Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
권호 표지
DOI QR코드

DOI QR Code

Prototypes of Panaxadiol and Panaxatriol Saponins Suppress LPS-mediated iNOS/NO Production in RAW264.7 Murine Macrophage Cells

RAW264.7 대식세포에서 LPS 매개 iNOS/NO 생성에 대한 protopanaxadiol saponin 및 protopanaxatriol saponin의 억제효과

  • Kim, Jin-Ik (Department of Bio Health Sciences, College of Natural Sciences, Changwon National University) ;
  • Narantuya, Nandintsetseg (Department of Bio Health Sciences, College of Natural Sciences, Changwon National University) ;
  • Choi, Yong-Won (Department of Bio Health Sciences, College of Natural Sciences, Changwon National University) ;
  • Kang, Dae-Ook (Department of Bio Health Sciences, College of Natural Sciences, Changwon National University) ;
  • Kim, Dong-Wan (Department of Bio Health Sciences, College of Natural Sciences, Changwon National University) ;
  • Lee, Kyoung (Department of Bio Health Sciences, College of Natural Sciences, Changwon National University) ;
  • Ko, Sung-Ryong (Bureau of General Affairs, The Korean Society of Ginseng) ;
  • Moon, Ja-Young (Department of Bio Health Sciences, College of Natural Sciences, Changwon National University)
  • Received : 2016.07.22
  • Accepted : 2016.08.16
  • Published : 2016.12.30
Download PDF
⟨ Previous Next ⟩

Abstract

This study was performed to investigate the modulatory effects of two prototypes of Panax ginseng saponin fractions, 20(S)-protopanaxadiol saponins (PDS) and 20(S)-protopanaxatriol saponins (PTS), on the induction of inflammatory mediators in lipopolysaccharide (LPS)-treated RAW264.7 murine macrophage cells. For this purpose, RAW264.7 cells were treated with LPS ($10{\mu}g/ml$) before, after, or simultaneously with PDS or PTS ($150{\mu}g/ml$), and the released level of nitric oxide (NO) and expression levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were evaluated. When RAW264.7 cells were treated with LPS and ginseng saponin fractions simultaneously for 24 hr, PTS, compared to PDS, more strongly attenuated the NO production induced by LPS treatment. When the cells were pretreated with LPS for 2 hr followed by PDS or PTS treatment for 24 hr, both ginseng saponins strongly reduced NO release. The pretreatment of RAW264.7 cells with PDS or PTS for 2 hr followed by LPS treatment for 24 hr significantly attenuated the LPS-induced production of NO. PTS showed stronger inhibitory potency to NO generation than PDS. Our western blot experiment showed that both PDS and PTS ($150{\mu}g/ml$) also significantly down-regulated the expressions of iNOS and COX-2 induced by LPS treatment. Our results suggest that both PDS and PTS possess strong protective effects against LPS-stimulated inflammation and that their protective effects are mediated by the suppression of NO synthesis via down-regulation of pro-inflammatory enzymes, iNOS, and COX-2 in the RAW264.7 cells.

본 연구는 RAW264.7 세포에서 lipopolysaccharide (LPS) 처리에 의한 염증매개인자의 유도에 대한 고려인삼 사포닌 분획인 20(S)-protopanaxadiol saponins (PDS)과 20(S)-protopanaxatriol saponins (PTS)의 조절효능을 탐구하였다. 이를 위해 RAW264.7 세포에 PDS 또는 PTS를 $150{\mu}g/ml$의 농도로 LPS ($10{\mu}g/ml$ 처리 이전이나 처리 이후 또는 LPS와 동시에 처리하였으며, 처리된 세포에서 nitric oxide (NO)의 방출량, 유도성 nitric oxide synthase (iNOS) 및 cyclooxygenase-2 (COX-2)의 발현 량을 분석하였다. PDS에 비하여 PTS는 RAW264.7 세포에 LPS와 동시에 처리하여 24시간 동안 배양했을 때 LPS 처리에 의해 유도된 NO의 생성을 강하게 감소시켰다. RAW264.7 세포에 LPS ($10{\mu}g/ml$를 2시간 동안 처리한 후에 PDS 또는 PTS를 $150{\mu}g/ml$ 농도로 24시간 동안 처리하면 두 인삼 사포닌 성분 모두 NO의 생성을 강하게 감소시켰다. RAW264.7 세포에 PDS 또는 PTS를 $150{\mu}g/ml$ 농도로 2시간 동안 처리한 후에 LPS ($10{\mu}g/ml$를 24시간 동안 처리했을 경우에도 두 인삼 사포닌 성분 모두 LPS 처리에 의해 유도된 NO 생성을 강하게 감소시켰다. LPS 처리에 의한 NO 생성을 저해하는 효과는 PDS에 비하여 PTS가 더 강하게 나타났다. PDS와 PTS 모두 $150{\mu}g/ml$ 처리농도에서 LPS ($10{\mu}g/ml$처리에 의해 유도된 iNOS와 COX-2의 발현 역시 상당히 감소시켰다. 따라서 본 연구의 결과는 RAW264.7 대식세포에서 PDS와 PTS 두 인삼 사포닌 성분은 LPS 처리에 의한 염증활성화에 강한 억제효과를 가지고 있음을 의미하며, 전염증성 효소인 iNOS와 COX-2 발현의 감소조절을 통하여 NO의 생성을 억제함으로써 항 염증효과가 나타남을 제시한다.

