Korean Journal of Food Science and Technology (한국식품과학회지)

Korean Society of Food Science and Technology (한국식품과학회)
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Immunostimulatory activity and intracellular signaling pathways of a rhamnogalcaturonan II polysaccharide isolated from ginseng berry

인삼열매로부터 분리한 Rhamnogalacturonan II 다당의 면역활성과 세포 내 신호전달 기작 규명

  • Cha, Ha Young (Department of Food Science and Biotechnology, Kyonggi University) ;
  • Son, Seung-U (Department of Food Science and Biotechnology, Kyonggi University) ;
  • Shin, Kwang-Soon (Department of Food Science and Biotechnology, Kyonggi University)
  • 차하영 (경기대학교 식품생물공학과) ;
  • 손승우 (경기대학교 식품생물공학과) ;
  • 신광순 (경기대학교 식품생물공학과)
  • Received : 2021.09.27
  • Accepted : 2021.11.19
  • Published : 2021.12.31
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Abstract

In this study, we aimed to elucidate the intracellular signaling pathways for macrophage activation by the polysaccharide GBW-II purified from ginseng berry. GBW-II consists of 14 different sugars, including rarely observed sugars such as 2-O-methyl-xylose, apiose, aceric acid, 2-keto-3-deoxy-D-manno-2-octulosonic acid, and 2-keto-3-deoxy-D-lyxo-2-heptulosaric acid, which are typical RG-II component sugars. GBW-II enhanced the production of IL-6 and TNF-α in RAW 264.7 cells. In experiments evaluating specific inhibitor activity, it was found that the production of IL-6 was suppressed by inhibitors of SB, PD, and BAY, and the production of TNF-α was suppressed by PD and BAY. The experiments with neutralizing antibodies showed that TLR4 was involved in the stimulation of IL-6 production by GBW-II in RAW 264.7 cells, whereas TNF-α production was regulated through SR and TLR2. These results suggest that GBW-II activates the MAPK and NF-κB pathways via several macrophage receptors, including SR, TLR2, and TLR4, and subsequently induces the secretion of IL-6 and TNF-α.

본 연구는 인삼열매로부터 RG-II 형태의 다당(GBW-II)을 분리하고 대식세포 활성화에 대한 세포 내 신호전달의 세부 기작을 규명함으로써 새로운 건강기능성식품 소재 개발을 위한 기초자료를 제시하고자 진행되었다. GBW-II의 구성당을 확인한 결과, 전형적인 RG-II의 구성당인 2-methyl-xylose, apiose, aceric acid, KDO 및 DHA와 같은 특이 구성당을 함유함을 확인할 수 있었다. GBW-II는 대식세포 유래 세포주인 RAW 264.7 cell에 처리하였을 경우, 어떠한 세포 독성도 확인되지 않았으나 IL-6와 TNF-α와 같은 cytokine의 분비는 농도 의존적으로 증가시키는 것으로 나타났다. 또한 RAW 264.7 cell을 이용한 세포 내 신호전달에 관한 실험 결과들을 종합해 볼 때, GBW-II는 대식세포 표면에 발현된 TLR2, TLR4 및 SR에 결합하여 MAPKs (p38, ERK) 및 NF-κB를 경유하여 IL-6와 TNF-α와 같은 cytokine의 분비를 증가시키는 것으로 최종 확인되었다. 한편, RG-I, RG-II, β-glucan, arabinoxylan 및 xyloglucan과 같은 식물체 유래 고분자 다당체의 약리활성은 그들의 구조적 차이에서 기원하는 것으로 알려져 있기 때문에 건강기능성식품 소재로의 개발을 위해서는 활성물질의 미세구조에 대한 해명이 필수적이라 할 수 있다. 따라서 본 연구진은 추후 연구에서 효소적 및 화학적 가수분해, methylation, sequencing 등을 이용하여 인삼열매 유래 정제 다당 GBW-II의 미세구조를 규명하고자 한다.

Keywords

Acknowledgement

본 연구는 2021학년도 경기대학교 대학원 연구원장학생 장학금 지원에 의하여 수행되었습니다. 또한 본 연구는 (주) AmorePacific의 연구비 지원을 받아 연구되었으며, 이에 감사드립니다.

