J Bacteriol Virol.  2019 Dec;49(4):153-161. 10.4167/jbv.2019.49.4.153.

Aralia cordata Extract Activates NF-κB and MAPK Signaling Pathways and Induces Pro-inflammatory Changes in RAW264.7 Macrophages

• Seo DW
• Cho YI
• Yi YJ
• Lee SM

• KMID: 2468022
• DOI: http://doi.org/10.4167/jbv.2019.49.4.153

Abstract

Macrophages play essential roles in innate immune responses by producing various immune mediators. Therefore, modulating macrophage function is an attractive strategy to treat immune disorders. Aralia cordata var. continentalis (AC), known as "Dokwhal" in Korea, possesses various biological and medicinal functions, including immunomodulation. The present study investigated the effect of the hot water extract of AC (HAC) on RAW264.7 murine macrophages. When these cells were treated with HAC, nitric oxide production and inducible nitric oxide synthase expression was induced dose-dependently. In addition, HAC treatment triggered the secretion of innate immune cytokines, such as TNF-α and IL-6. Phagocytosis, measured by FITC-dextran internalization showed that HAC stimulated the phagocytic activity of macrophages. Furthermore, HAC promoted the production of reactive oxygen species in RAW264.7 cells, determined by CM-H2DCFDA. In addition, the immunoblot analysis of intracellular signaling proteins revealed that NF-kB and MAPK signaling pathways, which are important signaling mediators of inflammation, are upregulated by HAC. In conclusion, these findings suggested that HAC can stimulate macrophage activity, and NF-kB and MAPK signaling pathways might be involved in the immunostimulatory effects of HAC.

Keyword

Aralia cordata; RAW264.7; Immunostimulatory effect; NF-κB; MAPK

MeSH Terms

Aralia*
Cytokines
Immune System Diseases
Immunity, Innate
Immunomodulation
Inflammation Mediators
Interleukin-6
Intracellular Signaling Peptides and Proteins
Korea
Macrophages*
NF-kappa B
Nitric Oxide
Nitric Oxide Synthase Type II
Phagocytosis
Reactive Oxygen Species
Water
Cytokines
Inflammation Mediators
Interleukin-6
Intracellular Signaling Peptides and Proteins
NF-kappa B
Nitric Oxide
Nitric Oxide Synthase Type II
Reactive Oxygen Species
Water

Figure

• 

  Figure 1 Effect of HAC on cell viability, nitrite accumulation, and iNOS expression in RAW264.7 cells. (A) Cells were treated with different concentrations of HAC for 24 h, and their viability was assessed by the MTT assay. The results of independent experiments were averaged and are expressed as the percentage of cell viability relative to the untreated control group. (B) Nitric oxide production was measured in the culture medium by the Griess reaction, and NaNO2 was used as a standard. (C) iNOS expression was determined in RAW264.7 cells treated with LPS or HAC by immunoblotting. Data are presented as the mean ± SEM of three independent experiments. **p<0.01, ***p<0.001 indicate significant difference from the untreated control group by the Student's t-test.

• 

  Figure 2 Effects of HAC on cell morphology, cytokine and ROS production, and phagocytosis. RAW264.7 cells were plated and then treated with the indicated concentration of HAC for 24 h. (A) Cell morphology was observed by light microscopy. Images were obtained with a 100×objective. (B) IL-6 and TNF-α levels were determined using an ELISA kit, as described in the text. (C) After incubation, the cells were washed twice with PBS and labeled with 20 µM CM-H2DCFDA for 30 min. The intracellular ROS level was analyzed by flow cytometry. (D) The cells were incubated with DMEM containing 1 mg/ml FITC-dextran, 2% FBS, and the indicated concentration of HAC for 6 h. After incubation, phagocytosis activity was measured by flow cytometry. The results are expressed as mean ± SEM of three independent experiments. *p<0.05, *p<0.01, ***p<0.001 indicate significant difference from the untreated control group by the Student's t-test.

