Korean Journal of Food Science and Technology (한국식품과학회지)

Korean Society of Food Science and Technology (한국식품과학회)
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Polysaccharide isolated from fermented barley extract activates macrophages via the MAPK and NF-κB pathways

보리발효추출물로부터 분리한 다당의 대식세포 활성화 및 신호 전달

  • Kim, Han Wool (Department of Food Science and Biotechnology, Kyonggi University) ;
  • Jee, Hee Sook (Food and Biotechnology Research Center, ILHWA Co., Ltd.) ;
  • Shin, Kwang-Soon (Department of Food Science and Biotechnology, Kyonggi University)
  • 김한울 (경기대학교 식품생물공학과) ;
  • 지희숙 ((주)일화 식품바이오연구센터) ;
  • 신광순 (경기대학교 식품생물공학과)
  • Received : 2018.09.20
  • Accepted : 2018.10.09
  • Published : 2018.10.31
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Abstract

Barley has nutritional benefits due to its high dietary fiber content; therefore, the intake of whole barley grains is recommended. However, barley is often consumed in the fermented form because of the improved texture and digestibility. The present study was designed to elucidate the intracellular signaling pathway for macrophage activation by the polysaccharide BF-CP from fermented barley. BF-CP is a neutral polysaccharide, composed of neutral sugars, including glucose (70.7%), xylose (11.4%), and arabinose (9.0%). BF-CP exhibited macrophage-stimulatory activity by inducing the production of interleukin (IL)-6, tumor necrosis factor $(TNF)-{\alpha}$, and nitric oxide in RAW 264.7 macrophages. Further, BF-CP treatment strongly increased the IL-6 and $TNF-{\alpha}$ gene expression in a concentration-dependent manner. Signal transduction experiments using immunoblotting showed that BF-CP phosphorylated mitogen-activated protein kinases (MAPKs), such as c-Jun N-terminal kinase, extracellular signal-regulated kinase, and p38, and nuclear factor $(NF)-{\kappa}B$, in RAW 264.7 cells in a concentration-dependent manner. These results suggest that BF-CP activates the macrophages via MAPK and $NF-{\kappa}B$ pathways, and also induces an increase in the production of cytokines.

보리는 높은 비율로 식이섬유를 함유하고 있어, 일반적으로 영양학적으로는 전곡 형태로의 섭취가 추천되고 있지만, 조직감과 소화율을 고려하여 발효물 형태의 소비가 바람직한 것으로 판단되고 있다. 본 연구는 효소당화 후, 효모 및 유산균 발효를 거쳐 조제한 보리발효물로부터 조다당 BF-CP를 분리하고, 대식세포에 대한 면역증강 효과 및 세포 내 신호전달을 규명하여 기능성 소재로의 이용방안을 모색하기 위해 계획되었다. BF-CP 획분의 일반화학적 특성을 분석한 결과 70.7% 글루코스 11.4% 자일로스 및 9.0% 아라비노스를 포함하여 91.1%의 중성당으로 이루어진 중성다당이었다. BF-CP는 RAW 264.7 대식세포주에서 농도의존적으로 IL-6, $TNF-{\alpha}$와 같은 사이토카인 및 NO의 생산능을 유도하는 등 높은 대식세포 활성능을 나타냈다. 또한 qPCR 분석을 통해, BF-CP 획분을 대식세포주에 처리하였을 때, 처리 농도에 비례하여 IL-6, $TNF-{\alpha}$ 및 iNOS의 mRNA 유전자 발현을 증가시킴을 확인할 수 있었다. 한편 Western blot을 활용한 신호전달 단백질 추적실험에서 BF-CP 획분을 대식세포주에 처리하였을 때, JNK, ERK 및 p38과 같은 MAPK 경로와 $NF-{\kappa}B$ 경로의 관련 단백질을 인산화시킴이 확인되었으며 그 활성은 BF-CP 농도에 의존적이었다. 이상의 결과로부터 보리발효물 유래 다당 BF-CP는 MAPK와 $NF-{\kappa}B$ 경로를 통해 대식세포를 활성화시키며, 이를 통하여 NO, IL-6 및 $TNF-{\alpha}$와 같은 면역활성화 관련 물질의 생산을 높은 비율로 유도시킨다는 것을 최종 확인할 수 있었다.

