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1 대한안과학회지 2013 년제 54 권제 3 호 J Korean Ophthalmol Soc 2013;54(3): pissn: eissn: = 증례보고 = 섬유주세포에서 Dipyridamole 이활성산소종과산화스트레스에미치는영향 이근우 김재우 대구가톨릭대학교의과대학안과학교실 목적 : 배양된섬유주세포에서 dipyridamole (DPD) 이활성산소종의생성과산화스트레스에미치는영향을알아보고자하였다. 대상과방법 : DPD 의항산화능은 DPPH assay 로조사하였다. 인체의섬유주세포를일차배양한후 0, 20, 50 μm DPD 에노출시켜 DCFHDA assay 로활성산소종의생성에미치는영향을조사하였으며, t-butyhlhydroperoxide (tbhp) 로유발한산화스트레스에대해세포활성도에미치는영향을 resazurin assay 로조사하였다. 결과 : DPD 은유의한항산화능을나타내었다 (p<0.05). 인체의섬유주세포에서 DPD 은유의하게활성산소종의생성을감소시켰으며 (p <0.05), tbhp 로유도된산화스트레스에대해 DPD 는유의하게세포활성도를증가시켰다 (p<0.05). DPD 은일산화질소의생성에는유의한영향을미치지않았다. 결론 : DPD 는섬유주세포에서항산화능력과세포보호작용을나타낼수있을것으로생각한다. < 대한안과학회지 2013;54(3): > 섬유주세포는방수유출로의조절에중요한역할을하는데, 섬유주의변성으로인해방수유출로의저항이증가되어개방각녹내장을유발하는것으로알려졌다. 1,2 따라서섬유주세포를보호하여그기능을유지또는회복할수있다면섬유주를통한방수유출을조절하는데많은도움이될것이다. 섬유주세포는내피세포와식세포로서의역할외에도형태학적연구와전기생리학적연구에서평활근과유사한성질을가진것으로알려졌으며 3,4 일산화질소 (Nitric oxide, NO) 는섬유주를이완시킴으로써섬유주를통한방수유출을촉진하는것으로알려졌다. 5,6 녹내장의경우 NO의생성저하에의해섬유주를수축시켜안압을상승시킬수있으며, 또한반응성산소종 (reactive oxygen species, ROS) 의생성을유발하여산화스트레스에의해섬유주의손상을유발할수있을것이다. 7 과다한 ROS의생성에의한산화스트레스는세포독성을유발할뿐만아니라, 세포변성, 노화등의여러병적손상을야기하게되는데, NO의생성이저하된경우에도유해한 ROS의생성이증가하게된다. 8,9 방수내에도항상일정한 정도의 ROS가생성되며이에대한다양한방어기전이존재한다. ROS 에노출된상태인섬유주는산화스트레스에의해병적인손상을받을가능성이매우높은조직이므로과다한 ROS의생성에의한산화스트레스를줄이거나방지할수있다면섬유주의손상을줄여섬유주의기능을유지또는회복할수있을것이다. 이러한섬유주세포를대상으로하여산화스트레스를줄이거나방지하는연구가많이시행되고있다. 10,11 Dipyridamole (DPD) 은혈소판저해제로서항혈전작용외에혈관이완을유발하여조직으로의혈류를증가시키는작용이있다. 12 또한 DPD는자유유리기를제거하는항산화제로의성질을가진것으로알려졌다. 13,14 따라서 DPD이섬유주세포에작용하는활성산소종을제거하는항산화작용을나타낼가능성이있으나인체의섬유주세포에대한 DPD의항산화작용에대해서는아직자세히알려져있지않다. 본연구에서는 DPD의항산화능을알아보고, 인체의섬유주세포를일차배양하여활성산소종의생성에미치는영향과산화스트레스를유발하여 DPD가섬유주세포의활성에미치는영향을알아보고자하였다. 접수일 : 2012년 7월 13일 게재허가일 : 2013년 2월 14일 심사통과일 : 2012 년 10 월 11 일 대상과방법 책임저자 : 김재우대구광역시남구두류공원로 17 길 33 대구가톨릭대학교병원안과 Tel: , Fax: jwkim@cu.ac.kr DPPH assay DPD (Sigma, St Louis, MO, USA) 의자유유리기제거 496

2 - 이근우 김재우 : Dipyridamole 과섬유주세포 - 효과를알아보기위하여 DPPH assay를시행하였다 자유유리기인 DPPH (2,2-Diphenyl-1-picrylhydrazyl, Sigma, St Louis, MO, USA) 를메탄올에녹인후 100 μm DPD에단계적으로희석한 DPD를섞은다음 517 nm에서흡광도를측정하였다. DPD의자유유리기제거효과는 DPD 를넣지않은대조군에대한 DPPH의소거정도를소거활성률 (%) 로나타내었다. 안구은행에서얻은사후 6시간이내에적출한안구의앞방각에서섬유주를벗겨내어폴리라이신 (Sigma, St. Louis, Mo. USA) 으로처리한배양접시에옮긴후항생제 (Gibco, Invitrogen, Carlsbad, CA, USA) 와 15% 우태아혈청 (Hyclone, USA) 이포함된 Dulbecco s modified Eagle s medium 배지 (DMEM, Gibco, Invitrogen, Carlsbad, CA, USA) 를사용하여 5% CO 2 배양기에서초대배양하였다. 