자외선에 의해 유도되는 단백질 손상에 미치는 효과

단백질을 구성하는 아미노산 중 lysine, arginine, proline은 산화반응에 의해 carbonyl유도체를 형성하며, 이 carbonyl group은 DNPH와 반응하여 안정한 형태의 hydrazone을 형성한다. 이렇게 형성된 hydrazone은 산화적 스트레스에 의한 단백 질의 손상 지표로 널리 사용되고 있다 (Levine RL, 2002). 자외선에 의한 단백질 손상에 미치는 FDP의 영향을 검토하기 위하여 anti-DNP 항체를 이용한 Western blot으로 피부 단백질의 손상정도를 측정하였다. 실험결과 UVB를 조사한 경우 표피에서 산화된 단백질이 유의적으로 증가하였으며, FDP를 도포한 경우에 표피 및 진피에서 산화된 단백질이 감소하였다 (Fig. 20).

피부에 자외선을 조사하면 생성되는 활성산소중의 하나인 H2O2를 hairless mouse 피부 파쇄액에 처리한 경우 대조군에 비해 산화된 단백질이 유의적으로 증가하였다 (149%). 항산화제로 알려진 ascorbic acid가 H2O2에 의한 산화단백질 생성을 유의적으로 억제한 반면, FDP는 H2O2에 의한 산화단백질 생성을 억제하 지 못하였다 (Fig. 21). 이는 DPPH 라디칼 소거효능에 대한 실험 결과와 마찬가지 로 자외선에 의해 유도되는 산화적 손상에 대한 FDP의 보호효과가 직접적인 라 디칼 소거에 의한 것이 아니라는 것을 의미한다.

Fig. 20. FDP prevents UVB-induced protein oxidation in hairless mouse skin.

Immunoblot analysis of carbonyl residues in protein extracts from epidermal (A) and dermal (B) tissues of hairless mouse skin. Protein extracts (20 µg) were incubated with DNPH, separated by SDS-PAGE, blotted onto a nitrocellulose membrane, and incubated with polyclonal rabbit anti-dinitrophenylhydrazone antibody. The -DNP lanes contain underivatized proteins for negative control. Lane 1, 4: non-irradiated control, lane 2, 5:

irradiated control, Lane 3, 6: UVB + FDP (1%).

Fig. 21. FDP can not prevent in vitro protein oxidation by hydrogen peroxide.

Immunoblot analysis of carbonyl residues in protein extracts from hydrogen-peroxide-treated (1 mM for 1 h at 37°C) mouse skin extracts. Protein extracts (10 µg) were incubated with

DNPH, and subjected to Western blot with polyclonal rabbit anti-dinitrophenylhydrazone antibody (A). Lane 1:non-treated control, Lane 2: H2O2 (1 mM), Lane 3: H2O2 + vit C (5 mM), Lane 4: H2O2 + FDP (5 mM). Total proteins were visualized by Coomassie Blue (B).

Densitometric analysis of total carbonylated proteins (C). The results are expressed as the mean ± S.E. of 3 different experiments. *p<0.05.

IV. 고 고 고 고 찰 찰 찰 찰

있음이 보고되었다 (Ahn SM 등, 2002). 본 연구에서 FDP는 각질형성세포 배양 모

하지 않았다 (Fig. 6). 자외선에 의해 hairless mouse 피부에서 MMP-2,9의 발현이

radical scavenging에 의한 작용인지 알아보기 위하여 실험해본 결과 FDP는 DPPH

라겐 합성을 감소시키고 MMP-1의 발현을 증가시켜 콜라겐 분해를 촉진시켜 피

ERK, JNK 및 p38이 인산화 되며, 이후 감소하기 시작하여 4시간 대에는 대조군

Keratinocyte에서 자외선에 의해 활성화되는 ERK에 의해 MMP-2의 발현이 조절된

얻었으나, 어떠한 기전에 의한 것인지는 확인할 수 없었다. 이상의 결과로 볼 때

적으로 UVB를 조사하여 산화적 stress를 유도하는 모델을 사용하였다. 100 mJ/cm²

생길 경우 산화적 stress나 활성산소의 평형상태에 영향을 주게 된다. 따라서 활

억제하여 MMP-2와 COX-2의 발현을 억제하는 것이라고 추정되지만 (Fig. 22) 좀 더 세밀한 연구가 진행되어야 할 것으로 생각된다. 또한 FDP는 피부에 도포하였 을 경우에 피부를 투과하여 자외선에 의해 유도되는 피부손상을 억제하는 효능 을 나타내므로 염증반응, 광노화, 피부암 등의 병리학적 증상을 예방하기 위한 목적으로 사용 가능할 것으로 생각된다.