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-κB 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. 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
  4. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal. Biochem. 72: 248-254 (1976) https://doi.org/10.1006/abio.1976.9999
  5. 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
  6. Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith FAJN. A colorimetric method for the determination of sugars. Nature 168: 167-167 (1951)
  7. Forman HJ, Torres M. Reactive oxygen species and cell signaling: respiratory burst in macrophage signaling. Am. J. Respir. Crit. Care Med. 166: S4-S8 (2002) https://doi.org/10.1164/rccm.2206007
  8. Gwag JE, Lee YG, Hwang-Bo J, Kim HG, Oh SM, Lee DY, Baek NI. Ginsenosides from the fruits of Panax ginseng and their cytotoxic effects on human cancer cell lines. J. Appl. Biol. Chem. 61: 371-377 (2018) https://doi.org/10.3839/jabc.2018.052
  9. 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
  10. Hayden MS, Ghosh S. Shared principles in NF-κB signaling. Cell 132: 344-362 (2008) https://doi.org/10.1016/j.cell.2008.01.020
  11. Hwang YC, Shin KS. Characterization of Immuno-stimulating Polysaccharides Isolated from Korean Persimmon Vinegar. Korean J. Food Sci. Technol. 40: 220-227 (2008)
  12. Janeway CA. Progress in immunology: Syndromes of diminished resistance to infection. J. Pediat. 72: 885-903 (1968) https://doi.org/10.1016/s0022-3476(68)80446-9
  13. Ji SY, Hwangbo H, Lee HS, Koo YT, Kim JS, Lee KW, Noh DJ, Choi YH. Immunostimulatory Activity of Agarwood through Activation of MAPK Signaling Pathway in RAW 264.7 Murine Macrophages. J. Life Sci. 31:745-754 (2021) https://doi.org/10.5352/JLS.2021.31.8.745
  14. Jones TM, Albersheim P. A gas chromatographic method for the determination of aldose and uronic acid constituents of plant cell wall polysaccharides. Plant Physiol. 49: 926-936 (1972) https://doi.org/10.1104/pp.49.6.926
  15. 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
  16. Kawai T, Akira S. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat. Immunol. 11: 373-384 (2010) https://doi.org/10.1038/ni.1863
  17. Kim EH, Gwak JY, Chung MJ. Immunomodulatory Activity of Platycodon grandiflorum, Codonopsis lanceolata, and Adenophora triphylla Extracts in Macrophage Cells. J. Korean Soc. Food Sci. Nutr. 47: 1069-1075 (2018) https://doi.org/10.3746/jkfn.2018.47.11.1069
  18. Ko SH, Park CH, Jekal YS. Effects of Exercise Intervention Program on the Level of Obesity, Physical Fitness Level and Metabolic Related Risk Factors among Hypertensive and Diabetic Patients. Korean Society for Wellness 12: 645-655 (2017) https://doi.org/10.21097/ksw.2017.02.12.1.645
  19. Kwon HW. Inhibitory Effects of PD98059, SB203580, and SP600125 on α-and δ-granule Release and Intracellular Ca2+ Levels in Human Platelets. Biomed. Sci. Lett. 24: 253-262 (2018) https://doi.org/10.15616/BSL.2018.24.3.253
  20. 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 α in mice. Cancer Immunol. Immunother. 45: 100-108 (1997) https://doi.org/10.1007/s002620050408
  21. Lee SJ, Hong HD, Shin KS. Rapid Isolation Method for Preparation of Immuno-Stimulating Rhamnogalacturonans in Citrus Peels. Korean J. Food Sci. Technol. 47: 286-292 (2015) https://doi.org/10.9721/KJFST.2015.47.3.286
  22. Lee KS, Kim GH, Seong BJ, Kim SI, Han SH, Lee SS, Yang H, Yoo YC. Anti-inflammatory Activity of Solvent Fractions from Ginseng Berry Extract in LPS-Induced RAW264.7 Cells. Korean J. Medicinal Crop Sci. 22: 449-456 (2014) https://doi.org/10.7783/KJMCS.2014.22.6.449
  23. Lee DY, Park CW, Lee SJ, Park HR, Seo DB, Park JY, Park JY, Shin KS. Immunostimulating and antimetastatic effects of polysaccharides purified from ginseng berry. Am. J. Chin. Med. 47: 823-839 (2019) https://doi.org/10.1142/s0192415x19500435