섬유주세포가이식된조직편주위로자라나온것을확인한후섬유주조직의이식편을제거하고배양을계속하였으며세포가배양접시에충만해지면 10% 우태아혈청 (Gibco, Invitrogen, Carlsbad, CA, USA) 을포함한배지로 1:3의비율로트립신처리하여계대배양하였다. Dichlorofluorescin diacetate assay 섬유주세포에서 DPD가활성산소종의생성에미치는영향을알아보기위하여 dichlorofluorescin diacetate assay 를시행하였다. 18 인체의섬유주세포를일차배양한후 0, 20, 50 μm의 DPD에노출시킨다음배지를제거하고 D-PBS로세척한후 10 μm의 dichlorofluorescin diacetate (Sigma, St Louis, MO, USA) 를넣어 30분간배양한다음 D-PBS 로씻어낸후산화된 dichlorofluorescin을형광분석계 (FLUOstar OPTIMA, BMG labtech, Germany) 를이용하여 excitation 488 nm, emission 535 nm의파장에서 2시간후형광도의변화를측정하였으며이때시간의변화에따라 8개의 well에서각각측정하여평균치를사용하였다. Bradford assay를이용하여세포단백질의양을측정한다음측정한형광도수치를정상화하였다. Resazurin assay 섬유주세포에서유도된산화스트레스에대해 DPD가미치는영향을알아보기위하여 resazurin assay를이용하여세포활성도를측정하였다 , 0.25 mm의 t-butyhlhydroperoxide (tbhp, Sigma, St Louis, MO, USA) 을배지에첨가하여산화스트레스를유발하였으며 0.5 μm의 DPD 를단독또는첨가하여 2시간동안노출시킨후 10% resazurin (Sigma, St Louis, MO, USA) 을넣은다음형광도를측정하였다 (excitation 550, emission 570 nm). Bradford assay를이용하여세포단백질의양을측정한다음측정한형광도수치를정상화하였다. 세포배양 MTT and Griess assays DPD가섬유주세포에서내인성일산화질소의생성에미치는영향을알아보기위하여 Griess assay를 20 시행하여배지에서의 nitrite 생성량을측정하였으며 DPD가섬유주세포에대한세포독성의정도를알아보기위하여 screening test로흔히이용되고있는발색검사법의일종인 MTT (3-[4,5 dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Sigma, St. Louis, MO, USA) assay를시행하였다. 21,22 MTT assay는약물처리한세포의배지에 MTT를각 well당 100 μl씩투여한후 4시간동안정치배양한다음염류용액으로씻어낸후 dimethylsulfoxide (Sigma, St. Louis, MO, USA) 를각 well당 0.5 ml씩넣어 10분이상흔든다음 96-well plate 에 200 μl씩옮겨 spectrophotometer (Fluostar Optima, BMG labtech, Offenberg, Germany) 로 570 nm에서흡광도를측정하였다. 이때세포의생존정도는실험군의값을약물처리를하지않은대조군의비로나누어백분율로나타내었다. Griess assay는 3 일동안약물처리한세포의배지에동량의 Griess 반응액 (Sigma, St. Louis, MO, USA) 을섞은후 96-well plate에옮겨 NO생성의반응물인아질산염의양을 spectrophotometer로 540 nm에서흡광도를측정하였다. 이때표준치를구하기위해 sodium nitrite (Sigma, St. Louis, MO, USA) 를단계적으로희석하여사용하였다. 통계적처리모든실험은 3계대에서 5계대사이의세포를이용하였고대조군은약물처리를하지않은군으로하였으며, 실험군과대조군의비교는 unpaired t-test를사용하였으며유의수준은 0.05% 로정하였다. 결과 DPPH assay 에서 DPD는 20, 50, 100 μm의농도에서대조군에비해농도에비례하여자유유리기인 DPPH를유의 497

3 - 대한안과학회지 2013 년제 54 권제 3 호 Radical scavenging activity (%) Fluorescence/mg protein Dipyridamole ( µ M) Figure 1. Antioxidant property of dipyridamole with DPPH assay. Dipyridamole reduced DPPH in a dose-dependent manner ( p < 0.05) Figure 3. Effect of dipyridamole on the tbhp-induced oxidative stress in trabecular meshwork cells with resazurin assay. Exposure to tbhp decreased cellular