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1 CANCER PREVENTION RESEARCH ORIGINAL ARTICLE 췌장베타세포에서 Streptozotocin 으로유도한산화적스트레스에대한 Phloroglucinol 의보호효과 1 제주대학교의과대학생화학교실, 2 제주중앙여자고등학교 김아름다슬 1 ㆍ강민지 2 ㆍ고미성 2 ㆍ양혜원 2 ㆍ장원이 2 ㆍ최현의 2 ㆍ현진원 1 Phloroglucinol Protects Cells against Streptozotocin-induced Oxidative Stress in Rat Pancreatic β-cell RINm5F Areum Daseul Kim 1, Min Ji Kang 2, Mi Sung Ko 2, Hye Won Yang 2, Won E Jang 2, Hyeon Ui Choi 2 and Jin Won Hyun 1 1 Department of Biochemistry, Jeju National University School of Medicine, Jeju , 2 Jeju Jungang Girls High School, Jeju , Korea Streptozotoxin-induced oxidative cell damage-mediated apoptosis plays an important role in the destruction of pancreatic β-cells and contributes to the development of diabetes. In the present study, protective effect of phloroglucinol (1,3,5-trihydroxybenzene) on streptozotocin (STZ)-induced oxidative cell damage was investigated in rat pancreatic β-cells, RINm5F. Phloroglucinol was significantly decreased the level of STZ-induced intracellular reactive oxygen species (ROS) and reduced STZ-induced loss of the mitochondrial membrane potential. Phloroglucinol enhanced cell viability against STZ-induced cell damage and inhibited STZ-induced apoptotic cell death, confirmed by formation of apoptotic bodies and sub G 1 phase. These results suggest that phloroglucinol exhibits protective effects on STZ-induced oxidative cell damage-mediated apoptosis in rat RINm5F pancreatic β-cells. (Cancer Prev Res 17, 95-99, 2012) Key Words: Phloroglucinol, Oxidative stress, Reactive oxygen species, RIN5mF cells 서론당뇨병은췌장의베타세포에서분비되는호르몬인인슐린의분비부족이나인슐린에대한저항성의증가로인해발병되는진행성질환으로당질대사이상을초래할뿐만아니라단백질, 지질및전해질등의대사조절기능에도이상이발생하게된다. 1) 당뇨병환자에서고혈당의지속화와당화과정에서자유라디칼은지질과산화를촉진하여체내의산화적스트레스로인한점진적인췌 장베타세포기능이상을초래하여인슐린저항성을더욱증가시킨다. 2,3) 이런산화적스트레스는체내활성산소종 (reactive oxygen species, ROS) 을많이생성하거나항산화시스템의기능을저하하면서, 이로인한췌장베타세포의손상을일으키게됨으로써당뇨병이심화되거나당뇨병성망막증, 당뇨병성신증, 말초신경병증등합병증을유발하게된다. 4,5) Streptozotocin (STZ) 은췌장랑게르한스섬의베타세포에선택적으로작용하여실험동물에당뇨병을유발하는데많이사용되고있는약물이며, 6) 당뇨유발기전은 ROS에의한베타세포손상때문으로 책임저자 : 현진원, , 제주시제주대학로 102 번지제주대학교의학전문대학원생화학교실 Tel: , Fax: jinwonh@jejunu.ac.kr 접수일 :2012 년 5 월 21 일, 1 차수정일 :2012 년 5 월 24 일, 2 차수정일 :2012 년 5 월 30 일, 게재승인일 :2012 년 6 월 4 일 Correspondence to:jin Won Hyun Department of Biochemistry, Jeju National University School of Medicine, 102, Jejudaehang-ro, Jeju , Korea Tel: , Fax: jinwonh@jejunu.ac.kr 95

