygenases for LT synthesis and products derived from these enzymes produce strong immune stimulatory effects [22, 23]. Arachidonic acid metabolism also leads to the synthesis of prostaglandins (PGs), which are one of the lipid mediators [22]. PGs are involved in the immune response via gene regulation of cytokine signaling such as TNF-α and IL-1β [24].

Codium fragile is a green seaweed belonging to the family Codiaceae, which is spread along the shores of East Asia, Oceania, and North Europe [25]. It has been shown that the extracted compounds of C. fragile show several biological functions, such as anti-coagulant [26], anti-viral [27], anti-inflammatory [28], and immunomodulatory activities [29]. Very recently, our group reported that anionic molecules extracted from C. fragile acted on cyclophosphamide immune-suppressed mice. The current study investigated if the anionic macromolecules (Codium) extracted from C. fragile enhanced immunity when co-treated with ARA on RAW264.7 cells, compared with treatment without ARA.

2. Materials and methods

2.1 Chemicals

Arachidonic acid was purchased from Nu-Check Prep, Inc. (USA). Griess reagent was purchased from Sigma-Aldrich (USA). The EZ-Cytox Cell Viability Assay Kit was purchased from Daeil Labservice (Seoul, South Korea). The Tri reagent® was purchased from Molecular Research Center, Inc (Ohio, USA). A high capacity cDNA reverse transcription kit was purchased from Applied Biosystems (California, USA). SYBR® Premix Ex Taq™ II was purchased from Takara Bio Inc. (Kusatsu, Japan). Radioimmunoprecipitation assay (RIPA) buffer was purchased from Tech & Innovation (Hebei, China). The Pierce™ BCA Protein Assay Kit and Pierce® ECL Plus Western Blotting Substrate were purchased from ThermoScientific (Waltham, USA). Specific antibodies for p-NF-κB p65, p-p38, p-ERK1/2 and p-JNK were purchased from Cell Signaling Technology (Danvers, USA) and α-Tubulin was purchased from Abcam (Cambridge, United Kingdom).

2.2 Isolation of crude anionic macromolecules

Anionic macromolecules from C. fragile (Codium) were isolated as described previously [30]. Briefly, Codium was extracted from the milled sample of C. fragile using 80% EtOH and then dried at room temperature in a fume hood. The dried biomass was extracted with distilled water at 65°C with stirring for 2 h. The supernatant was collected and then concentrated by evaporation under reduced pressure at 60°C to approximately 200 mL. EtOH (99%) was added into the supernatant to obtain a final EtOH concentration of 70% and then kept at 4°C overnight. The precipitate was obtained by filtration of the solution. The precipitate was washed with EtOH (99%), followed by acetone, and then dried at room temperature in a fume hood. The recovered precipitate was re-dissolved in distilled water, and then the removal of free-proteins in the precipitate was carried out using the Sevag method to confirm that the proteins included in the precipitate were covalently bound [31].

2.3 Macrophage proliferation and nitric oxide production

Murine macrophages (1×105 cells), RAW264.7 cells (Korean Cell Line Bank), in RPMI-1640 medium (supplemented with 10% FBS and 1% penicillin/streptomycin) were placed in a 96-well plate. The plate was incubated for 24 h at 37°C in an atmosphere of 5% CO2. The different concentrations of Codium (0, 0.5, 1, 2, and 4 μg/mL) augmented with 0.5 μM of ARA were added to the macrophages and the plate was incubated for another 24 h. Cells grown in the presence of 1 μg/mL LPS were used as a positive control. All experiments were performed in triplicate. Nitric oxide (NO) production was determined by measuring the quantity of nitrite in the cell culture medium using the Griess reagent [32] and compared with standard curve. The EZ-Cytox Cell Viability Assay kit was used to analyze cellular proliferation [33]. The cellular proliferation ratio (%) was calculated based on the following formula: Where; A450 = absorbance at 450 nm.

2.4 RNA extraction and cDNA synthesis

Total RNA was extracted from treated cells using the Tri reagent®. RNA was precipitated using 100% isopropanol and washed with 75% EtOH. The RNA concentration was analyzed using the nanophotometer (Implen, Germany). First strand cDNA was synthesized using the High Capacity cDNA Reverse Transcription kit according to the manufacturer’s instructions.