activity significantly compared to non-exposed control ( p < 0.05). Dipyridamole improved cellular activity significantly after treatment with tbhp ( p < 0.05). Fluorescence/mg protein Dipyridamole ( µ M) Figure 2. Effect of dipyridamole on the generation of reactive oxygen species (ROS) in trabecular meshwork cells with DCFHDA assay. Dipyridamole decreased ROS significantly in a dose-dependent manner ( p < 0.05). 하게감소시켜 DPD가자유유리기를제거하는항산화능을가지고있음을알수있었다 (p<0.05) (Fig. 1). 인체의섬유주세포를일차배양한결과초대배양 7일째부터섬유주조직의이식편주위로섬유주세포가자라나오기시작하였으며섬유주세포의확인은세포들이밀집해서단층을형성하며세포들사이에분지를내어서로연접하며약간길다란모양의세포체를가지는편평한모양의특징적형태학적인양상과섬유주조직의이식편주위에서위성양상으로자라나는섬유주세포의특징적인성장양상으로확인하였다. 23,24 DPD가가진항산화능이섬유주세포에서의 ROS 생성에영향을미치는지를알아보기위하여섬유주세포에서 DCFHD assay로활성산소종의생성에미치는영향을측정한결과 20 μm과 50 μm DPD를첨가하였을경우대조군에비해단위 mg당형광도가 32.34, 72.55로유의하게감소하였다 (p<0.05) (Fig. 2). 산화스트레스에의해미토콘드리아의호흡활성도가저 Nitrite ( M) µ Dipyridamole ( µ M) Figure 4. Effect of dipyridamole on the production of nitric oxide in trabecular meshwork cells with Griess assay. Dipyridamole did not affect the production of nitric oxide significantly (p > 0.05). 하되게되는데, 약물을처리하지않은대조군에비해서 tbhp는 0.1 mm과 0.25 mm의농도에서유의하게섬유주세포의세포활성도를감소시켰으며, 이때 5 μm DPD를첨가하였을경우 0.1 mm과 0.25 mm 의 tbhp 농도에서단위 mg 당형광도가각각 221, 214로유의하게증가하여 (p<0.05) DPD가 tbhp에의해유도된산화스트레스에대해세포의호흡활성도를증가시키는작용을나타내었다 (Fig. 3). 섬유주세포에서 DPD가일산화질소의생성과세포의생존에미치는영향 DPD는일산화질소의생성에는유의한영향을미치지않았으며 (Fig. 4), MTT assay의결과 25, 50 μm DPD는약물처리를하지않은대조군에비해섬유주세포에서유의한 498

4 - 이근우 김재우 : Dipyridamole 과섬유주세포 - 세포독성을나타내지않았다 (Data not shown). 고찰 자유유리기는산화스트레스를유발하여인체의노화를유발하는주요한요인으로알려졌으며녹내장의경우에도산화스트레스는섬유주세포의손상을유발할뿐만아니라섬유주세포의노화를촉진할수있는것으로알려졌고안압상승과시야손상의정도가섬유주에서의산화스트레스에의한핵산의손상정도와비례하는것으로도알려졌다. 25 혈관내피세포의경우고농도의포도당에노출되면 NO의생성이저하되며산화스트레스가유발되는것으로알려졌다. 26 섬유주세포에 ROS가지속적으로노출될경우섬유주세포에서의생리적 NO의생성에영향을미칠수있고산화스트레스도유발할수있다. 따라서산화스트레스는섬유주세포의기능에영향을줄뿐만아니라섬유주세포의노화를촉진시킬수있을것이다. 항혈전작용을가진 DPD은자유유리기를제거하는작용이있어세포막과미토콘드리아의과산화를방지하거나저밀도지방단백의산화변성을유발하여항산화작용을나타내는데 13,27-29 직접적으로내인성자유유리기또는질소종의생성을변화시키는지는아직자세히알려져있지않다. 섬유주세포는혈관내피세포의성질을가지고있으므로 DPD 가섬유주세포의산화스트레스에미치는영향을알아보기위해시행한본연구의결과에서 DPD의항산화능을확인하였으며, DPD 을추가하여섬유주세포를배양한경우 ROS 의생성이유의하게억제되었다. 또한산화스트레스를유발하여 DPD을추가한경우에도 DPD을추가하지않은대조군에비해유의하게세포의활성도를증가시켰으므로 DPD이산화스트레스에노출된섬유주세포에대해서도유의하게세포보호작용을나타냄을알수있었다. 이러한 DPD의세포보호작용은신경세포에서도나타나는것으로알려졌다. 30 본연구의결과에서 DPD은 NO의생성에는유의한영향을미치지않았다. 이러한결과는 DPD이 phosphodiesterase 저해제로서작용하며이는혈관에서 DPD이 NO의생성증가또는 cgmp 의강화작용보다는 adenosine 강화작용에의한것이라도볼수있다는기존의보고와일치한다. 