Fig. 22. Schematic diagram for the inhibitory role of FDP in UV-induced skin inflammation. FDP could reduce COX-2 and MMP-2 expression by regulating UVB-induced alteration of cellular signaling pathway. FDP also could reduce the cellular UV-induced ROS generation by increasing metabolic flux through PPP.

V. 결 결 결 론 결 론 론 론

Fructose 1,6-diphosphate (FDP)가 자외선에 의해 유도되는 염증반응 및 산화적 스트레스를 억제할 수 있는지를 피부세포배양 모델과 in vivo 동물시험을 통해 연 구하였다.

FDP는 HaCaT keratinocytes에서 UVB에 의해 유도되는 세포손상을 억제하고 COX-2, MMP-2의 발현을 억제하였다. 또한 직접적인 라디칼 소거효능 없이 UVB 에 의한 세포내 항산화계의 손상을 보호하였다.

UVB에 의해 변화되는 세포내 신호전달계에 미치는 FDP의 영향을 COX-2와 MMP-2의 발현을 중심으로 연구해 본 결과 ERK, JNK, Akt 및 GSK3β의 인산화를 억제하였으며, AP-1, NF-κB, CREB의 활성화를 억제하였다.

Franz-type diffusion cell을 이용한 피부투과 시험결과 FDP는 피부에 도포하였 을 경우 투과할 수 있는 물질이었다. 따라서 UVB에 의해 유도되는 피부손상을 억제할 수 있는지 hairless mice 피부에 FDP를 도포하여 시험해 본 결과 FDP는 UVB에 의해 유도되는 항산화계 손상을 억제하고 산화된 단백질 생성을 억제하 는 효과를 나타내었다. 이러한 결과로 유추해 볼 때 FDP는 자외선에 의해 유도 되는 세포내 신호전달계를 조절하거나 항산화계를 보호함으로써 자외선에 의한 피부염증반응을 억제함으로써 피부손상을 보호할 수 있을 것으로 생각된다.

참고문헌 참고문헌 참고문헌 참고문헌

1. Ahn SM, Yoon HY, Lee BG, Park KC, Chung JH, Moon CH, Lee SH: Fructose-1,6-diphosphate attenuates prostaglandin E2 production and cyclo-oxygenase-2 expression in UVB-irradiated HaCaT keratinocytes. Br J Pharmaco. 137: 497-503, 2002

2. Akerboom TP, and Sies H: Assay of glutathione, glutathione disulfide, and glutathione mixed disulfides in biological samples. Methods Enzymol 77: 373-382, 1981

3. Ashida M, Bito T, Budiyanto A, Ichihashi M and Ueda M: Involvement of EGF receptor activation in the induction of COX-2 in HaCaT keratinocytes after UVB. Exp Dermatol 12: 445-452, 2003

4. Athar M: Oxidative stress and experimental carcinogenesis. Indian J Exp Biol 40: 656–

667, 2002

5. Barber LA, Spandau DF, Rathman SC, Murphy RC, Johnson CA, Kelley SW, Hurwitz SA, Travers JB: Expression of the platelet-activating receptor results in enhanced ultraviolet B radiation-induced apoptosis in a human epidermal cell line. J Bio. Chem 273: 18891–

18897, 1988

6. Baudhuin P, Beaufay H, Rahman-Li Y, Sellinger OZ, Wattiaux R, Jacques P, De Duve C:

Intracellular distribution of monoamine oxidase, aspartate aminotransferase, alanine aminotransferase, D-amino acid oxidase and catalase in rat liver tissue. Biochem J 92:

179-184, 1964

7. Beals CR, Sheridan CM, Turck CW, Gardner P, Crabtree GR: Nuclear export of NF-ATc enhanced by glycogen synthase kinase-3. Science 275: 1930-1934, 1997

8. Belkhiri A; Richards C; Whaley M; McQueen SA; Orr FW: Increased expression of activated matrix metalloproteinase- 2 by human endothelial cells after sublethal H2O2

exposure. Lab Invest 77: 533– 539, 1997

9. Bender K, Blattner C, Knebel A, Iordanov M, Herrlich P and Rahmsdorf HJ; UV-induced signal transduction. J Photochem Photobiol B 37: 1-17, 1997

10. Bicker DR, and Athar M: Oxidative stress in Pathogenesis of skin disease. J Invest Dermatol 126: 2565-2575, 2006

11. Bonizzi G, Piette J, Merville MP, Bours V: Cell type-specific role for reactive oxygen species in nuclear factor-kappaB activation by interleukin-1. Biochem Pharmacol 59: 7-11, 2000

12. Brenneisen P, Wenk J, Klotz LO, Wlaschek M, Briviba K, Krieg T, Sies H, Scharffetter-Kochanek K: Central role of ferrous/ferric iron in the ultraviolet B irradiation-mediated signaling pathway leading to increased interstitial collagenase (matrix-degrading metalloproteinase (MMP)-1) and stromelysin-1 (MMP-3) mRNA levels in cultured human dermal fibroblasts. J Biol Chem 273: 5279–5287, 1988

13. Bullock BP, Habener JF: Phosphorylation of the cAMP response element binding protein CREB by cAMP-dependent protein kinase A and glycogen synthase kinase-3 alters DNA-binding affinity, conformation, and increases net charge. Biochemistry 37: 3795-3809, 1998

14. Casagrande R, Georgetti SR, Verri WA. Jr, Dorta DJ, dos Santos AC, Fonseca MJ:

Protective effect of topical formulations containing quercetin against UVB-induced oxidative stress in hairless mice. J Photochem Photobiol B 84: 21-27, 2006

15. Cejkova J, Stipek S, Crkovska J, Ardan T: Changes of superoxide dismutase, catalase and glutathione peroxidase in the corneal epithelium after UVB rays. Histochemical and biochemical study. Histol Histopathol 15: 1043-1050, 2000

16. Chen W, Tang Q, Gonzales MS, Bowden GT: Role of p38 MAP kinases and ERK in

mediating ultraviolet-B induced cyclooxygenase-2 gene expression in human keratinocytes. Oncogene 20: 3921-3926, 2001

17. Chilcott RP, Jenner J, Carrick W, Hotchkiss SA, Rice P: Human skin absorption of Bis-2-(chloroethyl)sulphide (sulphur mustard) in vitro. J Appl Toxicol 20: 349-355, 2000

18. Cohly HH, Stephens JW, Angel MF, Johnson JC, Markov AK: The role of fructose-1,6-diphosphate in cell migration and proliferation in an in vitro xenograft blood vessel model of vascular wound healing. In vitro Cell Dev Bio 35: 510-514, 1999

19. Cross CE, Halliwell B, Borish ET, Pryor WA, Ames BN, Saul RL, McCord JM, Harman D; Oxygen radical and human disease. Ann Intern Med 107: 526-545, 1987

20. Edde L, Zhou X, Eaton JW, Sherman MP: Induction of nitric oxide synthase in macrophages: inhibition by fructose-1,6-diphosphate. Biochem Biophys Res Commun 243: 683-687, 1998

21. Ehringer WD, Niu W, Chiang B, Wang OL, Gordon L, Chien S: Membrane permeability of fructose-1,6-diphosphate in lipid vesicles and endothelial cells. Mol Cell Biochem 210: 35-45, 2000

