Journal of Applied Biological Chemistry

  • Volume 66
  • /
  • Pages.519-526
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  • 2023
  • /
  • 1976-0442(pISSN)
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  • 2234-7941(eISSN)
The Korean Society for Applied Biological Chemistry (한국응용생명화학회)
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Immune stimulating effects of Astragalus membranaceus and Zanthoxylum schinifolium 1:1 mixture in Raw264.7 cells

Raw264.7 세포에서 황기와 산초 1:1 혼합물의 면역 증진 효과

  • Il Je Cho (Central Research Center, Okchundang Inc.) ;
  • Yeong Eun Yu (Central Research Center, Okchundang Inc.) ;
  • Sang Min Lee (Central Research Center, Okchundang Inc.) ;
  • Eun Ok Kim (Central Research Center, Okchundang Inc.) ;
  • Joon Heum Park (Central Research Center, Okchundang Inc.) ;
  • Sea Kwang Ku (Department of Histology and Anatomy, College of Korean Medicine, Daegu Haany University)
  • Received : 2023.12.12
  • Accepted : 2023.12.19
  • Published : 2023.12.31
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Abstract

Present study explored immunostimulatory effects of Astragalus membranaceus and Zanthoxylum schinifolium 1:1 (w:w) mixture (AZM-1:1) in Raw264.7 cells, mouse macrophage derived cells. Treatment with 100-400 ㎍/mL of AZM-1:1 in Raw264.7 cells significantly increased nitric oxide production in parallel with inducible nitric oxide synthase mRNA expression without affecting cytotoxicity. In addition, AZM-1:1 dose-dependently increased prostaglandin E2 production in conditioned medium along with cyclooxygenase-2 mRNA induction. Moreover, AZM-1:1 induces the transcription of tumor necrosis factor-α, interleukin-1β, interleukin-6, and monocyte chemoattractant protein-1. Immunoblot analyses revealed that AZM-1:1 significantly increased the phosphorylation of mitogen-activated protein kinases, provoked phosphorylation-mediated degradation of inhibitory-κBα, and phosphorylated p65. Furthermore, treatment with AZM-1:1 promoted phagocytosis of Escherichia coli particle labeled with green fluorescence. Taken together, AZM-1:1 may be a promising nutraceutical for stimulation the innate immune system, including macrophages.

본 연구는 마우스 대식세포 유래 Raw264.7 세포주에서 황기와 산초 1:1 혼합물(AZM-1:1)의 면역 증진 효능을 탐색하였다. Raw264.7 세포에 100-400 ㎍/mL의 A ZM-1:1 처치는 세포 생존율의 변화 없이 inducible nitric oxide synthase mRNA의 발현 증가와 함께 nitric oxide의 생성을 통계적으로 유의하게 증가시켰다. 더불어 A ZM-1:1은 처치 농도 의존적으로 cyclooxygenase-2 mRNA의 유도와 함께 세포 배양액 중 prostaglandin E2의 함량을 증가시켰다. 또한, AZM-1:1은 tumor necrosis factor-α, interleukin-1β, interleukin-6 및 monocyte chemoattractant protein-1의 전사를 촉진하였다. Immunoblot 분석을 통하여 AZM-1:1은 mitogen-activated protein kinase의 인산화를 증가시키고, inhibitory-κBα의 인산화를 매개한 분해를 촉진하며, p65의 인산화를 증가시킬 수 있음을 확인하였다. AZM-1:1의 처치는 녹색 형광으로 표지된 대장균 파편의 탐식작용을 촉진하였다. 따라서, 이상의 결과는 A ZM-1:1가 대식세포를 포함한 내재면역을 증진시키는 기능성 식의약 소재가 될 수 있음을 나타낸다.

Keywords

Acknowledgement

본 연구는 중소벤처기업부와 중소기업기술정보진흥원의 "지역특화산업 육성+(R&D, S3268061)" 사업의 지원을 받아 수행되었으며 이에 감사드립니다. Chemi-Doc을 통한 immunoblot 이미지와 슬라이드 스캐너를 통한 세포 이미지는 한국뇌연구원 첨단뇌연구장비센터 지원을 통해 얻었으며 이에 감사드립니다.