2 96 Cancer Prevention Research Vol. 17, No. 2, 세포내 ROS 양측정 Fig. 1. The chemical structure of phloroglucinol. 알려져있다. 7,8) 최근천연물로부터당뇨병을예방하거나또는억제할수있는천연물생리활성물질을이용한연구가활발히이루어지고있다. 특히해양생물중해조류의 alginic acid, fucoidan 및 laminaran 등의항당뇨성다당류와 9,10) 더불어 fucosterol 및 phlorotannins 11,12) 등의새로운당뇨치료제의개발이크게기대되고있다. 그중갈조류에속하는감태는제주연안에대부분서식하고있으며, phlorotannin 계열의화합물인 phloroglucinol (Fig. 1) 을함유하고있다. Phlorogucinol 화합물은라디칼을소거하는효과를나타내며, 13,14) ionizing radiation이유도하는 ROS의억제효과와세포손상에대한보호효과를나타내었다. 15) 따라서본연구에서는 phloroglucinol를이용하여췌장베타세포에서 streptozotocin이유발하는세포내산화적손상의억제와세포보호효과를살펴보고자한다. 1. 세포배양 재료및방법 Rat 췌장세포 RINm5F 세포들을 RPMI 1640 (10% FBS 첨가, 1% 항생제포함 ) 배지에현탁하여 37 o C, 5% CO 2 배양기에서배양하고실험에사용하였다. 2. 세포생존율측정 RINm5F 세포에 phloroglucinol를처리한후 37 o C, 5% CO 2 배양기에서 1시간배양후에 STZ (stock 1 M) 을 final concentration 10 mm가되도록가한후다시배양기에서 24시간동안배양하였다. MTT법은 mitochondria의호흡으로노란색의수용성 MTT가보라색의 formazan 결정이되는원리를이용한방법이다. 16) 즉, 세포에 MTT (3-(4,5- Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; stock 10 mg/ml) 을 10μg/ml가되도록처리하여 4시간동안배양후에배양액을제거하고유기용매인 Dimethyl sulfoxide (DMSO) 를 150μl 넣어 formazan을녹인후에 540 nm 파장에서흡광도를측정하였다. Well당세포수가약 cells/ml 되도록 96 well에각각접종한후에 10 mm STZ을처리한후다시 37 o C, 5% CO 2 배양기에서 12시간배양하였다. DCF-DA (dichlorofluorescin diacetate, stock 500μM) 시약을 20μl 씩처리한후분광광도계 (excitation 485 nm, emission 535 nm) 로측정하였다. 17) 또한유동세포분석기로측정하기위해 well당세포수가약 cells/ml 되도록 6 well에각각접종한후에 10 mm STZ 를가한후다시 37 o C, 5% CO 2 배양기에서 12시간배양한후 DCF-DA 시약을가한후유동세포분석기 (Becton Dikins, FL1-H) 로측정하였다. ROS의양을형광현미경으로측정하기위하여 well당세포수가약 cells/ml 되도록 4 well 배양용슬라이드에각각접종한후에 10 mm STZ 가한후다시 37 o C, 5% CO 2 배양기에서 12시간배양하였다. 그후에 DCF-DA 시약을가한후 Mounting medium을넣어 cover 슬라이드로덮은후 Laser Scanning Microscope 5 PASCAL program을이용하여형광현미경으로 (FITC-Ch2) 측정하였다. 4. 세포사멸측정 RINm5F 세포에 phloroglucinol를처리한후 37 o C, 5% CO 2 배양기에서 1시간배양후에 10 mm STZ을가한후다시배양기에서 24시간동안배양하였다. Propidium iodide (PI) 염색법은 Tripsin/EDTA로세포를떼어내고모은세포를 70% EtOH로고정한후에 PBS로두번세척한후 100μg/ml RNase로 RNA를제거하고 100μg/ml PI 염색시약을처리후 30분동안배양하여 DNA를염색하여유동세포분석기의 FL-2 파장으로 sub G 1 기를통해세포사멸을확인하였다. 18) 5. Hoechst 염색에의한세포핵의형태관찰 Well당약 cells/ml 세포수가되도록 24 well에각각접종한후에 phloroglucinol 처리한후 37 o C, 5% CO 2 배양기에서 1시간배양후에 STZ을가한후다시배양기에서 24시간동안배양하였다. Hoechst 형광염색약을처리하고 10분간배양한후 Laser Scanning Microscope 5 PASCAL program을이용하여형광현미경으로관찰하였다. 1. ROS 의소거효과 결과및고찰 RINm5F 세포에 Streptozotocin (STZ) 로유도된 ROS 의

김아름다슬외 6 인 : 췌장베타세포에서 Streptozotocin 으로유도한산화적스트레스에대한 Phloroglucinol 의보호효과 97 Fig. 2. The effect of phloroglucinol on STZ-induced intracellular ROS generation.

Next, cells were incubated for 24 h and intracellular ROS were detected using (A) a fluorescence spectrophotometer, (B) flow cytometer and (C) confocal microscope after DCF-DA staining.

2A). 다음으로유동세포분석기를이용하여세포내 ROS를측정한결과, STZ 처리시형광강도값이 233을나타냈으며, STZ과 phloroglucinol을병행처리시형광강도값이 131로감소함을확인할수있었으며, 이를통하여 phloroglucinol을처리하였을때, 유의적으로세포내 ROS를억제하는것을관찰할수있었다 (Fig. 2B).