22. Epstein JH: Photocarcinogenesis, skin cancer, and aging. J Am Acad Dermatol 9: 487-502, 1983

23. Espanol MT, Litt L, Hasegawa K, Chang L, Macdonald JM, Gregory G, James TL, Chan PH: Fructose-1,6-diphosphate preserves adenosine triphosphate but not intracellular pH during hypoxia in respiring neonatal rat brain slices. Anesthesiolog 88: 461-472, 1999

24. Farias LA, Smith EE, Markov AK: Prevention of ischemia-hypoxic brain injury and death in rabbits with fructose-1,6-diphosphate. Strok 21: 606-613. 1990

25. Firestein BL, Bredt DS: Interaction of neuronal nitric-oxide synthase and phosphor-fructokinase. J Biol Chem 274: 10545-10550, 1999

26. Franz T: Percutaneous absorption on the relevance of in vitro data. J Invest Dermatol 64:

190-195, 1975

27. Frost JA, Geppert TD, Cobb MH, Feramisco JR: A requirement for extracellular signal-regulated kinase (ERK) function in the activation of AP-1 by Ha-Ras, phorbol 12-myristate 13-acetate, and serum. Proc Natl Acad Sci 91: 3844–3848, 1994

28. Greaves MW, Sondergaard J: Pharmacological agents released in ultraviolet inflammation studies by continuous skin perfusion. J Invest Dermatol 54: 365-367, 1970

29. Haertel-Wiesmann M, Liang Y, Fantl WJ, Williams LT: Regulation of cyclooxygenase-2 and periostin by Wnt-3 in mouse mammary epithelial cells. J Biol Chem 257: 32046-32051, 2000

30. Han JH, Roh MS, Park CH, Park KC, Cho KH, Kim KH, Eun HC, Chung JH: Selective COX-2 inhibitor, NS-398, inhibits the replicative senescence of cultured dermal fibroblasts. Mech Ageing Dev 125: 359-366, 2004

31. Hruza L, Pentland AP: Mechanisms of UV-induced inflammation. J Invest Dermatol 100: 35S-41S, 1993

32. Hutcheson AE, Rao MR, Olinde KD, Markov AK: Myocardial toxicity of cyclosporin A:

inhibition of calcium ATPase and nitric oxide synthase activities and attenuation by fructose-1,6-diphosphate in vitro. Res Commun Mol Pathol Pharmacol 89: 17-26, 1995

33. Kamata H, Hirata H : Redox regulation and cellular signaling. Cell Signal 11: 1-14, 1999

34. Kang-Rotondo CH, Miller CC, Morrison AR, Pentland AP: Enhanced keratinocyte prostaglandin synthesis after UV injury is due to increased phospholipase activity. Am J Physiol 264: 396-401, 1993

35. Kelleher JA, Chan TYY, Chan PH, Gregory GA: Protection of astrocytes by fructose 1,6 diphosphate and citrate ameliorates neuronal injury under hypoxic conditions. Brain Res 726: 167-173, 1996

36. Kellher JA, Chan PH, Chan TYY and Gregory GA: Energy metabolism in hypoxic astrocytes: protective mechanis of fructose 1,6-bisphosphate. Neurochem Res 20: 785-792, 1995

37. Kim ES, Kim MS, Moon A: TGF-beta-induced upregulation of MMP-2 and MMP-9 depends on p38 MAPK, but not ERK signaling in MCF10A human breast epithelial cells.

Int J Oncol 25: 1375-1382, 2004

38. Lee YS, Jin DQ, Beak SM, Lee ES, Kim JA: Inhibition of ultraviolet-A-modulated signaling pathways by Asiatic acid and ursolic acid in HaCaT human keratinocytes. Eur J Pharmacol 476: 173-178, 2003

39. Levine RL, Williams JA, Stadtman ER, Shacter E: Carbonyl assays for determination of oxidatively modified proteins. Methods Enzymol 233: 346-357, 1994

40. Levine RL: Carbonyl modified proteins in cellular regulation, aging, and disease. Free Radic Biol Med 32: 790-796, 2002

41. Markov AK, Brumley MA, Figueroa A, Skelton TN, Lehan PH: Hemodynamic effects of fructose 1,6-diphosphate in patients with normal and impaired left ventricular function.

