Nitric oxide production and inducible nitric oxide synthase expression induced by Porphyromonas gingivalis lipopolysaccharide

Porphyromonas gingivalis의 세균내독소가 RAW264.7세포에서의 nitric oxide의 생성과 inducible nitric oxide synthase의 발현에 미치는 영향 및 기전

  • Paek, Eun-Young (Department of Periodontology, College of Dentistry, Pusan National University) ;
  • Choi, Eun-Young (Department of Life Science, College of Natural Science, Silla University) ;
  • Choi, Jeom-II (Department of Periodontology, College of Dentistry, Pusan National University) ;
  • Lee, Ju-Yun (Department of Periodontology, College of Dentistry, Pusan National University) ;
  • Kim, Sung-Jo (Department of Periodontology, College of Dentistry, Pusan National University)
  • 백은영 (부산대학교 치과대학 치주과학교실) ;
  • 최은영 (신라대학교 자연대학 생물과학과) ;
  • 최점일 (부산대학교 치과대학 치주과학교실) ;
  • 이주연 (부산대학교 치과대학 치주과학교실) ;
  • 김성조 (부산대학교 치과대학 치주과학교실)
  • Published : 2005.12.31

Abstract

본 연구는 치주질환 주요 병인균주 중의 하나인 Porphyromonas gingivalis의 세균내독소가 마우스 대식 세포주인 RAW264.7 세포에서의 nitric oxide의 생성과 iNOS의 발현에 미치는 영향을 분석하고 그 기전을 규명하기 위해 수행되었다. Butanol추출법과 phenol-water법에 의해 P. gingivalis 381로부터 세균내독소를 추출하였으며, NO의 생성은 배양 상층액 내의 nitrite 농도를 측정하여 결정하였다. 또한, iNOS의 western blot 분석과 reverse transcription (RT)-PCR 산물의 분석을 수행하였다. P. gingivalis의 세균내독소는 부가적인 자극이 없는 상태에서도 iNOS의 발현과 NO 생성을 유발하였으며, NF- ${\kappa}B$, microtubule polymerization, protein tyrosine kinase, 그리고 protein kinase C 등이 P. gingivalis 세균내독소에 의한 NO 생성에 간여하는 것으로 여겨진다. 또한, P. gingivalis 세균내독소에 의한 NO 생성에는 L-arginine이 요구되었다. P. gingivalis 세균내독소에 의한 NO 생성은 염증성 치주질환의 발병과 진행에 있어 중요한 역할을 하는 것으로 여겨진다.

