Glycoprotein Isolated from Morus indica Linne Has an Antioxidative Activity and Inhibits Signal Factors Induced by Bisphenol A in Raw 264.7 Cells

뽕잎 당단백질의 항산화능과 Raw 264.7 세포에 있어서 bisphenol A에 유도된 신호전달인자의 억제

  • Shim, Jae-Uoong (Molecular Biochemistry Laboratory, Biotechnology Research Institute & Center for the Control of Animal Hazards Using Biotechnology, Chonnam National University) ;
  • Lee, Sei-Jung (Molecular Biochemistry Laboratory, Biotechnology Research Institute & Center for the Control of Animal Hazards Using Biotechnology, Chonnam National University) ;
  • Oh, Phil-Sun (Molecular Biochemistry Laboratory, Biotechnology Research Institute & Center for the Control of Animal Hazards Using Biotechnology, Chonnam National University) ;
  • Lim, Kye-Taek (Molecular Biochemistry Laboratory, Biotechnology Research Institute, Chonnam National University)
  • 심재웅 (전남대학교 생물공학연구소 분자생화학 교실 & 동물유해인자 제어를 위한 전문인력 양성 사업팀) ;
  • 이세중 (전남대학교 생물공학연구소 분자생화학 교실 & 동물유해인자 제어를 위한 전문인력 양성 사업팀) ;
  • 오필선 (전남대학교 생물공학연구소 분자생화학 교실 & 동물유해인자 제어를 위한 전문인력 양성 사업팀) ;
  • 임계택 (전남대학교 생물공학연구소 분자생화학 교실)
  • Published : 2007.04.30

Abstract

The present study investigated anti-oxidative and anti-inflammatory activity of glycoprotein isolated from Morus Indica Linne (MIL glycoprotein). We found that MIL glycoprotein has a molecular weight of 32 kD and consists of carbohydrate (40.03%) and protein (59.97%), and that it has a strong scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl radical $({\cdot}OH)$, and superoxide anion $(O_2{\cdot}\;^-)$ radicals. In addition, MIL glycoprotein had a stable character and an optimal DPPH radical scavenging activity in the alkaline and neutral pH solution, and up to at 105. However, the results indicated that it has a minimal scavenging activity in the metal ionic solution ($Ca^{2+}$, $Mn^{2+}$, and $Mg^{2+}$) in the presence of EDTA. In addition, we further investigated whether MIL glycoprotein scavenges oxygen radicals and blocks inflammation-related signals in the bisphenol A (BPA)-stimulated Raw 264.7 cells. The results in this study showed that it has a character to scavenge the productions of reactive oxygen species (ROS) and nitric oxide (NO) dose-dependently. Also it blocked the activities of inflammation-related signals such as nuclear factor-kappa B ($NF-{\kappa}B$) and inducible nitric oxide synthase (iNOS). For example, it had an inhibitory effect on the activation of $NF-{\kappa}B$ (p50) and iNOS proteins at 200 ${\mu}g/mL$ MIL glycoprotein. Here, we speculate that MIL glycoprotein is one of natural antioxidants and of modulators of the BPA-induced inflammation.

본 연구에서는 뽕잎 당단백질의 활성을 알아보기 위하여 뽕잎 당단백질의 안정성 및 특성을 알아보고, hydroxyl 라디칼, superoxide anion 라디칼, DPPH 라디칼 등의 활성 산소종에 대한 항산화 효과를 살펴보았다. 또한 Raw 264.7 세포에 환경호르몬의 일종인 BPA와 뽕잎 당단백질(32 kDa)을 함께 처리 하여, 뽕잎 당단백질의 활성산소종과 NO의 소거 능력뿐만 아니라 염증 매개성 단백질들 [$NF-{\kappa}B(p50)$와 iNOS]의 활성 억제능력 대하여 평가하였다. 뽕이 당단백질은 금속이온에는 다소 약하지만 온도와 pH에는 안정적인 특징을 지니고 있었으며, 탁월한 hydroxyl 라디칼, superoxide anion 라디칼, DPPH 라디칼 소거 능력을 가지고 있었다. 이러한 항산화 능력을 지닌 뽕잎 당단백질을 BPA와 함께 Raw 264.7 세포에 처리한 결과, BPA만 처리된 Raw 264.7 세포의 활성 산소종 양은 8시간째에, 그리고 NO 양은 24시간째에 현저히 증가한데 반해, BPA와 함께 뽕잎 당단백질을 처리한 Raw 264.7 세포에서는 같은 시간 동안에 농도에 비례하여 활성 산소종 및 NO양이 유의적으로 감소한 것을 알 수 있었다. 또한 8시간 동안 BPA처리에 의해 활성화된 Raw 264.7 세포의 $NF-{\kappa}B(p50)$와 iNOS 단백질들은 함께 처리한 뽕잎 당단백질의 농도에 비례하여 현저히 억제되었다. 따라서, 이러한 결과를 종합해 보면, 뽕잎 당단백질은 높은 안정성과 강력한 항산화 효과를 가지고 있었으며, 이러한 항산화 효과가 환경 호르몬(BPA)에 의한 활성산소종 및 NO 생성을 저해할 뿐만 아니라, $NF-{\kappa}B(p50)$와 iNOS의 활성을 억제함으로써 Raw 264.7 세포의 염증 신호전달을 막는데 영향을 끼쳤을 것으로 생각 된다.