Keywords

References

  1. Byeon, S. E., Lee, J., Kim, J. H., Yang, W. S., Kwak, Y. S., Kim, S. Y., Choung, E. S., Rhee, M. H. and Cho, J. Y. 2012. Molecular mechanism of macrophage activation by red ginseng acidic polysaccharide from Korean red ginseng. Mediators Inflamm. 2012, 732860.
  2. Cario, E. and Podolsky, D. K. 2000. Differential alteration in intestinal epithelial cell expressionof toll-like receptor 3 (TLR3) and TLR4 in inflammatory bowel disease. Infect. Immun. 68, 7010-7017. https://doi.org/10.1128/IAI.68.12.7010-7017.2000
  3. Chu, S. F. and Zhang, J. T. 2009. New achievements in ginseng research and its future prospects. Chin. J. Integr. Med. 15, 403-408. https://doi.org/10.1007/s11655-009-0403-6
  4. Cuzzocrea, S. and Salvemini, D. 2007. Molecular mechanisms involved in the reciprocal regulation of cyclooxygenase and nitric oxide synthase enzymes. Kidney Int. 71, 290-297. https://doi.org/10.1038/sj.ki.5002058
  5. Friedl, R., Moeslinger, T., Kopp, B. and Spieckermann, P. G. 2001. Stimulation of nitric oxide synthesis by the aqueous extract of Panax ginseng root in RAW264.7 cells. Brit. J. Pharmacol. 134, 1663-1670. https://doi.org/10.1038/sj.bjp.0704425
  6. Hasegawa, H. 2004. Proof of the mysterious efficacy of ginseng: basic and clinical trials: metabolic activation of ginsenoside: deglycosylation by intestinal bacteria and esterification with fatty acid. J. Pharmacol. Sci. 95,153-157. https://doi.org/10.1254/jphs.FMJ04001X4
  7. Hoshino, K., Takeuchi, O., Kawai, T., Sanjo, H., Ogawa, T. and Takeda, Y. et al. 1999. Cutting edge: toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the LPS gene product. J. Immunol. 162, 3749-3752.
  8. Hui, D. Y. 2008. Intimal hyperplasia in murine models. Curr. Drug Targets 9, 251-260. https://doi.org/10.2174/138945008783755601
  9. Ichikawaa, T., Li, J., Nagarkatti, P., Nagarkatti, M., Hofsethc, L. J., Windust, A. and Cui, T. 2009. American ginseng preferentially suppresses STAT/iNOS signaling in activated macrophages. J. Ethnopharmacol. 125, 145-150. https://doi.org/10.1016/j.jep.2009.05.032
  10. Jeong, H. G., Pokharel, Y. R., Han, E. H. and Kang, K. W. 2007. Induction of cyclooxygenase-2 by ginsenoside Rd via activation of CCAAT-enhancer binding proteins and cyclic AMP response binding protein. Biochem. Biophys. Res. Commun. 359, 51-56. https://doi.org/10.1016/j.bbrc.2007.05.034
  11. Kim, D. H. 2012. Chemical diversity of Panax ginseng, Panax quinquifolium, and Panax notoginseng. J. Ginseng Res. 36, 1-15. https://doi.org/10.5142/jgr.2012.36.1.1
  12. Kim, H. S. and Moon, E. Y. 2009. Reactive oxygen species-induced expression of B cell activating factor (BAFF) is independent of Toll-like receptor 4 and myeloid differentiation primary response gene 88. Biomol. Ther. 17, 144-150. https://doi.org/10.4062/biomolther.2009.17.2.144
  13. Kim, T. W., Joh, E. H., Kim, B. and Kim, D. H. 2012. Ginsenoside Rg5 ameliorates lung inflammation in mice by inhibiting the binding of LPS to toll-like receptor-4 on macrophages. Int. Immunopharmacol. 12, 110-116. https://doi.org/10.1016/j.intimp.2011.10.023
  14. Kwak, Y. S., Kyung, J. S., Kim, J. S., Cho, J. Y. and Rhee, M. H. 2010. Anti-hyperlipidemic effects of red ginseng acidic polysaccharide from Korean red ginseng. Biol. Pharm. Bull. 33, 468-472. https://doi.org/10.1248/bpb.33.468