Keywords

References

  1. Aderem A, Underhill DM. Mechanisms of phagocytosis in macrophages. Annu. Rev. Immunol. 17: 593-623 (1999) https://doi.org/10.1146/annurev.immunol.17.1.593
  2. Ando I, Tsukumo Y, Wakabayashi T, Akashi S, Miyake K, Kataoka T, Nagai K. Safflower polysaccharides activate the transcription factor NF-${\kappa}B$ via Toll-like receptor 4 and induce cytokine production by macrophages. Int. Immunopharmacol. 2: 1155-1162 (2002) https://doi.org/10.1016/S1567-5769(02)00076-0
  3. Belardelli F. Role of interferons and other cytokines in the regulation of the immune response. Apmis 103: 161-179 (1995) https://doi.org/10.1111/j.1699-0463.1995.tb01092.x
  4. Beutler B. Endotoxin, toll-like receptor 4, and the afferent limb of innate immunity. Curr. Opin. Microbiol. 3: 23-28 (2000) https://doi.org/10.1016/S1369-5274(99)00046-6
  5. Beutler B. Innate immunity: an overview. Mol. Immunol.40: 845-859 (2004) https://doi.org/10.1016/j.molimm.2003.10.005
  6. Blumenkrantz N, Asboe-Hansen G. New method for quantitative determination of uronic acids. Anal. Biochem. 54: 484-489 (1973) https://doi.org/10.1016/0003-2697(73)90377-1
  7. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254 (1976) https://doi.org/10.1016/0003-2697(76)90527-3
  8. Cargnello M, Roux PP. Activation and function of the MAPKs and their substrates, the MAPK-activated protein kinases. Microbiol. Mol. Biol. Rev. 75: 50-83 (2011) https://doi.org/10.1128/MMBR.00031-10
  9. Cebra JJ, Shroff KE. Peyer's patches as inductive sites for IgA commitment. pp. 151-158. In: Handbook of Mucosal Immunology. Academic press, New York, NY, USA. (1994)
  10. Chihara G. Recent progress in immunopharmacology and therapeutic effects of polysaccharides. Dev. Biol. Stand. 77: 191-197 (1992)
  11. Cho CW, Han CJ, Rhee YK, Lee YC, Shin KS, Hong HD. Immunostimulatory effects of polysaccharides isolated from Makgeolli (traditional Korean rice wine). Molecules 19: 5266-5277 (2014) https://doi.org/10.3390/molecules19045266
  12. Di Renzo L, Yefeno E, Klein E. The function of human NK cells is enhanced by ${\beta}$-glucan, a ligand of CR3 (CD11b/CD18). Eur. J. Immunol. 21: 1755-1758 (1991) https://doi.org/10.1002/eji.1830210726
  13. Dubois, M, Gilles KA, Hamilton JK, Rebers PT, Smith F. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28: 350-356 (1956) https://doi.org/10.1021/ac60111a017
  14. Forman HJ, Torres M. Reactive oxygen species and cell signaling: respiratory burst in macrophage signaling. Am. J. Respir. Crit. Care Med. 166 (supplement_1): S4-S8 (2002) https://doi.org/10.1164/rccm.2206007
  15. Gordon S. Pattern recognition receptors: Doubling up for the innate immune response. Cell 111: 927-930 (2002) https://doi.org/10.1016/S0092-8674(02)01201-1
  16. Hamilton TA, Adams DO. Molecular mechanisms of signal transduction in macrophages. Immunol. Today 8: 151-158 (1987) https://doi.org/10.1016/0167-5699(87)90145-9
  17. Han SB, Yoon YD, Ahn HJ, Lee HS, Lee CW, Yoon WK, Park SK, Kim HM. Toll-like receptor-mediated activation of B cells and macrophages by polysaccharide isolated from cell culture of Acanthopanax senticosus. Int. Immunopharmacol. 3: 1301-1312 (2003) https://doi.org/10.1016/S1567-5769(03)00118-8
