Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Anti-Inflammatory Activity of Dichloromethane Fraction from Katsuwonus pelamis Heart in LPS-Induced RAW 264.7 Cells and Mouse Ear Edema

Lipopolysaccharide로 자극된 RAW 264.7 세포와 마우스 귀부종 모델에 대한 참치 심장 Dichloromethane 분획물의 항염증 효과

  • Kim, Min-Ji (Department of Food Science & Technology/Institute of Food Science, Pukyong National University) ;
  • Bae, Nan-Young (Department of Food Science & Technology/Institute of Food Science, Pukyong National University) ;
  • Choi, Hyeun-Deok (Institute of Fisheries Sciences, Pukyong National University) ;
  • Kim, Koth-Bong-Woo-Ri (Department of Food Science & Technology/Institute of Food Science, Pukyong National University) ;
  • Park, Sun-Hee (Department of Food Science & Technology/Institute of Food Science, Pukyong National University) ;
  • Sung, Nak-Yun (Department of Food Science and Technology, Kongju University) ;
  • Byun, Eui-Hong (Department of Food Science and Technology, Kongju University) ;
  • Nam, Hee-Sup (Research & Development Center, Dongwon F&B) ;
  • Ahn, Dong-Hyun (Department of Food Science & Technology/Institute of Food Science, Pukyong National University)
  • 김민지 (부경대학교 식품공학과/식품연구소) ;
  • 배난영 (부경대학교 식품공학과/식품연구소) ;
  • 최현덕 (부경대학교 수산과학연구소) ;
  • 김꽃봉우리 (부경대학교 식품공학과/식품연구소) ;
  • 박선희 (부경대학교 식품공학과/식품연구소) ;
  • 성낙윤 (공주대학교 식품공학과) ;
  • 변의홍 (공주대학교 식품공학과) ;
  • 남희섭 ((주)동원 F&B 동원식품과학연구원) ;
  • 안동현 (부경대학교 식품공학과/식품연구소)
  • Received : 2016.09.28
  • Accepted : 2016.12.15
  • Published : 2017.06.28
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Abstract

This study investigated the effect of the dichloromethane fraction form Katsuwonus pelamis heart on anti-inflammatory responses in lipopolysaccharide-stimulated RAW 264.7 cells and mouse models. Ethanol extract was partitioned with dichloromethane, ethyl acetate, butanol, and water. Among the fractions, the dichloromethane fraction showed a significant decrease in nitric oxide (NO) and pro-inflammatory cytokines [interleukin (IL)-6, $IL-1{\beta}$, and tumor necrosis $factor-{\alpha}$] production compared to ethanol extract. The dichloromethane fraction attenuated the expression of inducible nitric oxide synthase and nuclear $factor-{\kappa}B$ ($NF-{\kappa}B$) p65 proteins in a dose-dependent manner. In addition, the expression of phosphorylation of mitogen-activated protein kinases (MAPKs) was also inhibited by the dichloromethane fraction. Moreover, the administration of 10, 50, and 250 mg/kg body weight-dose dependently inhibited the formation of edema by croton-oil and the application of dichloromethane (2 mg/ear) significantly reduced epidermal and dermal thickness and the infiltrated mast cell numbers. Therefore, the dichloromethane fraction exhibited an anti-inflammation effect by inhibiting $NF-{\kappa}B$ and MAPK signaling activation in macrophages.