Keywords

References

  1. Kim SW, Kim JS, Ryu HM, Nam JS, Cheigh HS, Min BT, Park SH, Yoo M. Toxic effect of endocrine disruptors on the growth rate of lactic acid bacteria. J. Exp. Biomed. Sci. 10: 403-406 (2004)
  2. Bindhumol V, Chitra KC, Mathur PP. Bisphenol A induces reactive oxygen species generation in the liver of male rats. Toxicology 188: 117-124 (2003) https://doi.org/10.1016/S0300-483X(03)00056-8
  3. Bradley EG, Timothy RZ. Exoestrogens: Mechanisms of action and strategies for identification and assessment. Environ. Toxicol. Chem. 17: 3-14 (1998) https://doi.org/10.1897/1551-5028(1998)017<0003:EMOAAS>2.3.CO;2
  4. Kim JY, Jeong HG. Down-regulation of inducible nitric oxide synthase and tumor necrosis factor-${\alpha}$ expression by bisphenol A via nuclear factor-${\kappa}B$ inactivation in macrophages. Cancer Lett. 196: 69-76 (2003) https://doi.org/10.1016/S0304-3835(03)00219-2
  5. Min HY, Park EJ, Lee SK, Cho YJ. Effects of grape extracts on free radical scavenging activity and inhibition of pro-inflammatory mediator production in mouse macrophage cells. Korean J. Food Sci. Technol. 35: 132-137 (2003)
  6. Heo KS, Lee SJ, Ko JH, Lim K, Lim KT. Glycoprotein isolated from Solanum nigrum L. inhibits the DNA-binding activities of NF-${\kappa}B$ and AP-1, and increases the production of nitric oxide in TPA-stimulated MCF-7 cells. Toxicol. In Vitro 18: 755-763 (2004) https://doi.org/10.1016/j.tiv.2004.03.003
  7. Lim KT, Heo KS, Son YO. Antioxidative and microbial effects of glycoprotein isolated from solanum nigrum Linne. Food Sci. Biotechnol. 11:484-489 (2002)
  8. Lee SJ, Heo KS, Oh PS, Lim K, Lim KT. Glycoprotein isolated from Ulmus davidiana Nakai inhibits TPA-induced apoptosis through nuclear factor-kappa B in NIH/3T3 cells. Toxicol. Lett. 146: 159-174(2004) https://doi.org/10.1016/j.toxlet.2003.10.005
  9. Ko JH, Lee SJ, Lim KT. 36 kDa Glycoprotein isolated from Rhus verniciflua Stokes fruit has a protective activity to glucose/glucose oxidase-induced apoptosis in NIH/3T3 cells. Toxicol, In Vitro 19:353-363 (2005) https://doi.org/10.1016/j.tiv.2004.10.006
  10. Lim KT, Lee SJ, Ko JH, Oh PS. Hypolipidemic effects of glycoprotein isolated from Ficus Carica Linnoeus in mice. Korean J. Food Sci. Technol. 37: 624-630 (2005)
  11. Jin YS, Sa JH, Shim TH, Rhee HI, Wang MH. Hepatoprotective and antioxidant effects of Morus bombicis Koidzumi on $CCl_4$-indyced liver damage. Biochem. Bioph. Res. Co. 329: 991-995 (2005) https://doi.org/10.1016/j.bbrc.2005.02.076
  12. Basnet P, Kadota S, Terashima S, Simazu S, Namba T. Two new 2-arylbenzofuran derivatives from hypoglycemic activity-bearing fractions of Morus insignis. Chem. Pharm. Bull. 41: 1238-1243 (1993) https://doi.org/10.1248/cpb.41.1238
  13. Kim SY, Lee WC. The effect of mulberry on inhibition of HMG-Co A reductase activity. RDA J. Agr. Sci. 38: 133-139 (1996)
  14. Yen GC, Wu SC, Duh PD. Extraction and identification of antioxidant components from leaves of mulberry (Morus alba L.). J. Agr. Food Chem. 44: 1687-1690 (1996) https://doi.org/10.1021/jf9503725
  15. Kim SY, Lee WC, Kim HB, Kim AJ, Kim SK. Antihyperlipidemic effects of methanol extracts from mulberry leaves in cholesterol- induced hyperlipidemia rats. J. Korean Soc. Food Sci. Nutr. 27: 1217-1222 (1998)
  16. Chae JY, Lee JY, Hong IS, Whangbo D, Choi PW, Lee WC, Kim JW, Choi SW, Ree SJ. Analysis of functional components of leaves of different mulberry cultivars. J. Korean Soc. Food Sci. Nutr. 32: 15-21 (2003) https://doi.org/10.3746/jkfn.2003.32.1.015