3 김아름다슬외 6 인 : 췌장베타세포에서 Streptozotocin 으로유도한산화적스트레스에대한 Phloroglucinol 의보호효과 97 Fig. 2. The effect of phloroglucinol on STZ-induced intracellular ROS generation. The cells were treated with phloroglucinol at 10μg/ml. After 1 h, 10 mm of STZ was added to the plate. Next, cells were incubated for 24 h and intracellular ROS were detected using (A) a fluorescence spectrophotometer, (B) flow cytometer and (C) confocal microscope after DCF-DA staining. *Significantly different from control (p<0.05), and **significantly different from STZ treated cells (p<0.05). 양과 STZ을 phloroglucinol과병행처리시세포내 ROS 양을분광광도계로측정한결과, STZ에유도된세포내 ROS 생성량에비하여 STZ과 phloroglucinol을병행처리하였을때세포내 ROS 생성량이감소하는것을관찰할수있었다 (Fig. 2A). 다음으로유동세포분석기를이용하여세포내 ROS를측정한결과, STZ 처리시형광강도값이 233을나타냈으며, STZ과 phloroglucinol을병행처리시형광강도값이 131로감소함을확인할수있었으며, 이를통하여 phloroglucinol을처리하였을때, 유의적으로세포내 ROS를억제하는것을관찰할수있었다 (Fig. 2B). 형광현미경을이용하여세포내 ROS를측정한결과, 대조군또는 phloroglucinol을처리하였을때, ROS에의한형광이발현되지않는것을관찰할수있었으며, 반면에 STZ 처리시세포내 ROS가현저하게증가하는 것을확인할수있었다. 그리고, STZ와 phloroglucinol을병행처리하였을때, STZ만을처리하였을때보다현저히형광강도가감소하는것을확인하였다 (Fig. 2C). 따라서 phloroglucinol이 STZ에의해유도되는세포내 ROS를소거시킴으로써산화적손상을억제함을확인할수있었다. 2. 세포손상보호효과 Streptozotocin (STZ) 처리시세포손상으로인해세포생존에영향을나타내는지를 MTT 실험으로확인한결과, STZ 처리시세포생존율이 64% 로감소하였으나 STZ 과 phloroglucinol을병행처리하였을때, 세포생존율이 76% 로증가함에따라세포손상을억제함을확인할수있었다 (Fig. 3A). STZ 산화적손상에따른세포사멸을

98 Cancer Prevention Research Vol. 17, No. 2, 2012 Fig. 3. The effect of phloroglucinol on STZ-induced cell demage.

(A) The cell viability cells after STZ treatment was determined by MTT assay.

(C) Apoptotic body formation was observed under a fluorescence microscope after Hoechst 33342 staining and arrows indicate apoptotic bodies.

and **significantly different from STZ treated cells (p<0.05).

4 98 Cancer Prevention Research Vol. 17, No. 2, 2012 Fig. 3. The effect of phloroglucinol on STZ-induced cell demage. Cells were treated with phloroglucinol at 10μg/ml, followed by STZ at 1 h later. Next, the cells were incubated for 24 h. (A) The cell viability cells after STZ treatment was determined by MTT assay. (B) The apoptotic sub G 1 DNA content was detected by a flow cytometry after propidium iodide staining. (C) Apoptotic body formation was observed under a fluorescence microscope after Hoechst staining and arrows indicate apoptotic bodies. *Significantly different from control (p<0.05), and **significantly different from STZ treated cells (p<0.05). phloroglucinol이보호하는지를측정하기위하여 PI 염색방법을이용하여 sub G 1 세포를 FACS를통하여확인한결과, STZ처리시 sub G 1 세포가 35% 로나타났으며, STZ 과 phloroglucinol을병행처리하였을때, sub G 1 세포가 14% 감소되었다 (Fig. 3B). 또한 Hoechst 염색을통한세포핵의형태관찰한결과 STZ 처리시핵분절현상이현저히증가하였으나 STZ과 phloroglucinol을병행처리하였을때, 핵및 DNA 분절현상이감소되었다 (Fig. 3C). 따라서 STZ가유도하는세포손상에대하여 phloroglucinol이보호효과가있다는것을확인하였다. 이상의결과로부터췌장베타세포에서 STZ 처리시유도된산화적손상에대해 phloroglucinol이이를억제함으로서세포손상을보호하는역할을한다는것을알수있었다. 결론본연구에서는췌장베타세포 (RINm5F) 에서 STZ가유도하는산화적스트레스에의해세포내 ROS의생성과세포사멸을일으키는데있어서천연물보고해양생물중해조류의감태에서부터분리한 phlorotannin 계열인 phloroglucinol의세포내보호효과를관찰하였다. 췌장베타세포에서세포내 ROS의양을 DCF-DA시약처리후분광광도계, 유동세포분석기, 형광현미경으로측정한결과, STZ 처리시세포내 ROS의양이증가하였다. 반면에 STZ과 phloroglucinol을병행처리한결과, 세포내 ROS 의양이현저히감소함을알수있었다. 또한세포손상