본 실험에서는 참치 심장 70% ethanol 추출물을 dichloromethane으로 분획한 후 항염증 효과를 확인하기 위해 RAW 264.7 세포에 LPS로 염증을 유도시켜 염증 매개성 물질인 NO와 pro-inflammatory cytokine의 분비량의 변화를 확인하였다. 그 결과, 참치 심장 dichloromethane 분획물을 처리하였을 때, 농도 의존적으로 NO의 생성량을 감소시키는 것을 확인하였으며, 특히, $100{\mu}g/ml$에서 가장 높은 억제효과를 나타내었다. 따라서 dichloromethane 분획물의 억제 활성이 세포사멸에 의한 감소인지 알아보기 위해서 MTT assay를 하였을 때, 세포 생존율이 dichloromethane 분획물을 PBS 처리군과 비교하였을 때 유의적인 차이가 나타나지 않음을 확인하였고, 이를 통해 dichloromethane 분획물이 NO 및 전염증성 cytokine의 분비를 효과적으로 억제할 수 있는 물질임을 확인할 수 있었다. Dichloromethane 분획물을 처리하였을 때, 염증 관련 단백질 발현 정도를 western blot을 통해 확인한 결과, LPS에 의해 발현이 증가된 $NF-{\kappa}B$, iNOS 및 COX-2는 분획물을 처리함으로써 농도 의존적으로 감소되는 것을 확인할 수 있었다. 또한 dichloromethane 분획물의 처리가 인산화된 MAPKs의 발현을 저해함을 확인하여 참치 심장 dichloromethane 분획물이 $NF-{\kappa}B$와 MAPKs의 발현을 억제시킴으로써 NO 및 pro-inflammatory cytokine의 분비량을 감소시킴을 확인할 수 있었다. 동물 모델에서는, dichloromethane 분획물을 처리하였을 때 croton oil에 의한 귀 부종이 농도 의존적으로 감소함을 확인하였고, 특히, $250mg/kg{\cdot}body\;weight$ 농도로 투여시 시판 항염증제인 predinisolone을 $50mg/kg{\cdot}body\;weight$ 농도로 투여한 그룹과 유사한 효과를 나타내었다. 조직학적 변화를 확인한 결과에서는, 진피와 경피의 두께가 감소하였으며 진피내 mast cell 침윤이 감소되는 것을 확인할 수 있었다. 따라서 참치심장 dichloromethane 분획물이 효과적인 염증 예방 및 부종 완화를 위한 치료제로서 활용 가능성을 확인하였다.

