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Inhibitory Efficacy of Dystaenia takeshimana Extract on iNOS, COX-2 Protein and mRNA Expression in Raw 264.7 Cell

Raw 264.7 세포에서 섬바디나물 추출물의 iNOS, COX-2 단백질 및 mRNA 발현 억제 효과

  • Lee, Jin-Young (Department of Herbal Cosmetic Science, Hoseo University) ;
  • Yoo, Dan-Hee (Department of Herbal Cosmetic Science, Hoseo University) ;
  • Joo, Da-Hye (Department of Herbal Cosmetic Science, Hoseo University) ;
  • Chae, Jung-Woo (Gyeonggi-do Forest Environment Research Institute)
  • 이진영 (호서대학교 한방화장품과학과) ;
  • 유단희 (호서대학교 한방화장품과학과) ;
  • 주다혜 (호서대학교 한방화장품과학과) ;
  • 채정우 ((재)경기도산림환경연구소)
  • Received : 2016.10.27
  • Accepted : 2016.11.23
  • Published : 2016.12.28

Abstract

In this study, the anti-inflammatory activities of the 80% ethanol extract of Dystaenia takeshimana (DT) were investigated using Raw 264.7 cells treated with lipopolysaccharide (LPS). The effect of DT extract on the production of pro-inflammatory factors (iNOS, COX-2) in LPS-stimulated Raw 264.7 macrophages was examined. The cytotoxic effect of DT extract on macrophage cells (Raw 264.7) was examined by the 3-[4, 5-dimethyl-thiazol-2-yl]-2, 5-diphenyl-tetrazoliumbromide (MTT) assay. Treatment with DT extract showed 100% or more cell viability at the concentration $1,000{\mu}g/ml$. The inhibitory effect of DT extract on protein expression of inducible NOS (iNOS) and cyclooxygenase-2 (COX-2) was measured by western blotting using the concentrations 50, 100, and $500{\mu}g/ml$, with ${\beta}-actin$ used as the positive control. Consequently, the protein expression of iNOS, and COX-2 as observed by western blotting, was decreased by 56%, 61.6%, respectively with $500{\mu}g/ml$ DT extract. Inhibition of iNOS and COX-2 mRNA expression was measured by reverse transcription- polymerase chain reaction (PCR) using DT extract concentrations 50, 100, and $500{\mu}g/ml$, with GAPDH used as a positive control. Consequently, the mRNA expression of iNOS and COX-2 as observed by reverse-transcription-PCR was decreased by 77.9% and 83.3%, respectively at $500{\mu}g/ml$ concentration of DT extract. In conclusion, DT extract may affect inflammatory factors as a potential anti-inflammatory agent.

본 연구에서는 섬바디 나물의 항염증 효과를 알아보기 위하여 LPS로 염증을 유도한 Raw 264.7 세포에 대한 섬바디나물 80% 에탄올 추출물의 효과를 살펴보았다. 섬바디 나물 추출물을 LPS로 유도된 Raw 264.7 대식세포에서 전염증성인자(iNOS, COX-2)들을 생성하여 측정하였다. 섬바디 나물 추출물의 대식세포에서의 세포 독성 측정을 MTT를 수행하였다. 섬바디 나물 추출물의 세포 독성을 측정한 결과, $1,000{\mu}g/ml$의 농도에서 100% 이상의 세포 생존율을 보였다. 섬바디 나물 추출물의 50, 100, $500{\mu}g/ml$ 농도에서 iNOS와 COX-2의 단백질 발현 억제 효과를 측정하기 위해 western blot을 통해 측정하였고, 양성대조군으로는 ${\beta}-actin$을 사용하였다. 그 결과, 섬바디 나물 추출물을 western blot을 통해 측정한 iNOS, COX-2의 단백질 발현 억제 효과는 $500{\mu}g/ml$ 농도에서 각각 56%, 61.6%로 감소하였다. 섬바디 나물 추출물의 50, 100, $500{\mu}g/ml$ 농도에서 iNOS, COX-2의 mRNA의 발현억제 효과를 측정하기 위해 RT-PCR을 통해 측정하였으며, 양성 대조군으로 GAPDH를 사용하였다. 그 결과, 섬바디 나물 추출물을 RT-PCR을 통해 iNOS, COX-2의 mRNA 발현 억제 효과를 측정한 결과는 $500{\mu}g/ml$에서 각각 77.9%, 83.3%로 감소하였다. 이를 통해, 섬바디 나물 추출물은 염증을 억제시켜 주는 가능성이 있는 항염증 물질로써의 효과가 있을 것으로 보여진다.