2.5 Quantitative Real-Time PCR

Quantification of RAW264.7 immune gene expression was performed using SYBR® Premix Ex Taq™ II. The reaction mixture consisted of 0.4 μM of each specific primer pair (Table 1) and 0.1 ng of cDNA template. Quantitative Real-Time PCR was performed and analyzed with the QuantStudio 3 Flex real‐time PCR system (Applied Biosystems, Foster City, CA) using a relative standard curve method compared with β-actin as an internal control of immune gene expression.

Table 1. Oligonucleotide primers used in real-time PCR for evaluating immune gene expression.

2.6 Western blot assay

RAW264.7 cells were harvested with RIPA buffer. The protein concentration was measured using the Pierce™ BCA Protein Assay kit and proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were transferred to a polyvinylidene fluoride (PVDF) membrane, and a western blot assay was performed, as described by Narayanan et al. [34]. Specific antibodies were used for p-NF-κB p65, p-p38, p-ERK1/2, p-JNK, and α-Tubulin. Signals were recognized using the Pierce® ECL Plus Western Blotting Substrate. The blot was quantitatively analyzed using the ChemiDoc XRS+ imaging system (Bio-Rad) and ImageLab software (version 4.1, Bio-Rad).

2.7 Statistics

Statistics Software, Statistix 8.1, was used for statistical analysis and values were evaluated by one-way analysis of variance, followed by post-hoc Duncan’s multiple range tests. The differences between the two groups were compared using t-tests (p < 0.05).

3. Results

3.1 Codium coupled with ARA enhanced NO production in RAW264.7 cells without any cytotoxicity

The Codium composition was previously reported from our research group [30]. The main consist of Codium is carbohydrates (54.6%) and consisted of protein (15.7%), sulfates (13.0%), and uronic acid (1.4%). The major sugar of Codium was measured using monosaccharide composition analysis and the highest was galactose (59.5%). Fig 1A showed no obvious cytotoxicity of Codium (up to 4 μg/mL) in RAW264.7 cells irrespective of presence of ARA. As shown in Fig 1B, treatment of Codium coupled with ARA significantly increased NO production, depending on the Codium concentration. Moreover, the treatment of Codium with 0.5 μM ARA also increased NO production, compared with treatment of Codium or ARA alone.

Fig 1. Co-operative effect of Codium and ARA in RAW264.7 cells.

(A) Effect on macrophage proliferation (*, p < 0.05 compared to the RPMI group) and (B) effect on nitric oxide production (The letters a,b,c,d,e indicate a significant difference (p < 0.05) between the amount of NO production).

3.2 Codium coupled with ARA enhanced immune associated gene expression in RAW264.7 cells

Treatment of 4 μg/mL Codium to RAW264.7 cells resulted in enhanced production of immune-associated genes (Fig 2). Furthermore, these gene expression results indicated that incubation of RAW264.7 cells with Codium in addition to 0.5 μM ARA, led to an increase in expression of most immune-associated genes, depending on the concentration of Codium. Finally, co-treatment of Codium and ARA led to a higher target gene expression than treatment with Codium alone. Most of all, GPR120 which is a receptor for polyunsaturated fatty acids, was expressed in higher amounts than with Codium alone.

Fig 2. Relative quantitation of immune gene expression (fold-change).

Expression of (A) iNOS, (B) IL-1β, (C) IL-6, (D) TNF-α, (E) IFN-γ, (F) COX-2, (G) GPR120, and (H) TLR-4. The letters a,b,c,d,e,f indicate a significant difference (p < 0.05) between the treatments.

3.3 Codium coupled with ARA enhanced immunity through the MAPK and NF-κB signaling pathways in RAW264.7 cells

The production of NF-κB and MAPK associated proteins were analyzed through western blotting to determine the contribution of Codium and ARA on the immune-enhancement in RAW264.7 cells. Treatment of Codium stimulated the phosphorylation of NF-κB p-65, compared to normal cells (Fig 3). Codium also increased the phosphorylation levels of ERK1/2 and p38 of the MAPK pathway. Interestingly, phosphorylation of NF-κB p-65, ERK1/2, and p38 were increased in RAW264.7 cells with the treatment of Codium coupled with ARA.