Keywords

References

  1. Kindt TJ, Goldsby RA, Osborne BA. 2007. Innate immunity, pp. 52-73. In. Tenney S (6th ed.), Kuby Immunology Freeman press, New York.
  2. Stuehr DJ, Cho HJ, Kwon NS, Weise MF, Nathan CF. 1991. Purification and characterization of the cytokine-induced macrophage nitric oxide synthase: an FAD- and FMN-containing flavoprotein. Proc. Natl. Acad. 1: 7773-7777.
  3. Murray PJ, Wynn TA. 2011. Protective and pathogenic functions of macrophage subsets. Nat. Rev. Immunol. 11: 723-737. https://doi.org/10.1038/nri3073
  4. Carter AB, Knudtson KL, Monick MM, Hunninghake GW. 1999. The p38 mitogen-activated protein kinase is required for NF-${\kappa}B$-dependent gene expression. The role of TATA-binding protein (TBP). J. Biol. Chem. 274: 30858-30863. https://doi.org/10.1074/jbc.274.43.30858
  5. Sung MJ, Davaatseren M, Kim W, Park SK, Kim SH, Hur HJ, et al. 2009. Vitisin A suppresses LPS-induced NO production by inhibiting ERK, p38, and NF-${\kappa}B$ activation in RAW 264.7 cells. Int. Immunopharmacol. 9: 319-323. https://doi.org/10.1016/j.intimp.2008.12.005
  6. Kang BK, Kim KBWR, Kim MJ, Bark SW, Pak WM, Kim BR, et al. 2014. Anti-inflammatory activity of an ethanol extract of Laminaria japonica root on lipopolysaccharide-induced inflammatory responses in RAW 264.7 cells. Korean J. Food Sci. Technol. 46: 729-733. https://doi.org/10.9721/KJFST.2014.46.6.729
  7. Kim KH, Choi MW, Lim SY. 2013. Effect of tuna extract on production of nitric oxide and inflammatory cytokines. Korean J. Food Sci. Technol. 45: 385-390. https://doi.org/10.9721/KJFST.2013.45.3.385
  8. Jang JR, Kim KK, Mun SB, Lim SY. 2009. In vitro anticancer and antioxidant effect of solvent extracts from tuna dried at low temperature vacuum. J. Life Sci. 19: 633-638. https://doi.org/10.5352/JLS.2009.19.5.633
  9. Pedersen MH, Molgaard C, Hellgren LI, Lauritzen L. 2010. Effects of fish oil supplementation on markers of the metabolic syndrome. J. Pediatr. 157: 395-400. https://doi.org/10.1016/j.jpeds.2010.04.001
  10. Tenore GG, Calabrese G, Ritieni A, Campiglia P, Gianetti D, Novellino E. 2014. Functional food potentially safer than commercial fish oil based pharmaceutical formulations. Food Chem. Toxicol. 71: 231-235. https://doi.org/10.1016/j.fct.2014.06.016
  11. Jung HS. 2007. Antioxidant effect of histidine containing low molecular weight peptide isolated from skipjack boiled extract. Korean J. Food Cookery Sci. 23: 221-226.
  12. Park SE, Kim HW, Lee SR, Kim BK. 2000. Effects of nucleic acids complex of tuna testis on immunological activities. J. Korean Assoc. Cancer Prev. 5: 15-23.
  13. Shin MO, Ku MJ, Bae SJ. 2007. Cytotoxicity and quinone reductase activity stimulating effects of fin of Thunnus thynnus extracts in various cancer cells. Korean J. Nutr. 40: 147-153.
  14. Park YM, Won JH, Yun KJ, Ryu JH, Han YN, Choi SK, et al. 2006. Preventive effect of Ginkgo biloba extract (GBB) on the lipopolysaccharide-induced expressions of inducible nitric oxide synthase and cyclooxygenase-2 via suppression of nuclear factor-${\kappa}B$ in RAW 264.7 cells. Biol. Pharm. Bull. 29: 985-990. https://doi.org/10.1248/bpb.29.985
  15. Lee ST, Jeong YR, Ha MH, Kim SH, Byun MW. 2000. Induction of nitric oxide and TNF-${\alpha}$ by herbal plant extract in mouse macrophage. J. Korean Soc. Food Sci. Nutr. 29: 342-348.
  16. Kim JY, Jung KS, Jeong HG. 2004. Suppressive effects of the kahweol and cafestol on cyclooxygenase-2 expression in macrophages. FEBS Lett. 569: 321-326. https://doi.org/10.1016/j.febslet.2004.05.070
  17. Han MH, Lee MH, Hong SH, Choi YH, Moon JS, Song MK, et al. 2014. Comparison of anti-inflammatory activities among ethanol extracts of Sophora flavescens, Glycyrrhiza uralensis and Dictamnus dasycarpus and their mixtures in RAW 264.7 murine macrophages. Korean J. Life Sci. 24: 329-335. https://doi.org/10.5352/JLS.2014.24.3.329
  18. Lim HR, Shin SW. 2010. Effects of the essential oil components from Ligusticum chuanxiong on proinflammatory mediators of RAW 264.7 macrophage cells. Korean Sci. Pharm. 16: 259-264.
  19. Pruett SB, Fan R, Zheng Q. 2003. Characterization of glucocorticoid receptor translocation, cytoplasmic $I{\kappa}B$, nuclear $NF{\kappa}B$, and activation of $NF{\kappa}B$ in T lymphocytes exposed to stressinducible concentrations of corticosterone in vivo. Int. Immunopharmacol. 3: 1-16. https://doi.org/10.1016/S1567-5769(02)00081-4
  20. Robinson MJ, Cobb MH. 1997. Mitogen-activated protein kinase pathways. Curr. Opin. Cell Biol. 9: 180-186. https://doi.org/10.1016/S0955-0674(97)80061-0
  21. Lee ES, Ju HK, Moon TC, Lee E, Jahng Y, Lee SH, et al. 2004. Inhibition of nitric oxide and tumor necrosis factor-alpha (TNF-alpha) production by prepenone compound through blockade of nuclear factor (NF)-${\kappa}B$ activation in cultured murine macrophages. Biol. Pharm. Bull. 27: 617-620. https://doi.org/10.1248/bpb.27.617