References

  1. Moncada S, Palmer RMJ, Higgs EA. Nitric oxide: physiology, pathology, and pharmacology. Pharmacol Rev 1991:43: 109-142
  2. Nathan C, Xie QW. Nitric oxide synthases: roles. tolls and controls. Cell 1994: 78: 915-918 https://doi.org/10.1016/0092-8674(94)90266-6
  3. Geller DA. Nussler AK, Di Silvio M et al. Cytokines, endotoxin, and glucocorticoids regulate the expression of inducible nitric oxide synthase in hepatocytes, Proc Natl Acad Sci USA 1993:90 :522-526
  4. Nathan C, Xie QW. Regulation of biosynthesis of nitric oxide. J Biol Chem 1994:269:13725-13728
  5. Williams RC. Periodontal disease. N Engl J Med 1990:322:373-381 https://doi.org/10.1056/NEJM199002083220606
  6. Socransky SS, Haffajee AD. The bacterial etiology of destructive periodontal disease: current concepts. J Periodontol 1992:63: 322-331 https://doi.org/10.1902/jop.1992.63.4s.322
  7. Slots J, Bragd L, Wikstrom M, Dahlen G. The occurrence of Actinobacillus ectinomycetemcomitans, Bacteroides gingjvslis and Bacteroides intermedius in destructive periodontal disease in adults. J Clin Periodontol 1986: 13: 570-577 https://doi.org/10.1111/j.1600-051X.1986.tb00849.x
  8. Morrison DC. Ryan JL. Endotoxins and disease mechanisms. Annu Rev Med 1987:38:417-432 https://doi.org/10.1146/annurev.me.38.020187.002221
  9. Honig J, Rordorf-Adam C, Siegmund C, Wiedemann W, Erard F. Increased interleukin-1 beta concentration in gingival tissue from periodontitis patients. J Periodont Res 1989: 24: 362-367 https://doi.org/10.1111/j.1600-0765.1989.tb00883.x
  10. Stashenko P, Jandinski JJ, Fujiyioshi P, Rynar J, Socranski SS. Tissue levels of bone resorptive cytokines in periodontal disease. J Periodontol 1991:62:504-509 https://doi.org/10.1902/jop.1991.62.8.504
  11. Southey A. Tanaka S, Murakami T et al. Pathophysiological role of nitric oxide in rat experimental colitis. Int J Immunopharmacol 1997: 19:669-676 https://doi.org/10.1016/S0192-0561(97)00107-0
  12. Weinberg JB, Granger DL, Pisetsky DS et al. The role of nitric oxide in the pathogenesis of spontaneous murine autoimmune disease: increased nitric oxide production and nitric oxide synthase expression in MRL-lpr/lpr mice, and reduction of spontaneous glomerulonephritis and arthritis by orally administered $N^G$-monomethyl-L-arginine. J Exp Med 1994; 179: 651-660 https://doi.org/10.1084/jem.179.2.651
  13. Matejka M. Partyka L, Ulm C, Solar P, Sinzinger H. Nitric oxide synthesis is increased in periodontal disease. J Periodont Res 1998:33:517-518 https://doi.org/10.1111/j.1600-0765.1998.tb02352.x
  14. Batista AC, Silva TA. Chun JH. Lara VS. Nitric oxide synthesis and severity of human periodontal disease. Oral Dis 2002; 8: 254-260 https://doi.org/10.1034/j.1601-0825.2002.02852.x
  15. Hirose M, Ishihara K. Saito A. Expression of cytokines and inducible nitric oxide synthase in inflamed gingival tissue. J Periodontol 2001: 72: 590-597 https://doi.org/10.1902/jop.2001.72.5.590
  16. Kendall HK. Haase HR, Li H. Xiao Y, Bartold PM. Nitric oxide synthase type-II is synthesized by human gingival tissue and cultured human gingival fibroblasts. J Periodont Res 2000;35: 194-200 https://doi.org/10.1034/j.1600-0765.2000.035004194.x
  17. Lappin DF, Kjeldsen M, Sander L, Kinane DF, Inducible nitric oxide synthase expression in periodontitis. J Periodont Res 2000;35:369-373 https://doi.org/10.1034/j.1600-0765.2000.035006369.x
  18. Blix IJ, Helgeland K. LPS from Actinobacillus actinomycetemcomitans and production of nitric oxide in murine macrophages J774. Eur J Oral Sci 1998; 106: 576-581 https://doi.org/10.1046/j.0909-8836.1998.eos106107.x