31 또한세포의생존에는유의한영향을미치지않아세포에대한독성은낮을것으로생각한다. 따라서섬유주세포에서 DPD는 NO의생성에는유의한영향을미치지않아방수유출작용은나타내지않을것으로생각하나항산화작용을나타내어개방각녹내장의병인중하나로생각하는섬유주세포의변성내지노화를억제하는작용을나타낼수있을것이다. 결론적으로 DPD은배양된사람의섬유주세포에서유해한산화물질의생성을억제하였으며산화스트레스에대하여섬유주세포를보호하는효과도나타내었으므로비록생체내에서의연구는아니지만본연구의결과는 DPD이항산화작용을통한섬유주세포보호작용을나타낼가능성이있음을시사하며향후그기전과임상적효과에대해보다자세한연구가필요할것으로생각한다. 참고문헌 1) Alvarado J, Murphy C, Juster R. Trabecular meshwork cellularity in primary open-angle glaucoma and nonglaucomatous normals. Ophthalmology 1984;91: ) Rohen JW, LÜtjen-Drecoll E, FlÜgel C, et al. Ultrastructure of the trabecular meshwork in untreated cases of primary open-angle glaucoma (POAG). Exp Eye Res 1993;56: ) Wiederholt M, Dörschner N, Groth J. Effect of diuretics, channel modulators and signal interceptors on contractility of the trabecular meshwork. Ophthalmologica 1997;211: ) Wiederholt M, Stumpff F. The trabecular meshwork and aqueous humor reabsorption. In: Civan MM, ed. Current topics in membranes. The eye's aqueous Humor: from secretion to glaucoma. v. 45. San Diego: Academic Press; 1998: ) Wiederholt M, Sturm A, Lepple-Wienhues A. Relaxation of trabecular meshwork and ciliary muscle by release of nitric oxide. Invest Ophthalmol Vis Sci 1994;35: ) Behar-Cohen FF, Goureau O, D Hermies F, Courtois Y. Decreased intraocular pressure induced by nitric oxide donors is correlated to nitrite production in the rabbit eye. Invest Ophthalmol Vis Sci 1996;37: ) Saccà SC, Izzotti A, Rossi P, Traverso C. Glaucomatous outflow pathway and oxidative stress. Exp Eye Res 2007;84: ) Klein JA, Ackerman SL. Oxidative stress, cell cycle, and neurodegeneration. J Clin Invest 2003;111: ) Ischiropoulos H, Beckman JS. Oxidative stress and nitration in neurodegeneration: cause, effect, or association? J Clin Invest 2003;111: ) Hong JH, Kim YY, Kim JW. Effect of genistein on the survival and production of nitric oxide in trabecular meshwork cells. J Korean Ophthalmol Soc 2011;52: ) Lee SH, Kim JW. Effect of erythropoietin on the production of nitric oxide in trabecular meshwork cells. J Korean Ophthalmol Soc 2011;52: ) Chakrabarti S, Vitseva O, Iyu D, et al. The effect of dipyridamole on vascular cell-derived reactive oxygen species. J Pharmacol Exp Ther 2005;315: ) Iuliano L, Ghiselli A, Alessandri C, et al. Superoxide anion scavenging property of dipyridamole. Thromb Haemost 1989;61: ) Iuliano L, Piccheri