• 

  Figure 3 Effects of HAC on NF-κB and MAPK pathways. RAW264.7 cells were incubated at a density of 1×106 cells/well in a 12-well plate and stimulated with 200 µg/ml of HAC or 1 µg/ml of LPS for different times. Total cell lysates were collected and hybridized with a specific antibody detecting the phosphorylated form or total amount of NF-κB and MAPK. The results are representative of three independent experiments.

Reference

1. Park CW. The genera of vascular plants of Korea. Seoul: Academic Publishing;2007. p. 1482.

2. Perry LM, Metzger J. Medicinal plants of east and southeast Asia: attributed properties and uses MIT press. 1980.

3. Hwang YP, Choi JH, Jeong HG. Protective effect of the Aralia continentalis root extract against carbon tetrachlorideinduced hepatotoxicity in mice. Food Chem Toxicol. 2009; 47:75–81.

4. Park HJ, Hong MS, Lee JS, Leem KH, Kim CJ, Kim JW, et al. Effects of Aralia continentalis on hyperalgesia with peripheral inflammation. Phytother Res. 2005; 19:511–513.

5. Han GJ, Shin DS, Jang MS. A study of the nutritional composition of Aralia continentalis Kitagawa and Aralia continentalis Kitagawa leaf. Korean J Food Sci Technol. 2008; 40:680–685.

6. Seo DW, Cho YI, Gu S, Kim DH, Park JH, Yi YJ, et al. A hot water extract of Aralia cordata activates bone marrow-derived macrophages via a myeloid differentiation protein 88-dependent pathway and protects mice from bacterial infection. Microbiol Immunol. 2016; 60:343–355.

7. Janeway Jr CA, Medzhitov R. Innate immune recognition. Annu Rev Immunol. 2002; 20:197–216.

8. Fujiwara N, Kobayashi K. Macrophages in inflammation. Curr Drug Targets Inflamm Allergy. 2005; 4:281–286.

9. Bogdan C. Nitric oxide and the immune response. Nat Immunol. 2001; 2:907–916.

10. Clancy RM, Amin AR, Abramson SB. The role of nitric oxide in inflammation and immunity. Arthritis Rheum. 1998; 41:1141–1151.

11. Chan J, Tanaka K, Carroll D, Flynn J, Bloom BR. Effects of nitric oxide synthase inhibitors on murine infection with Mycobacterium tuberculosis. Infect Immun. 1995; 63:736–740.

12. Ambs S, Hussain SP, Harris CC. Interactive effects of nitric oxide and the p53 tumor suppressor gene in carcinogenesis and tumor progression. FASEB J. 1997; 11:443–448.

13. Mantovani A, Sica A, Sozzani S, Allavena P, Vecchi A, Locati M. The chemokine system in diverse forms of macrophage activation and polarization. Trends Immunol. 2004; 25:677–686.

14. Lee J, Choi JW, Sohng JK, Pandey RP, Park YI. The immunostimulating activity of quercetin 3-O-xyloside in murine macrophages via activation of the ASK1/MAPK/NF-kappaB signaling pathway. Int Immunopharmacol. 2016; 31:88–97.

15. Li J, Qian W, Xu Y, Chen G, Wang G, Nie S, et al. Activation of RAW 264.7 cells by a polysaccharide isolated from Antarctic bacterium Pseudoaltermonas sp. S-5. Carbohydr Polym. 2015; 130:97–103.

16. Catchpole B, Hamblin AS, Staines NA. Autologous mixed lymphocyte responses in experimentally-induced arthritis of the Lewis rat. Autoimmunity. 2002; 35:111–117.

17. Brito LA, Singh M. Acceptable levels of endotoxin in vaccine formulations during preclinical research. J Pharm Sci. 2011; 100:34–37.