Fig 3. Co-operative effect of Codium and ARA on proteins associated with NF-κB and MAPK pathways.

(A) Expression of proteins by western blot and (B) relative band intensity of proteins.

4. Discussion

Macrophages play important roles in host defense, inflammation control [4], combating infection, and removing tumor cells in hosts [35]. Increased production of nitric oxide (NO) by macrophages protects host cells from foreign infection [14]. The nitrite-derived NO production also activates immune cells for the production of pro-inflammatory cytokines, such as TNF-α and IL-1β [36]. It was confirmed that the mixture of Codium (up to 4 μg/mL) and 0.5 μM ARA did not affect the growth of RAW264.7 cells in terms of cell proliferation, revealing that there was no synergistic or individual cytotoxicity among these two supplements against RAW264.7 cell division (Fig 1). Codium is known to stimulate one of the important immune regulatory biomarkers, NO production, in macrophage cells [30], however the action of Codium coupled with ARA has not been reported. The current study showed that the combination of Codium and ARA significantly enhanced the production of NO in a dose-dependent manner and also resulted in higher NO production than with treatment of Codium alone.

Cytokine expression is another critical factor for the investigation of immunity, inflammation and hematopoiesis [37]. Activated macrophages secrete cytokines such as TNF-α, IL-1β and IL-6 [3739] to defend against intracellular pathogens [40]. Normally, n-6 fatty acid plays an important role in pro-inflammatory processes by stimulating the production of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) [16, 17, 41]. The current results showed that most of the immune-associated genes were significantly enhanced with the supplementation of ARA, compared to treatment of Codium alone (Fig 3), suggesting that ARA helped Codium to increase the expression of immune-associated genes in RAW264.7 cells. Interestingly, the expression of TLR-4, an important polysaccharide receptor [42, 43], did not show significant difference between the treatment of Codium alone or in combination with ARA, whereas the expression of GPR120, the receptor for polyunsaturated fatty acids, was significantly increased [21, 44, 45]. These results indicated that the increased expression of immune-associated genes was affected by the incorporation of ARA.

Codium fragile, one of the popular algae, has been employed for oriental therapy medicine to treat diseases such as enterobiosis, dropsy, and dysuria [25]. Moreover, this algae was demonstrated to have immune-regulatory activities such as anti-inflammatory, and immune-stimulatory activities [20, 33]. In addition, ARA is capable of activating the GPR120 receptor, which is related to calcium mobilization and ERK [46] and p38 stimulation [47], thus leading to health-promoting effects [21, 48, 49]. To regulate the immune response in macrophage cells, NF-κB, and MAPK pathways are coordinated via the phosphorylation of the associated proteins in these pathways [50]. That is, NF-κB coordinates the expression of pro-inflammatory mediators and pro-inflammatory cytokines [51], and MAPK-related molecules play critical roles in regulating cell growth and differentiation, and controlling cellular responses to cytokines [52]. Therefore, our results demonstrated that the synergistic effect between Codium and ARA led to activation of NF-κB, p-65, and MAPK such as ERK1/2 and p38, and thus promoted the immune response in RAW 264.7 cells.

5. Conclusions

In conclusion, it was demonstrated that the coordination of Codium and ARA synergistically enhanced the expression and production of biomarkers such as production of NO and the expression of immune-associated genes, such as iNOS, IL-1β, TNF-α, IFN-γ, and COX-2 in RAW264.7 cells. Furthermore, these increases were triggered by activation of NF-κB p-65 and MAPK, including ERK1/2 and p38, thus regulating the immune responses for immune-enhancement. These results suggested that incorporating ARA into Codium would contribute to an improved immune response in RAW264.7 cells.