Am Heart J 133: 541-549, 1997

42. Mathews-Roth MM, Krinsky NI: Carotenoid dose level and protection against UVB-induced skin tumor. Photochem Photobiol 42: 35-38, 1985

43. McCawley LJ, Matrisian LM: Matrix metalloproteinases: they're not just for matrix anymore! Curr Opin Cell Biol 13: 534-540, 2001

44. McCord J. and Fridovich I: Superoxide dismutase; An enzymatic function for erythrocuprein. J Biol Chem 244: 6049-6055, 1969

45. Miller CC, Hale P, Pentland AP: Ultraviolet B injury increases prostaglandin synthesis

through a tyrosine kinase-dependent pathway. Evidence for UVB-induced epidermal growth factor receptor activation. J Biol Chem 269: 3529-3533, 1994

46. Moan J, Dahlback A: The relationship between skin cancers, solar radiation and ozone depletion. Br J Cancer 65: 916-921, 1992

47. Mukhtar H., Elmets CA: Photocarcinogenesis: mechanisms, models and human health implications. Photochem Photobiol 63: 355-447, 1996

48. Munger MA, Botti RE, Grinblatt MA, Kasmer RJ: Effect of intravenous fructose-1,6-diphosphate on myocardial contractility in patients with left ventricular dysfunction.

Pharmacotherapy 14: 522-528, 1994

49. Nikolakaki E, Coffer PJ, Hemesoet R, Woodgett JR, Defize LH: Glycogen synthase kinase 3 phosphorylates Jun family members in vitro and negatively regulates their transactivating potential in intact cells. Oncogene 8: 833-840, 1993

50. Niu W, Zhang F, Ehringer W, Tseng M, Gray L, Chien S: Enhancement of hypothermic heart preservation with fructose 1, 6-diphosphate. J Surg Res 85: 120-129, 1999

51. Nomura M, Kaji A, He Z, Ma W-Y, Myamoto K, Yang CS, Dong Z: Inhibitory mechanisms of tea polyphenols on ultraviolet B-activated phosphatidylinositol 3-kinase-dependent pathway. J Biol Chem 276: 46624-46631, 2001

52. Okada K, Takahashi Y, Ohnishi K, Ishikawa O, Miyachi Y: Time-dependent effect of chronic UV irradiation on superoxide dismutase and catalase activity in hairless mice skin. J Dermatol Sci 8: 183-186, 1994

53. Onoue S, Kobayashi T, Takemoto Y, Sasaki I, Shinkai H: Induction of matrix metalloproteinase-9 secretion from human keratinocytes in culture by Ultraviolet B irradiation. J Dermatol Sci 33: 105-111, 2003

54. Pamela CC, Richard AH: Lippincott’s Illustrated Reviews Biochemestry 2^nd ed.

Lippincott-Raven, Hampton, New jersey, pp113-117, 1994

55. Park JY, Kim EJ, Kwon KJ, Jung YS, Moon CH, Lee SH, Baik EJ: Neuroprotection by fructose-1,6-bisphosphate involves ROS alterations via p38 MAPK/ERK. Brain Res 1026: 295-301, 2004

56. Pentland AP, Schoggins JW, Scott GA, Kahn KNM, Han R: Reduction of UV-induced skin tumors in hairless mice by selective COX-2 inhibition. Carcinogenesis 20:

1939-1944, 1999

57. Ranganathan AC, Nelson KK, Rodriguez AM, Kim KH, Tower GB, Rutter JL, Brinckerhoff CE, Epstein CJ, Huang TT, Jeffrey JJ, Melendez JA: Manganese superoxide dismutase signals matrix metalloproteinase expression via H2O2-dependent ERK1,2 activation. J Biol Chem 276: 14264– 14270, 2001

58. Rao MR, Olinde KD, Markov AK: Protection from amphotericin B-induced lipid peroxidation in rats by fructose-1,6-diphosphate. Res Commun Mol Pathol Pharmacol 95: 217-220, 1997