  19. Sosroseno W, Barid I. Herminajeng E, Susilowati H. Nitric oxide production by a murine macrophage cell line (RAW 264.7) stimulated with lipopolysaccharide from Actinobacillus actinomycetem-comitans. Oral Microbiol Immunol 2002; 17:72-78 https://doi.org/10.1046/j.0902-0055.2001.00091.x
  20. Kim SJ, Ha MS, Choi EY, Choi JI. Choi IS. Prevotella intermedia lipopolysaccharide stimulates release of nitric oxide by inducing expression of inducible nitric oxide synthase. J Periodont Res 2004: 39:424-431 https://doi.org/10.1111/j.1600-0765.2004.00757.x
  21. Kim SJ, Ha MS, Choi EY, Choi JI. Choi IS. Nitric oxide production and inducible nitric oxide synthase expression induced by Prevotella nigrescens lipopolysaccharide. FEMS Immunol Med Microbiol 2005 ;43: 51-58 https://doi.org/10.1016/j.femsim.2004.07.001
  22. Morrison DC, Leive L. Fractions of lipopolysaccharides from E. coli 0111: B4 prepared by two extraction procedures. J Biol Chem 1975:250:2911-2919
  23. Westphal O, Jann K. (1965) Bacterial lipopolvsaccharides: extraction with phenol-water and further applications of the procedure. In: RL Whistler eds. Methods in carbohydrate chemistry. New York: Academic Press. 83-91
  24. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983:65: 55-63 https://doi.org/10.1016/0022-1759(83)90303-4
  25. Green LC, Wagner DA, Glogowski J et al. Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal Biochem 1982;126:131-138. https://doi.org/10.1016/0003-2697(82)90118-X
  26. Rappolee DA, Wang A, Mark D. Werb Z. Novel method for studying mRNA phenotypes in single or small numbers of cells. J Cell Biochem 1989:39:1-11 https://doi.org/10.1002/jcb.240390102
  27. Sakuma I, Stuehr DJ, Gross SS. Nathan C. Levi R. Identification of arginine as a precursor of endothelium-derived relaxing factor. Proc Natl Acad Sci USA 1988:85:8664-8667
  28. Kaur H, Halliwell B. Evidence for nitric oxide-mediated oxidative damage in chronic inflammation. Nitrotyrosine in serum and synovial fluid from rheumatoid patients. FEBS Lett 1994:350: 9-12 https://doi.org/10.1016/0014-5793(94)00722-5
  29. Rachmilewitz D. Stamler JS, Bachwich D et al. Enhanced colonic nitric oxide generation and nitric oxide synthase activity in ulcerative colitis and Crohn's disease. Gut 1995:6:718-723
  30. Stuehr DJ, Nathan CF. Nitric oxide: a macrophage product responsible for cytostasis and respiratory inhibition in tumor target cells. J Expt Med 1989: 169 : 1543-1555 https://doi.org/10.1084/jem.169.5.1543
  31. Baeuerle PA, Henkel T. Function and activation of NF-kappa B in the immune system. Annu Rev Immunol 1994: 12: 141-179 https://doi.org/10.1146/annurev.iy.12.040194.001041
  32. Baldwin AS Jr. The NF-$\kappa$B and I-$\kappa$B proteins: new discoveries and insights. Annu Rev Immunol 1996: 14:649-683 https://doi.org/10.1146/annurev.immunol.14.1.649
  33. Xie QW, Kashiwabara Y, Nathan C. Role of transcription factor NF-KB/Rel in induction of nitric oxide synthase. J Biol Chem 1994:269:4705-4708
  34. Paquette DW, Williams RC. Modulation of host inflammatory mediators as a treatment strategy for periodontal diseases. Periodontol 2000 2000:24:239-252 https://doi.org/10.1034/j.1600-0757.2000.2240112.x
  35. Sosroseno W, Herminajeng E, Bird PS, Seymour GJ. L-arginine-dependent nitric oxide production of a murine macrophage-like RAW264. 7 cell line stimulated with Porphyromonas gjngjvaljs lipopolysaccharide. Oral Microbiol Immunol 2004: 19:65-70 https://doi.org/10.1046/j.0902-0055.2003.00108.x
  36. Shapira L, Champagne C, Van Dyke TE, Amar S. Strain-dependent activation of monocytes and inflammatory macrophages by lipopolysaccharide of Porphyromonas gjngivalis. Infect Immun 1998: 66: 2736-2742
  37. Brennan PA, Thomas GJ, Langdon JD. The role of nitric oxide in oral diseases. Arch Oral Biol 2003:48:93-100 https://doi.org/10.1016/S0003-9969(02)00183-8