  1. 1. Ganapathy S. Long chain polyunsaturated fatty acids and immunity in infants. Indian Pediatr. 2009;46(9): 785–790. pmid:19812424
  2. 2. Calder PC. Omega-3 fatty acids and inflammatory processes. Nutrients. 2010;2(3): 355–374. pmid:22254027
  3. 3. Gordon S, Martinez FO. Alternative activation of macrophages: Mechanism and functions. Immunity. 2010;32(5): 593–604. pmid:20510870
  4. 4. Mosser DM, Edwards JP. Exploring the full spectrum of macrophage activation. Nat. Rev. Immunol. 2008;8(12): 958–969. pmid:19029990
  5. 5. Martinez FO, Sica A, Mantovani A, Locati M. Macrophage activation and polarization. Front Biosci. 2008;13: 453–461. pmid:17981560
  6. 6. Xue J, Schmidt Susanne V, Sander J, Draffehn A, Krebs W, Quester I, et al. Transcriptome-based network analysis reveals a spectrum model of human macrophage activation. Immunity. 2014;40(2): 274–288. pmid:24530056
  7. 7. Mantovani A, Sozzani S, Locati M, Allavena P, Sica A. Macrophage polarization: tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes. Trends Immunol. 2002;23(11): 549–555. pmid:12401408
  8. 8. Lv R, Bao Q, Li Y. Regulation of M1type and M2type macrophage polarization in RAW264.7 cells by Galectin9. Mol Med Rep. 2017;16(6): 9111–9119. pmid:28990062
  9. 9. Lee JH, Moon SH, Kim HS, Park E, Ahn DU, Paik HD. Immune-enhancing activity of phosvitin by stimulating the production of pro-inflammatory mediator. Poult. Sci. 2017;96(11): 3872–3878. pmid:29050435
  10. 10. Seo JY, Shin IS, Lee SM. Effect of various protein sources in formulated diets on the growth and body composition of juvenile sea cucumber Apostichopus japonicus (Selenka). Aquacult. Res. 2010;42(4): 623–627.
  11. 11. Lee SG, Kim MM. Anti-inflammatory effect of scopoletin in RAW264.7 macrophages. J. Life Sci. 2015;25(12): 1377–1383.
  12. 12. van Horssen R, Ten Hagen TL, Eggermont AM. TNF-alpha in cancer treatment: molecular insights, antitumor effects, and clinical utility. Oncologist. 2006;11(4): 397–408. pmid:16614236
  13. 13. Xia Y, Zhai Q. IL-1beta enhances the antibacterial activity of astrocytes by activation of NF-kappaB. Glia. 2010;58(2): 244–252. pmid:19672968
  14. 14. Rahat MA, Hemmerlein B. Macrophage-tumor cell interactions regulate the function of nitric oxide. Front Physiol. 2013;4: 144. pmid:23785333
  15. 15. Jablonski KA, Amici SA, Webb LM, Ruiz-Rosado Jde D, Popovich PG, Partida-Sanchez S, et al. Novel markers to delineate murine M1 and M2 macrophages. PLoS One. 2015;10(12): e0145342. pmid:26699615
  16. 16. Bell JG, Castell JD, Tocher DR, Macdonald FM, Sargent JR. Effects of different dietary arachidonic acid: docosahexaenoic acid ratios on phospholipid fatty acid compositions and prostaglandin production in juvenile turbot (Scophthalmus maximus). Fish Physiol. Biochem. 1995;14(2): 139–151. pmid:24197361
  17. 17. Dantagnan P, Gonzalez K, Hevia M, Betancor MB, Hernández AJ, Borquez A, et al. Effect of the arachidonic acid/vitamin E interaction on the immune response of juvenile Atlantic salmon (Salmo salar) challenged against Piscirickettsia salmonis. Aquac. Nutr. 2017;23(4): 710–720.
  18. 18. Calder PC. Dietary modification of inflammation with lipids. Proc. Nutr. Soc. 2002;61(3): 345–358. pmid:12296294
  19. 19. Lee H, Park WJ. Unsaturated fatty acids, desaturases, and human health. J. Med. Food. 2014;17(2): 189–197. pmid:24460221