  18. Honda S, Akao E, Suzuki S, Okuda M, Kakehi K, Nakamuraa J. High-performance liquid chromatography of reducing carbohydrates as strongly ultraviolet-absorbing and electrochemically sensitive 1-phenyl-3-methyl5-pyrazolone derivatives. Anal. Biochem. 180: 351-357 (1989) https://doi.org/10.1016/0003-2697(89)90444-2
  19. Hwang YC, Shin KS. Characterization of immuno-stimulating polysaccharides isolated from Korean persimmon vinegar. Korean J. Food Sci. Technol. 40: 220-227 (2008)
  20. Iwasaki A, Medzhitov R. Regulation of adaptive immunity by the innate immune system. Science 327: 291-295 (2010) https://doi.org/10.1126/science.1183021
  21. Izydorczyk MS, Dexter JE. Barley ${\beta}$-glucans and arabinoxylans: Molecular structure, physicochemical properties, and uses in food products-a Review. Food Res. Int. 41: 850-868 (2008) https://doi.org/10.1016/j.foodres.2008.04.001
  22. James SP, Zeitz M. Human gastrointestinal Mucosal T cells. pp. 275-285. In: Handbook of Mucosal Immunology. Academic press, New York, NY, USA. (1994)
  23. Karkhanis YD, Zeltner JY, Jackson JJ, Carlo DJ. A new and improved microassay to determine 2-keto-3-deoxyoctonate in lipopolysaccharide of Gram-negative bacteria. Anal. Biochem. 85: 595-601 (1978) https://doi.org/10.1016/0003-2697(78)90260-9
  24. Kawai T, Akira S. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat. Immunol. 11: 373 (2010) https://doi.org/10.1038/ni.1863
  25. Kim SR, Seog HM, Choi HD, Park YK. Cholesterol lowering effects in rat liver fed barley and ${\beta}$-glucan enriched barley fraction with cholesterol. Korean J. Food Sci. Technol. 34: 319-324 (2002)
  26. Kroncke KD, Fehsel K, Kolb-Bachofen V. Inducible nitric oxide synthase in human diseases. Clin. Exp. Immunol. 113: 147-156 (1998) https://doi.org/10.1046/j.1365-2249.1998.00648.x
  27. Kuno T, Hirose Y, Hata K, Kato K, Qiang SH, Kitaori N. Preventive effect of fermented brown rice and rice bran on N-nitrosomethylbenzylamine-induced esophageal tumorigenesis in rats. Int. J. Oncol. 25: 1809-1815 (2004)
  28. Lasek W, Feleszko W, Golab J, Stoklosa T, Marczak M, Dabrowska A, Malejczyk M, Jakobisiak M. Antitumor effects of the combination immunotherapy with interleukin-12 and tumor necrosis factor alpha in mice. Cancer Immunol. Immunother. 45: 100-108 (1997) https://doi.org/10.1007/s002620050408
  29. Lee EH, Park HR, Shin MS, Cho SY, Choi HJ, Shin KS. Antitumor metastasis activity of pectic polysaccharide purified from the peels of Korean citrus hallabong. Carbohydr. Polym. 111: 72-79 (2014) https://doi.org/10.1016/j.carbpol.2014.04.073
  30. Lee DY, Shin MS, Shin KS. Characterization of macrophage-activating polysaccharide isolated from fermented brown rice. J. Med. Food 19: 1147-1154 (2016) https://doi.org/10.1089/jmf.2016.3742
  31. Lee CH, Youn Y, Song GS, Kim YS. Immunostimulatory effects of traditional doenjang. J. Korean Soc. Food Sci. Nutr. 40: 1227-1234 (2011) https://doi.org/10.3746/jkfn.2011.40.9.1227