  17. Neville DM Jr, Glossmann H. Molecular weight determination of membrane protein and glycoprotein subunits by discontinuous gel electrophoresis in dodecyl sulfate. Method Enzymol. 32: 92-102 (1974) https://doi.org/10.1016/0076-6879(74)32012-5
  18. Lowry OH, Rosebrough NT, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J. BioI. Chem. 193: 265-275(1951)
  19. Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28: 350-356 (1956) https://doi.org/10.1021/ac60111a017
  20. Maffei Facino R, Carini M, Aldini G, Berti F, Rossoni G. Panax ginseng administration in the rat prevents myocardial ischemiareperfusion damage induced by hyperbaric oxigen: evidence for an antioxidant intervention. Planta Med. 65: 614-619 (1999) https://doi.org/10.1055/s-1999-14034
  21. Gotoh N, Niki E. Rates of interaction of superoxide with vitamin E, vitamin C, and related compounds as mesured by chemiluminescence. Biochim. Biophys. Acta 1115: 201-207 (1992) https://doi.org/10.1016/0304-4165(92)90054-X
  22. Halliwell B, GutteridgeJM, Aruoma OI. The deoxyribose method: a simple 'testtube' assay for determination of rate constants for reactions of hydroxyl radicals. Anal. Biochem. 29: 289-299 (1987)
  23. Wang H, Joseph JA. Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader. Free Radical Bio. Med. 27: 612-616 (1999) https://doi.org/10.1016/S0891-5849(99)00107-0
  24. Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR. Analysis of nitrate, nitrite,and [15N] nitratein biological fluids. Anal. Biochem. 126: 131-138 (1982) https://doi.org/10.1016/0003-2697(82)90118-X
  25. Ooi VEC. Liu F. Immunomodulation and anti-cancer activity of polysaccharide-protein complexes. Curr. Med. Chem. 7: 715-729 (2000) https://doi.org/10.2174/0929867003374705
  26. Oh JH, Kim EH, Kim JL, Moon YI, Kang YH, and Kang JS. Study on antioxidant potency of green tea by DPPH method. J. Korean Soc. Food Sci. Nutr. 33: 1079-1084 (2004) https://doi.org/10.3746/jkfn.2004.33.7.1079
  27. Lee SJ, Lim KT. Antioxidative effects of glycoprotein isolated from Solanum nigrum Linne on oxygen radicals and its cytotoxic effects on the MCF-7 cell. J. Food Sci. 68: 466-470 (2003) https://doi.org/10.1111/j.1365-2621.2003.tb05695.x
  28. Oh PS, Lee SJ, Lim KT. Antioxidative activity of 90 kDa glycoprotein isolated from Opuntia ficus-indica var. saboten Makino. Food Sci. Biotechnol. 13: 781-789 (2004)
  29. Lee SJ, Oh PS, Ko JH, Lim K, Lim KT. Glycoprotein isolated from Gardenia jasminoides Ellis has a scavenging activity against oxygen radicals and inhibits the oxygen radical-induced protein kinase C alpha and nuclear factor-kappa B in NIH/3T3 cells. Environ. Toxicol. Pharmacol. 21: 8-21 (2006) https://doi.org/10.1016/j.etap.2005.04.003
  30. Nguyen T, Brunson D, Crespy CL, Penman BW, Wishnok JS, Tannenbaum SR. DNA damages and mutation in human cells exposed to nitric oxide in vitro. P. Natl. Acad. Sci. USA 89: 3030-3034 (1992) https://doi.org/10.1073/pnas.89.7.3030
  31. Nichols TC, Fischer TH, Deliargyris EN, Baldwin AS Jr. Role of nuclear factor-${\kappa}B$ (NF-${\kappa}B$) in inflammtion, periodontitis, and atherogenesis. Ann. Periodontol. 6: 20-29 (2001) https://doi.org/10.1902/annals.2001.6.1.20
  32. Lee JH, Choi YH, Choi BT. The aqueous extract from Platycodi radix attenuates lipopolysaccharide-induced NF-${\kappa}B$ activation in Raw 264.7 cell and acute lung injury in rats. J. Korean Assoc. Cancer Prevention 7: 87-93 (2002)
  33. Oh PS, Lee SJ, Lim KT. Glycoprotein isolated from Rhus verniciflua Stokes inhibits inflammation-related protein and nitric oxide production in LPS-stimulated RAW 264.7 cells. Biol. Pharm. Bull. 30: 111-116 (2007) https://doi.org/10.1248/bpb.30.111