References

  1. Abbas AK, Lichtman AH, Phillai S (2012) Cellular and molecular immunology. 7th edn. Saunders, Philadelphia, pp 41-88 
  2. Doan T, Melvold R, Susan V, Waltenbaugh C (2008) Lippincott's Illustrated Reviews: Immunology. 1st edn. Lippincott Williams & Wilkins, Baltimore, pp 41-53 
  3. Fu J, Wang Z, Huang L, Zheng S, Wang D, Chen S, Zhang H, Yang S (2014) Review of the botanical characteristics, phytochemistry, and pharmacology of Astragalus membranaceus (Huangqi). Phytother Res 28(9): 1275-1283. doi: 10.1002/ptr.5188 
  4. Latour E, Arlet J, Latour EE, Juszkiewicz A, Luczkowska K, Marcinkiewicz A, Basta P, Trzeciak J, Machalinski B, Skarpanska-Stejnborn A (2021) Standardized astragalus extract for attenuation of the immunosuppression induced by strenuous physical exercise: randomized controlled trial. J Int Soc Sports Nutr 18(1): 57. doi: 10.1186/s12970-021-00425-5 
  5. D'Avino D, Cerqua I, Ullah H, Spinelli M, Di Matteo R, Granato E, Capasso R, Maruccio L, Ialenti A, Daglia M, Roviezzo F, Rossi A (2023) Beneficial Effects of Astragalus membranaceus (Fisch.) Bunge Extract in Controlling Inflammatory Response and Preventing Asthma Features. Int J Mol Sci 24(13): 10954. doi: 10.3390/ijms241310954 
  6. Lin Y, Wang B, Luo XQ (2011) Clinical study of astragalus's preventing the recurrence of asthma in children. Zhongguo Zhong Xi Yi Jie He Za Zhi 31(8): 1090-1092 
  7. Dong Q, Pu J, Du T, Xu S, Liu W, Liu L, Wang Z, Cai C (2021) Astragalus-mediated stimulation on antigen-presenting cells could result in higher IL-21 production from CXCR5+ Tfh-like cells and better IL-21-mediated effector functions. Hum Immunol 82(6): 429-437. doi: 10.1016/j.humimm.2021.03.012 
  8. Bao W, Zhang Q, Zheng H, Li L, Liu M, Cheng H, Wong T, Zhang G, Lu A, Bian Z, Ma D, Leung C, Han Q (2021) Radix Astragali polysaccharide RAP directly protects hematopoietic stem cells from chemotherapy-induced myelosuppression by increasing FOS expression. Int J Biol Macromol 183: 1715-1722. doi: 10.1016/j.ijbiomac.2021.05.120 
  9. Li CX, Liu Y, Zhang YZ, Li JC, Lai J (2022) Astragalus polysaccharide: a review of its immunomodulatory effect. Arch Pharm Res 45(6): 367-389. doi: 10.1007/s12272-022-01393-3 
  10. Kim TJ (1998) Korean resources plants. Publishing center of Seoul National University, Seoul, p 266 
  11. Chen IS, Lin YC, Tsai IL, Teng CM, Ko FN, Ishikawa T, Ishii H (1995) Coumarins and anti-platelet aggregation constituents from Zanthoxylum schinifolium. Phytochemistry 39(5): 1091-1097. doi: 10.1016/0031-9422(95)00054-b 
  12. Chon SU, Heo BG, Park YS, Kim DK, Gorinstein S (2009) Total phenolics level, antioxidant activities and cytotoxicity of young sprouts of some traditional Korean salad plants. Plant Foods Hum Nutr 64(1): 25-31. doi: 10.1007/s11130-008-0092-x 
  13. Min BK, Hyun DG, Jeong SY, Kim YH, Ma ES, Woo MH (2011) A new cytotoxic coumarin, 7-[(E)-3',7'-dimethyl-6'-oxo-2',7'-octadienyl] oxy coumarin, from the leaves of Zanthoxylum schinifolium. Arch Pharm Res 34(5): 723-726. doi: 10.1007/s12272-011-0504-6 