Keywords

References

  1. Carmichael J, DeGraff WG, Gazdar AF, Minna JD, Mitchell JB. 1987. Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res. 47: 936-942.
  2. Editor of Ulleunggun. 2002. Wild plants of Ulleung Island, Ulleunggun.
  3. Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR. 1982. Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids. Anal. Biochem. 126: 131-138. https://doi.org/10.1016/0003-2697(82)90118-X
  4. Heinzemann A, Daser A. 2002. Mouse models for the genetic dissection of atopy. Int. Arch. Allergy Immunnol. 127: 170-180. https://doi.org/10.1159/000053861
  5. Higuchi MN, Hisgahi J, Taki H, Osawa T. 1990. Cytolytic mechanism of activetaed macrophages. Tumor necrosis factor and Larginine- dependent mechanism act synergistically as the major cytolytic mechanism of activated macrophages. J. Immunol. 144: 1425-1431.
  6. Jung TH. 1965. Hanguksikmuldogam, pp.897-898.
  7. Kim HJ, Lee DJ, Ku JJ, Choi K, Park KW, Kang SH, et al. 2013. Anti-inflammatory effect of extracts from folk plants in Ulleung island. Korean J. Plant Res. 26: 169-177. https://doi.org/10.7732/kjpr.2013.26.2.169
  8. Kim JS, Kim JC, Shim SH, Lee EJ, Jin WY, Bae KH, et al. 2006. Chemical constituents of the root of Dystaenia takeshimana and thier anti-inflammatory activity. Arch. Pharm. Res. 26: 671-623.
  9. Kim MY. 2004. Endemic plants of Korea, Solgwahak.
  10. Kim Y, Min HY, Park HJ, Lee EJ, Park EJ, Hwang HJ, et al. 2004. Suppressive effects of nitric oxide production and inducible nitric oxide synthase (iNOS) gene expression by Calystegia soldanella methaxl extract on lipopolysaccharide-activated RAW 264.7 cells. Eur. J. Cancer Prev. 13: 419-424. https://doi.org/10.1097/00008469-200410000-00010
  11. Ko KS, Jeon ES. 2003. Wild plants of Korea. Iljinsa.
  12. Lebert DC, Huttenlocher A. 2014. Inflammation and wound repair. Semin Immunol. 26: 315-320. https://doi.org/10.1016/j.smim.2014.04.007
  13. Lee CB. 1980. Daehansikmuldogam 2, Hyangmunsa.
  14. Lee CN, An HS. 1963. Hanguksikmulmyunggam, pp.144. Bumhaksa.
  15. Lee TH, Kwak HB, Kim HH, Lee ZH, Chung DK, Baek NI, et al. 2007. Methanol extracts of Stewartia koreana inhibit cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) gene expression by blocking $NF-{\kappa}B$ transactivation in LPS-activated raw264.7 cells. Mol. Cells 23: 398-404.
  16. Lee YM. 2003. Wild flowers of Korea, Darumsaesang.
  17. Namkoong S, Jang SA, Sohn EH, Bak JP, Sohn ES, Koo HJ, et al. 2015. Comparative study of Litsea japonica leaf and fruit extract on the anti-inflammatory effects. Korean J. Plant Res. 28: 145-152. https://doi.org/10.7732/kjpr.2015.28.2.145
  18. Nathan C. 1997. Inducible nitric oxide synthase: what difference does it make?. J. Clin. Invest. 100: 2417-2423. https://doi.org/10.1172/JCI119782
  19. Rainsford KD. 2007. Anti-inflammatory drugs in the 21st century. Sub-cell Biochem. 42: 3-27.
  20. Reuter S, Gupta SC, Chaturvedi MM, Aggarwal, BB. 2010. Oxidative stress, inflammation and cancer: How are they linked?. Free Radic. Biol. Med. 49: 1603-1616. https://doi.org/10.1016/j.freeradbiomed.2010.09.006
  21. Sung YY, Kim DS, Yang WK, Nho KJ, Seo HS, Kim YS, et al. 2012. Inhibitory effects of Drynaria fortunci extracts on house dist mite antigen-induced atopic dermatitis in NC/Nca mice. J. Ethnopharmacol. 144: 94-100. https://doi.org/10.1016/j.jep.2012.08.035