  32. Lewis TS, Paul SS, Natalie GA. Signal transduction through MAP kinase cascades. Adv. Cancer Res. 74: 49-114 (1998)
  33. Liacini A, Sylvester J, Li WQ, Zafarullah M. Inhibition of interleukin-1-stimulated MAP kinases, activating protein-1 (AP-1) and nuclear factor kappa B (NF-${\kappa}B$) transcription factors down-regulates matrix metalloproteinase gene expression in articular chondrocytes. Matrix Biol. 21: 251-262 (2002) https://doi.org/10.1016/S0945-053X(02)00007-0
  34. MacMicking J, Xie QW, Nathan C. Nitric oxide and macrophage function. Annu. Rev. Immunol. 15: 323-350 (1997) https://doi.org/10.1146/annurev.immunol.15.1.323
  35. Martensson O, Maite DC, Ana I, Olle H. Effects of fermented, ropy, non-dairy, oat-based products on serum lipids and the faecal excretion of cholesterol and short chain fatty acids in germfree and conventional rats. Nutr. Res. 22: 1461-1473 (2002) https://doi.org/10.1016/S0271-5317(02)00474-8
  36. Oh HJ, Lee SR. Physiological function in vitro of ${\beta}$-glucan isolated from barley. Korean J. Food Sci. Technol. 28: 689-695 (1996)
  37. Park KY. Increased health functionality of fermented foods. Food Ind. Nutr. 17: 1-8 (2012)
  38. Park HM, Hong JH. Immune-Enhancing Effects of Polysaccharides Isolated from Phellinus linteus Mycelium on Mori ramulus. J. Korean Soc. Food Sci. Nutr. 46: 26-33 (2017) https://doi.org/10.3746/jkfn.2017.46.1.026
  39. Park HR, Lee MS, Jo SY, Won HJ, Lee HS, Lee H, Shin KS. Immuno-stimulating activities of polysaccharides isolated from commercial soy sauce and traditional Korean soy sauce. Korean J. Food Sci. Technol. 44: 228-234 (2012) https://doi.org/10.9721/KJFST.2012.44.2.228
  40. Park JY, Shin MS, Kim SN, Kim HY, Kim KH, Shin KS, Kang KS. Polysaccharides from Korean Citrus hallabong peels inhibit angiogenesis and breast cancer cell migration. Int. J. Biol. Macromol. 85: 522-529 (2016) https://doi.org/10.1016/j.ijbiomac.2016.01.015
  41. Paulsen B. Plant polysaccharides with immunostimulatory activities. Curr. Org. Chem. 5: 939-950 (2001) https://doi.org/10.2174/1385272013374987
  42. Pennini ME, Perkins DJ, Salazar AM, Lipsky M, Vogel SN. Complete dependence on IRAK4 kinase activity in TLR2, but not TLR4, signaling pathways underlies decreased cytokine production and increased susceptibility to Streptococcus pneumoniae infection in IRAK4 kinase-inactive mice. J. Immunol. 1201644 (2012)
  43. Rice PJ, Kelley JL, Kogan G, Ensley HE, Kalbfleisch JH, Browder IW, Williams DL. Human monocyte scavenger receptors are pattern recognition receptors for ($1{\rightarrow}3$)-${\beta}$-D-glucans. J. Leukocyte Biol. 72: 140-146 (2002)
  44. Roubroeks JP, Skjak-Braek G, Ryan L, Christensen BE. Molecular Weight Dependency on the Production of the TNF Stimulated by Fractions of rye ($1{\rightarrow}3$),($1{\rightarrow}4$)-${\beta}$-D-Glucan. Scand. J. Immunol. 52: 584-587 (2000) https://doi.org/10.1046/j.1365-3083.2000.00826.x
  45. Shao BM, Xu W, Dai H, Tu P, Li Z, Gao XM. A study on the immune receptors for polysaccharides from the roots of Astragalus membranaceus, a Chinese medicinal herb. Biochem. Biophys. Res. Commun. 320: 1103-1111 (2004) https://doi.org/10.1016/j.bbrc.2004.06.065