  24. Lewis TS, Shapiro PS, Ahn NG. Signal transduction through MAP kinase cascades. Adv. Cancer Res. 74: 49-139 (1998) https://doi.org/10.1016/S0065-230X(08)60765-4
  25. 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-κB) 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
  26. 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
  27. Mercken M, Vandermeeren M, Lubke U, Six J, Boons J, Vanmechelen E, Van De Voorde A, Gheuens J. Affinity Purification of Human τ Proteins and the Construction of a Sensitive Sandwich Enzyme-Linked Immunosorbent Assay for Human τ Detection. J. Neurochem. 58: 548-553 (1992) https://doi.org/10.1111/j.1471-4159.1992.tb09754.x
  28. Mohnen D. Pectin structure and biosynthesis. Curr. Opin. Plant Biol. 11: 266-277 (2008) https://doi.org/10.1016/j.pbi.2008.03.006
  29. Nam KY. Clinical Applications and Efficacy of Korean Ginseng. J. Ginseng Res. 26: 111-131 (2002) https://doi.org/10.5142/JGR.2002.26.3.111
  30. Park WY, Sung NY, Byun EH, Oh KH, Byun MW, Yoo YC. Immuno-Modulatory Activities of Polysaccharides Separated from Jubak in Macrophage Cells. J. Korean. Soc. Food. Sci. Nutr. 44:1079-1083 (2015) https://doi.org/10.3746/JKFN.2015.44.7.1079
  31. Paulsen B. Plant polysaccharides with immunostimulatory activities. Curr. Org. Chem. 5: 939-950 (2001) https://doi.org/10.2174/1385272013374987
  32. 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. 190: 307-316 (2013) https://doi.org/10.4049/jimmunol.1201644
  33. Rice PJ, Kelley JL, Kogan G, Ensley HE, Kalbfleisch JH, Browder IW, Williams DL. Human monocyte scavenger receptors are pattern recognition receptors for (1→3)-β-D-glucans. J. Leukoc. Biol. 72: 140-146 (2002) https://doi.org/10.1189/jlb.72.1.140
  34. 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
  35. Shin HS, Lee SY. Research Trends in Immune Function of Korea Traditional Foods. Food Industry and Nutrition 25: 19-28 (2020)
  36. Shin MS, Lee H, Hong HD, Shin KS. Characterization of immunostimulatory pectic polysaccharide isolated from leaves of Diospyros kaki Thumb.(Persimmon). J. Funct. Food. 26: 319-329 (2016) https://doi.org/10.1016/j.jff.2016.07.025
  37. Singleton VL, Rossi JA. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am. J. Enol. Vitic. 16: 144-158 (1965)
  38. 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
  39. Stevenson TT, Darvill AG, Albersheim P. 3-Deoxy-D-lyxo-2-heptulosaric acid, a component of the plant cell-wall polysaccharide rhamnogalacturonan-II. Carbohydr. Res. 179: 269-288 (1988) https://doi.org/10.1016/0008-6215(88)84124-7
  40. Taylor PR, Brown GD, Reid DM, Willment JA, Martinez-Pomares L, Gordon S, Wong SY. The β-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
  41. Vidal S, Doco T, Williams P, Pellerin P, York WS, O'Neill MA, Glushka J, Darvill AG, Albersheim P. Structural characterization of the pectic polysaccharide rhamnogalacturonan II: evidence for the backbone location of the aceric acid-containing oligoglycosyl side chain. Carbohydr. Res. 326: 277-294 (2000) https://doi.org/10.1016/S0008-6215(00)00036-7
  42. YAMADA H. Chemical and pharmacological studies on efficacy of Japanese and Chinese herbal medicines. Kitasato Arch. Exp. Med. 65: 159-180 (1992)
  43. Yang SH, Sharrocks AD, Whitmarsh AJ. Transcriptional regulation by the MAP kinase signaling cascades. Gene 320: 3-21 (2003) https://doi.org/10.1016/S0378-1119(03)00816-3
  44. York WS, Darvill AG, McNeil M, Albersheim P. 3-Deoxy-D-manno-2-octulosonic acid (KDO) is a component of rhamnogalacturonan II, a pectic polysaccharide in the primary cell walls of plants. Carbohydr. Res. 138: 109-126 (1985) https://doi.org/10.1016/0008-6215(85)85228-9