18. Foresti R, Clark JE, Green CJ, Motterlini R. Thiol compounds interact with nitric oxide in regulating heme oxygenase-1 induction in endothelial cells. Involvement of superoxide and peroxynitrite anions. J Biol Chem. 1997; 272:18411–18417.

19. Aderem A, Underhill DM. Mechanisms of phagocytosis in macrophages. Annu Rev Immunol. 1999; 17:593–623.

20. Kim JS, Kang SS. Saponins from the aerial parts of Aralia continentalis. Nat Prod Sci. 1998; 4:45–50.

21. Adachi Y, Okazaki M, Ohno N, Yadomae T. Enhancement of cytokine production by macrophages stimulated with (1-->3)-beta-D-glucan, grifolan (GRN), isolated from Grifola frondosa. Biol Pharm Bull. 1994; 17:1554–1560.

22. Wang ZM, Peng X, Lee KLD, Tang JC, Cheung PCC, Wu JY. Structural characterisation and immunomodulatory property of an acidic polysaccharide from mycelial culture of Cordyceps sinensis fungus Cs-HK1. Food Chem. 2011; 125:637–643.

23. Farrell AJ, Blake DR. Nitric oxide. Ann Rheum Dis. 1996; 55:7–20.

24. Gorbunov N, Esposito E. Nitric oxide as a mediator of inflammation. Int J Immunopathol Pharmaool. 1993; 6:67–75.

25. Yu Q, Nie SP, Li WJ, Zheng WY, Yin PF, Gong DM, et al. Macrophage immunomodulatory activity of a purified polysaccharide isolated from Ganoderma atrum. Phytother Res. 2013; 27:186–191.

26. Oppenheim JJ. Cytokines: past, present, and future. Int J Hematol. 2001; 74:3–8.

27. Han EH, Choi JH, Hwang YP, Park HJ, Choi CY, Chung YC, et al. Immunostimulatory activity of aqueous extract isolated from Prunella vulgaris. Food Chem Toxicol. 2009; 47:62–69.

28. Yue GG, Chan BC, Hon PM, Kennelly EJ, Yeung SK, Cassileth BR, et al. Immunostimulatory activities of polysaccharide extract isolated from Curcuma longa. Int J Biol Macromol. 2010; 47:342–347.

29. Baeuerle PA, Henkel T. Function and activation of NF-kappaB in the immune system. Annu Rev Immunol. 1994; 12:141–179.

30. Lee JY, Kim JY, Lee YG, Rhee MH, Hong EK, Cho JY. Molecular mechanism of macrophage activation by Exopolysaccharides from liquid culture of Lentinus edodes. J Microbiol Biotechnol. 2008; 18:355–364.

31. Xie QW, Kashiwabara Y, Nathan C. Role of transcription factor NF-kappa B/Rel in induction of nitric oxide synthase. J Biol Chem. 1994; 269:4705–4708.

32. Sun H, Zhang J, Chen F, Chen X, Zhou Z, Wang H. Activation of RAW264. 7 macrophages by the polysaccharide from the roots of Actinidia eriantha and its molecular mechanisms. Carbohydr Polym. 2015; 121:388–402.

33. Kim YS, Kim EK, Nawarathna WPAS, Dong X, Shin WB, Park JS, et al. Immune-Stimulatory Effects of Althaea rosea Flower Extracts through the MAPK Signaling Pathway in RAW264.7 Cells. Molecules. 2017; 22.

34. Bai Y, Jiang Y, Liu T, Li F, Zhang J, Luo Y, et al. Xinjiang herbal tea exerts immunomodulatory activity via TLR2/4-mediated MAPK signaling pathways in RAW264.7 cells and prevents cyclophosphamide-induced immunosuppression in mice. J Ethnopharmacol. 2019; 228:179–187.

35. Johnson GL, Lapadat R. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science. 2002; 298:1911–1912.

36. Rao KM. MAP kinase activation in macrophages. J Leukoc Biol. 2001; 69:3–10.