  20. 20. Mullen A, Loscher CE, Roche HM. Anti-inflammatory effects of EPA and DHA are dependent upon time and dose-response elements associated with LPS stimulation in THP-1-derived macrophages. J. Nutr. Biochem. 2010;21(5): 444–450. pmid:19427777
  21. 21. Oh DY, Talukdar S, Bae EJ, Imamura T, Morinaga H, Fan W, et al. GPR120 is an omega-3 fatty acid receptor mediating potent anti-inflammatory and insulin-sensitizing effects. Cell. 2010;142(5): 687–698. pmid:20813258
  22. 22. Haeggström JZ, Funk CD. Lipoxygenase and leukotriene pathways: biochemistry, biology, and roles in disease. Chem. Rev. 2011;111(10): 5866–5898. pmid:21936577
  23. 23. Ricciotti E, FitzGerald GA. Prostaglandins and inflammation. Arterioscler. Thromb. Vasc. Biol. 2011;31(5): 986–1000. pmid:21508345
  24. 24. Kawahara K, Hohjoh H, Inazumi T, Tsuchiya S, Sugimoto Y. Prostaglandin E2-induced inflammation: relevance of prostaglandin E receptors. Biochim. Biophys. Acta, Mol. Cell. Biol. Lipids. 2015;1851(4): 414–421. pmid:25038274
  25. 25. Verbruggen H, Leliaert F, Maggs CA, Shimada S, Schils T, Provan J, et al. Species boundaries and phylogenetic relationships within the green algal genus Codium (Bryopsidales) based on plastid DNA sequences. Mol. Phylogenet Evol. 2007;44(1): 240–254. pmid:17346993
  26. 26. Ciancia M, Quintana I, Vizcarguenaga MI, Kasulin L, de Dios A, Estevez JM, et al. Polysaccharides from the green seaweeds Codium fragile and C. vermilara with controversial effects on hemostasis. Int. J. Biol. Macromol. 2007;41(5): 641–649. pmid:17920674
  27. 27. Ohta Y, Lee JB, Hayashi K, Hayashi T. Isolation of sulfated galactan from Codium fragile and its antiviral effect. Biol. Pharm. Bull. 2009;32(5): 892–898. pmid:19420760
  28. 28. Kang CH, Choi YH, Park SY, Kim GY. Anti-inflammatory effects of methanol extract of Codium fragile in lipopolysaccharide-stimulated RAW 264.7 cells. J. Med. Food. 2012;15(1): 44–50. pmid:22082064
  29. 29. Lee JB, Ohta Y, Hayashi K, Hayashi T. Immunostimulating effects of a sulfated galactan from Codium fragile. Carbohydr. Res. 2010;345(10): 1452–1454. pmid:20362278
  30. 30. Tabarsa M, Karnjanapratum S, Cho M, Kim JK, You S. Molecular characteristics and biological activities of anionic macromolecules from Codium fragile. Int. J. Biol. Macromol. 2013;59: 1–12. pmid:23597705
  31. 31. Sevag M, Lackman DB, Smolens J. The isolation of the components of streptococcal nucleoproteins in serologically active form. J. Biol. Chem. 1938;124(2): 425–436.
  32. 32. Cao RA, Lee Y, You SG. Water soluble sulfated-fucans with immune-enhancing properties from Ecklonia cava. Int. J. Biol. Macromol. 2014;67: 303–311. pmid:24661888
  33. 33. Kim JK, Cho ML, Karnjanapratum S, Shin IS, You SG. In vitro and in vivo immunomodulatory activity of sulfated polysaccharides from Enteromorpha prolifera. Int. J. Biol. Macromol. 2011;49(5): 1051–1058. pmid:21907732
  34. 34. Narayanan BA, Narayanan NK, Simi B, Reddy BS. Modulation of inducible nitric oxide synthase and related proinflammatory genes by the omega-3 fatty acid docosahexaenoic acid in human colon cancer cells. Cancer Res. 2003;63(5): 972–979. pmid:12615711
  35. 35. Manosroi A, Saraphanchotiwitthaya A, Manosroi J. Immunomodulatory activities of Clausena excavata Burm. f. wood extracts. J. Ethnopharmacol. 2003;89(1): 155–160. pmid:14522448
  36. 36. Mocca B, Wang W. Bacterium-generated nitric oxide hijacks host tumor necrosis factor alpha signaling and modulates the host cell cycle in vitro. J. Bacteriol. 2012;194(15): 4059–4068. pmid:22636782