C, Coppola I, et al. Fluorescence quenching of dipyridamole associated to peroxyl radical scavenging: a versatile probe to measure the chain breaking antioxidant activity of biomolecules. Biochim Biophys Acta 2000;1474: ) Liu F, Ng TB. Antioxidative and free radical scavenging activities of selected medicinal herbs. Life Sci 2000;66: ) Lee SE, Hwang HJ, Ha JS, et al. Screening of medicinal plant ex

5 - 대한안과학회지 2013 년제 54 권제 3 호 - tracts for antioxidant activity. Life Sci 2003;73: ) Parejo I, Viladomat F, Bastida J, et al. Investigation of Bolivian plant extracts for their radical scavenging activity and antioxidant activity. Life Sci 2003;73: ) Wang H, Joseph JA. Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader. Free Radic Biol Med 1999;27: ) Abu-Amero KK, Bosley TM. Detection of mitochondrial respiratory dysfunction in circulating lymphocytes using resazurin. Arch Pathol Lab Med 2005;129: ) Green LC, Wagner DA, Glogowski J, et al. Analysis of nitrate, nitrite and [15N]nitrate in biological fluids. Anal Biochem 1982; 126: ) Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65: ) Freimoser FM, Jakob CA, Aebi M, Tuor U. The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay is a fast and reliable method for colorimetric determination of fungal cell densities. Appl Environ Microbiol 1999;65: ) Polansky JR, Weinreb RN, Baxter JD, Alvarado J. Human trabecular cells. I. Establishment in tissue culture and growth characteristics. Invest Ophthalmol Vis Sci 1979;18: ) Alvarado JA, Wood I, Polansky JR. Human trabecular cells. II. Growth pattern and ultrastructural characteristics. Invest Ophthalmol Vis Sci 1982;23: ) Saccà SC, Pascotto A, Camicione P, et al. Oxidative DNA damage in the human trabecular meshwork: clinical correlation in patients with primary open-angle glaucoma. Arch Ophthalmol 2005;123: ) El-Remessy AB, Abou-Mohamed G, Caldwell RW, Caldwell RB. High glucose-induced tyrosine nitration in endothelial cells: role of enos uncoupling and aldose reductase activation. Invest Ophthalmol Vis Sci 2003;44: ) Iuliano L, Colavita AR, Camastra C, et al. Protection of low density lipoprotein oxidation at chemical and cellular level by the antioxidant drug dipyridamole. Br J Pharmacol 1996;119: ) Iuliano L, Piccheri C, Coppola I, et al. Fluorescence quenching of dipyridamole associated to peroxyl radical scavenging: a versatile probe to measure the chain breaking antioxidant activity of biomolecules. Biochim Biophys Acta 2000;1474: ) Selley ML, Czeti AL, McGuiness JA, Ardlie NG. Dipyridamole inhibits the oxidative modification of low density lipoprotein. Atherosclerosis 1994;111: ) Farinelli SE, Greene LA, Friedman WJ. Neuroprotective actions of dipyridamole on cultured CNS neurons. J Neurosci 1998;18: ) Gamboa A, Abraham R, Diedrich A, et al. Role of adenosine and nitric oxide on the mechanisms of action of dipyridamole. Stroke 2005;36:

6 - 이근우 김재우 : Dipyridamole 과섬유주세포 - =ABSTRACT= Effect of Dipyridamole on the Reactive Oxygen Species and Oxidative Stress in Trabecular Meshwork Cells Keun Woo Lee, MD, Jae Woo Kim, MD, PhD Department of Ophthalmology, Catholic University of Daegu School of Medicine, Daegu, Korea Purpose: To investigate the effects of dipyridamole (DPD) on the production of reactive oxygen species (ROS) and oxidative stress in cultured human trabecular meshwork cells (HTMC). Methods: Antioxidant activity of DPD was determined by DPPH assay. Primarily cultured HTMC were exposed to 0, 20, and 50 μm DPD using serum-deprived media. The effect of DPD on the production of ROS was assessed with the DCHFDA assay. The effect of DPD on the t-butyl hydroperoxide (tbhp)-induced oxidative stress was assessed with resazurin assay. Results: DPD showed significant antioxidant activity. DPD significantly decreased the production of ROS (p < 0.05) and improved cellular activity significantly after treatment with t-bhp (p < 0.05). DPD did not affect the generation of nitric oxides. Conclusions: DPD suppressed the formation of ROS and possessed cytoprotective activity against the oxidative stress in HTMC. J Korean Ophthalmol Soc 2013;54(3): Key Words: Antioxidant, Dipyridamole, Reactive oxygen species, Trabecular meshwork cells Address reprint requests to Jae Woo Kim, MD, PhD Department of Ophthalmology, Daegu Catholic University Medical Center #33 17-gil Duryugongwon-ro, Nam-gu, Daegu , Korea Tel: , Fax: , jwkim@cu.ac.kr 501

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<31362D30372D333528B1E8C0E7BFEC2DC8ABC1A4C8EC E687770> 대한안과학회지 214 년제 55 권제 3 호 J Korean Ophthalmol Soc 214;55(3):416-421 pissn: 378-6471 eissn: 292-9374 http://dx.doi.org/1.3341/jkos.214.55.3.416 = 증례보고 = 섬유주세포에서국소탄산탈수효소억제제가일산화질소의생성에미치는영향에대한연구 홍정흠 김세은 김재우