  46. Shin MS, Lee H, Hong HD, Shin KS. Characterization of immunostimulatory pectic polysaccharide isolated from leaves of Diospyros kaki Thumb. (Persimmon). J. Funct. Foods 26: 319-329 (2016) https://doi.org/10.1016/j.jff.2016.07.025
  47. Shin MS, Park SB, Shin KS. Molecular mechanisms of immunomodulatory activity by polysaccharide isolated from the peels of Citrus unshiu. Int. J. Biol. Macromol. 112: 576-583 (2018) https://doi.org/10.1016/j.ijbiomac.2018.02.006
  48. Singleton V, Rossi JA. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am. J. Enol. Vitic. 16: 144-158 (1965)
  49. Smiderle FR, Alquini G, Tadra-Sfeir MZ, Iacomini M, Wichers HJ, Van Griensven LJ. Agaricus bisporus and Agaricus brasiliensis ($1{\rightarrow}6$)-${\beta}$-D-glucans show immunostimulatory activity on human THP-1 derived macrophages. Carbohydr. Polym. 94: 91-99 (2013) https://doi.org/10.1016/j.carbpol.2012.12.073
  50. Starr R, Willson TA, Viney EM, Murray LJ, Rayner JR, Jenkins BJ, Gonda TJ, Alexander WS, Metcalf D, Nicola NA, Hilton DJ. A family of cytokine-inducible inhibitors of signalling. Nature 387: 917-921 (1997) https://doi.org/10.1038/43206
  51. Stuehr DJ. Mammalian nitric oxide synthases. Biochim. Biophys. Acta 1411: 217-230 (1999) https://doi.org/10.1016/S0005-2728(99)00016-X
  52. Tada R, Ikeda F, Aoki K, Yoshikawa M, Kato Y, Adachi Y, Tanioka A, Ishibashi K, Tsubaki K, Ohno N. Barley-derived ${\beta}$-D-glucan induces immunostimulation via a dectin-1-mediated pathway. Immunol. Lett. 123: 144-148 (2009) https://doi.org/10.1016/j.imlet.2009.03.005
  53. Tanoue T, Nishida E. Molecular recognitions in the MAP kinase cascades. Cell. Signalling 15: 455-462 (2003) https://doi.org/10.1016/S0898-6568(02)00112-2
  54. Taylor PR, Brown GD, Reid DM, Willment JA, Martinez-Pomares L, Gordon S, Wong SY. The b-glucan receptor, dectin-1, is predominantly expressed on the surface of cells of the monocyte/macrophage and neutrophil lineages. J. Immunol. 169: 3876-3882 (2002) https://doi.org/10.4049/jimmunol.169.7.3876
  55. Valitutti S, Muller S, Cella M, Padovan E, Lanzavecchia A. Serial triggering of many T-cell receptors by a few peptide-MHC complexes. Nature 375: 148 (1995) https://doi.org/10.1038/375148a0
  56. Wang SY, Hsu ML, Hsu HC, Tzeng CH, Lee SS, Shiao MS, Ho CK. The antitumor effect of Ganoderma lucidum is mediated by cytokines released from activated macrophages and T lymphocytes. Int. J. Cancer 70: 699-705 (1997) https://doi.org/10.1002/(SICI)1097-0215(19970317)70:6<699::AID-IJC12>3.0.CO;2-5
  57. Wasser SP. Medicinal mushrooms as a source of antitumor and immunomodulating polysaccharides. Appl. Microbiol. Biotechnol. 60: 258-274 (2002) https://doi.org/10.1007/s00253-002-1076-7
  58. Yamada H. Chemical and pharmacological studies on efficacy of Japanese and Chinese herbal medicines. Kitasato Arch. Exp. Med. 65: 1-22 (1992)
  59. Yang SH, Andrew DS, Alan JW. Transcriptional regulation by the MAP kinase signaling cascades. Gene 320: 3-21 (2003) https://doi.org/10.1016/S0378-1119(03)00816-3