  14. Lee HY, Park YM, Lee YH, Kang YG, Lee HM, Park DS, Yang HJ, Kim MJ, Lee YR (2018) Immunostimulatory Effect of Zanthoxylum schinifolium-Based Complex Oil Prepared by Supercritical Fluid Extraction in Splenocytes and Cyclophosphamide-Induced Immunosuppressed Rats. Evid Based Complement Alternat Med 2018: 8107326. doi: 10.1155/2018/8107326 
  15. Cho WC, Leung KN (2007) In vitro and in vivo immunomodulating and immunorestorative effects of Astragalus membranaceus. J Ethnopharmacol 113(1): 132-141. doi: 10.1016/j.jep.2007.05.020 
  16. Jung CJ, Park SM, Lee DG, Yu YE, Ku TH, La IJ, Cho IJ, Ku SK (2023) Adenophora Stricta Root Extract Alleviates Airway Inflammation in Mice with Ovalbumin-Induced Allergic Asthma. Antioxidants (Basel) 12(4): 922. doi: 10.3390/antiox12040922 
  17. Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 3(6): 1101-1108. doi: 10.1038/nprot.2008.73 
  18. MacMicking J, Xie QW, Nathan C (1997) Nitric oxide and macrophage function. Annu Rev Immunol 15: 323-350. doi: 10.1146/annurev.immunol.15.1.323 
  19. Lane TE, Otero GC, Wu-Hsieh BA, Howard DH (1994) Expression of inducible nitric oxide synthase by stimulated macrophages correlates with their antihistoplasma activity. Infect Immun 62(4): 1478-1479. doi: 10.1128/iai.62.4.1478-1479.1994 
  20. Karupiah G, Xie QW, Buller RM, Nathan C, Duarte C, MacMicking JD (1993) Inhibition of viral replication by interferon-gamma-induced nitric oxide synthase. Science 261(5127): 1445-1448. doi: 10.1126/science.7690156 
  21. Liew FY, Li Y, Moss D, Parkinson C, Rogers MV, Moncada S (1991) Resistance to Leishmania major infection correlates with the induction of nitric oxide synthase in murine macrophages. Eur J Immunol 21(12): 3009-3014. doi: 10.1002/eji.1830211216 
  22. Wallace JL (2008) Prostaglandins, NSAIDs, and gastric mucosal protection: why doesn't the stomach digest itself? Physiol Rev 88(4): 1547-1465. doi: 10.1152/physrev.00004.2008 
  23. Wang W, Liang M, Wang L, Bei W, Rong X, Xu J, Guo J (2023) Role of prostaglandin E2 in macrophage polarization: Insights into atherosclerosis. Biochem Pharmacol 207: 115357. doi: 10.1016/j.bcp.2022.115357 
  24. Nakanishi M, Rosenberg DW (2013) Multifaceted roles of PGE2 in inflammation and cancer. Semin Immunopathol 35(2): 123-137. doi: 10.1007/s00281-012-0342-8 
  25. Bradley JR (2008) TNF-mediated inflammatory disease. J Pathol 214(2): 149-160. doi: 10.1002/path.2287 
  26. 26. Dinarello CA (2011) Interleukin-1 in the pathogenesis and treatment of inflammatory diseases. Blood 117(14): 3720-3732. doi: 10.1182/blood-2010-07-273417 
  27. Tanaka T, Narazaki M, Kishimoto T (2018) Interleukin (IL-6) Immunotherapy. Cold Spring Harb Perspect Biol 10(8): a028456. doi: 10.1101/cshperspect.a028456 
  28. Deshmane SL, Kremlev S, Amini S, Sawaya BE (2009) Monocyte chemoattractant protein-1 (MCP-1): an overview. J Interferon Cytokine Res 29(6): 313-326. doi: 10.1089/jir.2008.0027 