59. Rittie L, Fisher: GJ UV-light-induced signal cascades and skin aging. Aging Res Rev 1:

705-720, 2002

60. Salvemini F, Franze A, Iervolino A, Filosa S, Salzano S, Ursini MV: Enhanced glutathione levels and oxidoresistance mediated by increased glucose-6-phosphate dehydrogenase expression. J Biol Chem 274: 2750-2757, 1999

61. Sander CS, Chang H, Salzmann S, Muller CS, Ekanayake-Muduyanselage S, Elsner P, Thiele JJ: Photoaging is associated with protein oxidation in human skin in vivo. J

Invest Dermatol 118: 618-662, 2002

62. Sano W, Watanabe F, Tamai H, Furuya E, Mino M: Beneficial effect of fructose-1,6-bisphosphate on mitochondrial function during ischemia-reperfusion of rat liver.

Gastroenterology 108: 1785-1792, 1995

63. Satrino J, Schlondorff D: Activation and attenuation of transcription factor NF-κB in mouse glomerular mesangial cells in response to tumor necrosis factor-α, immunoglubin γ, and adenosine 3’,5’-cyclic monophosphate. J Clin Invest 94: 1629-1636, 1994

64. Shindo Y, Witt E, Han D, Packer L: Dose-response effects of acute ultraviolet irradiation on antioxidants and molecular markers of oxidation in murine epidermis and dermis. J Invest Dermatol 102: 470-475, 1994

65. Shindo Y, Witt E, Packer L: Antioxidant defense mechanisms in murine epidermis and dermis and their response to ultraviolet light. J Invest Dermatol 102: 260-265, 1993

66. Sola A, Berrios M, Sheldon RA, Ferreiro DM, Gregory GA: Fructose 1,6 bisphosphate after hypoxic ischemic injury is protective to the neonatal rat brain. Brain Res 742: 294-299, 1996

67. Soriani M, Luscher P, Tyrrell RM: Direct and indirect modulation of ornithine decarboxylase and cyclooxygenase by UVB in human skin cells. Carcinogenesis 20:

727-732, 1999

68. Stadtman ER; Protein oxidation in aging and age-related diseases. Ann N Y Acad Sci 928: 22-38, 2001

69. Sun JX, Farias LA, Markov AK: Fructose 1,6 diphosphate prevents intestinal ischemia reperfusion injury and death in rats. Gastroenterology 98: 117-126, 1990

70. Suomela S, Kariniemi AL, Impola U, Karvonen SL, Snellman E, Uurasmaa T, Peltonen J, Saarialho-kere U: Matrix metalloproteinase-19 is expressed by keratinocytes in psoriasis.

Acta Derm Venereol 83: 108-114, 2003

71. Takeuchi K, Cao-Danh H, Friehs I, Glynn P, D’Agostino D, Simplaceanu E, McGowan FX, del Nido PJ; Administration of fructose-1,6-diphosphate during early reperfusion significantly improves recovery of contractile function in the postischemic heart. J Thorac Cardiovasc Surg 116: 335-343, 1998

72. Tang Q, Gozales M, Inoue H, Bowden GT: Roles of Akt and glycogen kinase 3beta in the

ultraviolet B induced cyclooxygenase-2 transcription in human keratinocyte, Cancer Res 61: 4329-4332, 2001

73. Thiele JJ, Hsieh SN, Briviba K, Sies H; Protein oxidation in human stratum corneum:

susceptibility of keratins to oxidation in vitro and presence of keratin oxidation gradient in vivo. J Invest Dermatol 113: 335-339, 1999

74. Van Dross RT, Hong X, Pelling JC: Inhibition of TPA-induced COX-2 expression by apigenin through downregulation of Akt signal transduction. Mol carcinogenesis 44: 83-91, 2005

75. Vayaili PK, Mittal A, Hara Y, Elmets CA, Katiyar SK: Green tea polyphenols prevent ultravilolet light-induced oxidative damage and matrix metalloproteinase expression in mouse. J Invest Dermatol 122: 1480-1487, 2004

76. Vayalil PK, Elmets CA, Katiyar SK: Treatment of green tea polyphenols in hydrophilic

76. Vayalil PK, Elmets CA, Katiyar SK: Treatment of green tea polyphenols in hydrophilic