5 김아름다슬외 6 인 : 췌장베타세포에서 Streptozotocin 으로유도한산화적스트레스에대한 Phloroglucinol 의보호효과 99 보호효과를관찰하기위하여세포생존율, sub G 1 세포, 핵분절현상을관찰한결과, STZ 로유도된세포손상이 STZ 과 phloroglucinol 을병행처리후세포손상에대하여 보호효과를나타냄을알수있었다. 참고문헌 1) Sakurai T, Tsuchiya S. Superoxide production from nonenzymatically glycated protein. FEBS Lett 236, , ) West IC. Radical and oxidative stress in diabetes. Diabet Med 17, , ) Coyle JT, Puttfarcken P. Oxidative stress, glutamate and neurodegenerative disorders. Science 262, , ) Lones TJ. Oxidized low density lipoproteins: a role in the pathogenesis of atherosclerosis in diabetes. Diabet Med 8, , ) Tai ES, Lim SC, Tan BY, Chew SK, Heng D, Tan CE. Screening for diabetes mellitus: a two-step approach in individuals with impaired fasting glucose improves in detection of those at risk of complications. Diabet Med 17, , ) Takasawa S, Yamamoto H, Terazono K, Okamoto H. Novel gene activated in rat insulinomas. Diabetes 35, , ) Baynes JW. Role of oxidative stress in development of complications in diabetes. Diabetes 40, , ) Steer KA, Sochor M, Mclean P. Renal hypertrophy in experimental diabetes changes in pentose phosphate pathway activity. Diabetes 34, , ) Lee KS, Seo JS, Choi YS. Effect of sea tangle and hypoglycemic agent on lipid metabolism in diabetic rats. J Korean Soc Food Sci Nutr 27, , ) Jin DQ, Li Q, Kim JS, Yong CS, Kim JA, Hun K. Preventive effects of Laminaria japonica aqueous extract on the oxidative stress and xanthine oxidase activity in streptozotocin induced diabetic rat liver. Biol Pharm Bull 27, , ) Lee YS, Shin KH, Kim BK, Lee SH. Anti-diabetic activities of fucosterol from Pelvetia siliquosa. Arch Pharm Res 27, , ) Okada Y, Ishimaru A, Suzuki R, Okuyama T. A new phloroglucinol derivatives from the brown alga Eisenia bicyclis potential for the effective treatment of diabetic complications. J Nat Prod 67, , ) Kang K, Park Y, Hwang HJ, Kim SH, Lee JG, Shin HC. Antioxidative properties of brown algae polyphenolics and their perspectives as chemopreventive agents against vascular risk factors. Arch Pharm Res 26, , ) Kang HS, Chung HY, Kim JY, Son BW, Jung HA, Choi JS. Inhibitory phlorotannins from the edible brown alga Ecklonia stolonifera on total reactive oxygen species (ROS) generation. Arch Pharm Res 27, , ) Kang KA, Zhang R, Chae S, Lee SJ, Kim J, Kim J, Jeong J, Lee J, Shin T, Lee NH, Hyun JW. Phloroglucinol (1,3,5- trihydroxybenzene) protects against ionizing radiation-induced cell damage through inhibition of oxidative stress in vitro and in vivo. Chem Biol Interact 185, , ) Carmichael J, DeGraff WG, Gazdar AF, Minna JD, Mitchell JB. Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res 47, , ) Rosenkranz AR, Schmaldienst S, Stuhlmeier KM, Chen W, Knapp W, Zlabinger GJ. A microplate assay for the detection of oxidative products using 2,7 -dichlorofluorescin-diacetate. J Immunol Meth 156, 39-45, ) Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Meth 139, , 1991.

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경락경혈학회지 Vol.27, No.1, pp.87 106, 2010 Journal of Meridian & Acupoint Dept. of 1 Meridian & Acupoint, 3 Acupuncture & Moxibustion, College of Oriental Medicine, Daejeon University 2 Division of Clinical