  15. Lee, K. W., Jung, S. Y., Choi, S. M. and Yang, E. J. 2012. Effects of ginsenoside Re on LPS-induced inflammatory mediators in BV2 microglial cells. BMC Complement. Altern. Med. 12, 196. https://doi.org/10.1186/1472-6882-12-S1-P196
  16. Lee, S. H., Soyoola, E., Chanmugam, P., Hart, S., Sun, W., Zhong, H., Liou, S., Simmons, D. and Hwang, D. 1992. Selective expression of mitogen-inducible cyclooxygenase in macrophages stimulated with lipopolysaccharide. J. Biol. Chem. 267, 25934-25938.
  17. Mariotto, S., Suzuki, Y., Persichini, T., Colasanti, M., Suzuki, H. and Cantoni, O. 2007. Cross-talk between NO and arachidonic acid in inflammation. Curr. Med. Chem. 14, 1940-1944. https://doi.org/10.2174/092986707781368531
  18. Mathrani, V. C., Kenyon, N. J., Zeki, A. and Last, J. A. 2007. Mouse models of asthma: can they give us mechanistic insights into the role of nitric oxide? Curr. Med. Chem. 14, 2204-2213. https://doi.org/10.2174/092986707781389628
  19. Moynagh, P. N. 2005. The NF-kappaB pathway. J. Cell Sci. 118, 4589-4592. https://doi.org/10.1242/jcs.02579
  20. Nathan, C. 1992. Nitric oxide as a secretory product of mammalian cells. FASEB J. 6, 3051-3064. https://doi.org/10.1096/fasebj.6.12.1381691
  21. Nathan, C. and Xie, Q. W. 1994. Nitric oxide synthases: roles, tolls, and controls. Cell 78, 915-918. https://doi.org/10.1016/0092-8674(94)90266-6
  22. Pacher, P., Beckman, J. S. and Liaudet, L. 2007. Nitric oxide and peroxynitrite in health and disease. Physiol. Rev. 87, 315-424. https://doi.org/10.1152/physrev.00029.2006
  23. Ran, S. and Montgomery, K. E. 2012. Macrophage-mediated lymphangiogenesis: the emerging role of macrophages as lymphatic endothelial progenitors. Cancers (Basel) 4, 618-657. https://doi.org/10.3390/cancers4030618
  24. Rossol, M., Heine, H., Meusch, U., Quandt, D., Klein, C., Sweet, M. J. and Hauschildt, S. 2011. LPS-induced cytokine production in human monocytes and macrophages. Crit. Rev. Immunol. 31, 379-446. https://doi.org/10.1615/CritRevImmunol.v31.i5.20
  25. Salinas, G., Rangasetty, U. C., Uretsky, B. F. and Birnbaum, Y. 2007. The cycloxygenase 2 (COX-2) story: it's time to explain, not inflame. J. Cardiovasc. Pharmacol. Ther. 12, 98-111. https://doi.org/10.1177/1074248407301172
  26. Tachikawa, E., Kudo, K., Harada, K., Kashimoto, T., Miyate, Y., Kakizaki, A. and Takahashi, E. 1999. Effects of ginseng saponins on responses induced by various receptor stimuli. Eur. J. Pharmacol. 369, 23-32. https://doi.org/10.1016/S0014-2999(99)00043-6
  27. Tak, P. P. and Firestein, G. S. 2001. NF-kappaB: a key role in inflammatory diseases. J. Clin. Invest. 107, 7-11. https://doi.org/10.1172/JCI11830
  28. Tsatsanis, C., Androulidaki, A., Venihaki, M. and Margioris, A. N. 2006. Signaling networks regulating cyclooxygenase-2. Int. J. Biochem. Cell Biol. 38, 1654-1661. https://doi.org/10.1016/j.biocel.2006.03.021
  29. Tsoyi, K., Kim, H. J., Shin, J. S., Kim, D. H., Cho, H. J., Lee, S. S., Ahn, S. K., Yun-Choi, H. S., Lee, J. H., Seo, H. G. and Chang, K. C. 2008. HO-1 and JAK-2/STAT-1 signals are involved in preferential inhibition of iNOS over COX-2 gene expression by newly synthesized tetrahydroisoquinoline alkaloid, CKD712, in cells activated with lipopolysacchride. Cell. Signal. 10, 1839-1847.
  30. Wu, C. F., Bi, X. L. and Yang, J. Y., et al. 2007. Differential effects of ginsenosides on NO and TNF-${\alpha}$ production by LPS-activated N9 microglia. Int. Immunopharmacol. 7, 313-320. https://doi.org/10.1016/j.intimp.2006.04.021