  29. Kawasaki T, Kawai T (2014) Toll-like receptor signaling pathways. Front Immunol. 5: 461. doi: 10.3389/fimmu.2014.00461 
  30. Manzoor Z, Koh YS (2012) Mitogen-activated protein kinases in inflammation. J Bacteriol Virol 42(3): 189-195. doi: 10.4167/jbv.2012.42.3.189 
  31. Viatour P, Merville MP, Bours V, Chariot A (2005) Phosphorylation of NF-kappaB and IkappaB proteins: implications in cancer and inflammation. Trends Biochem Sci 30(1): 43-52. doi: 10.1016/j.tibs.2004.11.009 
  32. Giridharan S, Srinivasan M (2018) Mechanisms of NF-κB p65 and strategies for therapeutic manipulation. J Inflamm Res 11: 407-419. doi: 10.2147/JIR.S140188 
  33. Cho IJ, Kim SG (2009) A novel mitogen-activated protein kinase phosphatase-1 and glucocorticoid receptor (GR) interacting protein-1-dependent combinatorial mechanism of gene transrepression by GR. Mol Endocrinol 23(1): 86-99. doi: 10.1210/me.2008-0257 
  34. Ohlsson BG, Englund MC, Karlsson AL, Knutsen E, Erixon C, Skribeck H, Liu Y, Bondjers G, Wiklund O (1996) Oxidized low density lipoprotein inhibits lipopolysaccharide-induced binding of nuclear factor-kappaB to DNA and the subsequent expression of tumor necrosis factor-alpha and interleukin-1beta in macrophages. J Clin Invest 98(1): 78-89. doi: 10.1172/JCI118780 
  35. Rovin BH, Dickerson JA, Tan LC, Hebert CA (1995) Activation of nuclear factor-kappa B correlates with MCP-1 expression by human mesangial cells. Kidney Int 48(4): 1263-1271. doi: 10.1038/ki.1995.410 
  36. Xie QW, Whisnant R, Nathan C (1993) Promoter of the mouse gene encoding calcium-independent nitric oxide synthase confers inducibility by interferon gamma and bacterial lipopolysaccharide. J Exp Med 177(6): 1779-1784. doi: 10.1084/jem.177.6.1779 
  37. Droogmans L, Cludts I, Cleuter Y, Kettmann R, Burny A (1992) Nucleotide sequence of the bovine interleukin-6 gene promoter. DNA Seq 3(2): 115-117. doi: 10.3109/10425179209034005 
  38. Rhoades KL, Golub SH, Economou JS (1992) The regulation of the human tumor necrosis factor alpha promoter region in macrophage, T cell, and B cell lines. J Biol Chem 267(31): 22102-22107  https://doi.org/10.1016/S0021-9258(18)41641-9
  39. Libermann TA, Baltimore D (1990) Activation of interleukin-6 gene expression through the NF-kappa B transcription factor. Mol Cell Biol 10(5): 2327-2334. doi: 10.1128/mcb.10.5.2327-2334.1990 
  40. Timmers HT, Pronk GJ, Bos JL, van der Eb AJ (1990) Analysis of the rat JE gene promoter identifies an AP-1 binding site essential for basal expression but not for TPA induction. Nucleic Acids Res 18(1): 23-34. doi: 10.1093/nar/18.1.23 
  41. He S, Yao L, Li J (2023) Role of MCP-1/CCR2 axis in renal fibrosis: Mechanisms and therapeutic targeting. Medicine (Baltimore) 102(42): e35613. doi: 10.1097/MD.0000000000035613 
  42. Jin K, Qian C, Lin J, Liu B (2023) Cyclooxygenase-2-Prostaglandin E2 pathway: A key player in tumor-associated immune cells. Front Oncol 13: 1099811. doi: 10.3389/fonc.2023.1099811