  37. 37. Li Y, Meng T, Hao N, Tao H, Zou S, Li M, et al. Immune regulation mechanism of Astragaloside IV on RAW264.7 cells through activating the NF-κB/MAPK signaling pathway. Int. J. Immunopharmacol. 2017;49: 38–49. pmid:28550733
  38. 38. Chow JC, Young DW, Golenbock DT, Christ WJ, Gusovsky F. Toll-like receptor-4 mediates lipopolysaccharide-induced signal transduction. J. Biol. Chem. 1999;274(16): 10689–10692. pmid:10196138
  39. 39. Uemura E, Yoshioka Y, Hirai T, Handa T, Nagano K, Higashisaka K, et al. Relationship between size and surface modification of silica particles and enhancement and suppression of inflammatory cytokine production by lipopolysaccharide- or peptidoglycan-stimulated RAW264.7 macrophages. J. Nanopart. Res. 2016;18(6): 165–172.
  40. 40. Apetoh L, Ghiringhelli F, Tesniere A, Obeid M, Ortiz C, Criollo A, et al. Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy. Nat. Med. 2007;13(9): 1050–1059. pmid:17704786
  41. 41. Hwang D. Essential fatty acids and immune response. FASEB J. 1989;3(9): 2052–2061. pmid:2501132
  42. 42. Kim HS, Shin BR, Lee HK, Park YS, Liu Q, Kim SY, et al. Dendritic cell activation by polysaccharide isolated from Angelica dahurica. Food Chem. Toxicol. 2013;55: 241–247. pmid:23246459
  43. 43. Zhang X, Qi C, Guo Y, Zhou W, Zhang Y. Toll-like receptor 4-related immunostimulatory polysaccharides: Primary structure, activity relationships, and possible interaction models. Carbohydr. Polym. 2016;149: 186–206. pmid:27261743
  44. 44. Dragano NRV, Solon C, Ramalho AF, de Moura RF, Razolli DS, Christiansen E, et al. Polyunsaturated fatty acid receptors, GPR40 and GPR120, are expressed in the hypothalamus and control energy homeostasis and inflammation. J. Neuroinflamm. 2017;14(1): 91–106. pmid:28446241
  45. 45. Im DS. Functions of omega-3 fatty acids and FFA4 (GPR120) in macrophages. Eur. J. Pharmacol. 2016;785: 36–43. pmid:25987421
  46. 46. Villegas-Comonfort S, Takei Y, Tsujimoto G, Hirasawa A, García-Sáinz JA. Effects of arachidonic acid on FFA4 receptor: Signaling, phosphorylation and internalization. Prostaglandins Leukot. Essent. Fatty Acids. 2017;117: 1–10. pmid:28237082
  47. 47. Haller I, Hausott B, Tomaselli B, Keller C, Klimaschewski L, Gerner P, et al. Neurotoxicity of lidocaine involves specific activation of the p38 mitogen-activated protein kinase, but not extracellular signal-regulated or c-jun N-terminal kinases, and is mediated by arachidonic acid metabolites. Anesthesiology. 2006;105(5): 1024–1033. pmid:17065898
  48. 48. Hirasawa A, Tsumaya K, Awaji T, Katsuma S, Adachi T, Yamada M, et al. Free fatty acids regulate gut incretin glucagon-like peptide-1 secretion through GPR120. Nat. Med. 2005;11(1): 90–94. pmid:15619630
  49. 49. Ichimura A, Hirasawa A, Poulain-Godefroy O, Bonnefond A, Hara T, Yengo L, et al. Dysfunction of lipid sensor GPR120 leads to obesity in both mouse and human. Nature. 2012;483(7389): 350–354. pmid:22343897
  50. 50. Baeuerle PA, Baltimore D. NF-kappa B: ten years after. Cell. 1996;87(1): 13–20. pmid:8858144
  51. 51. Tak PP, Firestein GS. NF-kappaB: a key role in inflammatory diseases. J. Clin. Invest. 2001;107(1): 7–11. pmid:11134171
  52. 52. Kim JB, Han AR, Park EY, Kim JY, Cho W, Lee J, et al. Inhibition of LPS-induced iNOS, COX-2 and cytokines expression by poncirin through the NF-kappaB inactivation in RAW 264.7 macrophage cells. Biol. Pharm. Bull. 2007;30(12): 2345–2351. pmid:18057724