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1 대한류마티스학회지 Vol. 17, No. 2, June, 2010 DOI: /jkra 원저 류마티스관절염활막세포에서 Peroxisome Proliferator-activated Receptor-γ Agonist 처치의염증매개인자감소및활막세포증식의억제효과 연세대학교의과대학내과학교실 권용진ㆍ정수진ㆍ김태연ㆍ박민찬 =Abstract= Peroxisome Proliferator-activated Receptor-γ Agonist Inhibits Pro-inflammatory Gene Expressions and Cellular Proliferation of Fibroblast Like Synoviocytes from Patients with Rheumatoid Arthritis by Down-regulation of NF-kappaB Yong-Jin Kwon, Soo-Jin Chung, Tae-Yeon Kim, Min-Chan Park Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Korea Objective: This study investigated the effect of rosiglitazone, a synthetic peroxisome proliferatoractivated receptor-γ (PPAR-γ) agonist, on pro-inflammatory gene expressions and cellular proliferation of fibroblast like synoviocyte (FLS) from patients with rheumatoid arthritis (RA), and to determine whether these actions are mediated by nuclear factor-kappab (NF-B) downregulation. Methods: Synovial tissues from patients with RA were obtained during total knee replacement surgery, and FLS were isolated. RA FLS were subsequently treated with 10 μm, 50 μm and 150 μm rosiglitazone with or without TNF-α (10 ng/ml) stimulation. FLS proliferation in response to rosiglitazone treatment was measured by MTT assay, and mrna expressions of IL-1β, IL-6, CCL-2, CCL-7, COX-2 and MMP-9 were determined by real-time quantitative RT-PCR. The effects of rosiglitazone on NF-κB activation were evaluated using electrophoretic mobility shift assay (EMSA). Results: Rosiglitazone treatment without TNF-α induced a dose-dependent reduction in mrna expressions of IL-1β, IL-6, CCL-2, CCL-7, COX-2 and MMP-9 from RA FLS. When TNF-α < 접수일 :2010 년 5 월 3 일, 수정일 (1 차 :2010 년 5 월 17 일, 2 차 :2010 년 5 월 19 일 ) 심사통과일 :2010 년 5 월 19 일 > 통신저자 : 박 민 찬 서울시강남구도곡동강남세브란스병원내과 Tel:02) , Fax:02) , mcpark@yuhs.ac 153

2 대한류마티스학회지제 17 권제 2 호 2010 were treated with rosiglitazone, mrna expressions of COX-2, MMP-9 were reduced dosedependently. But mrna expressions of IL-1β, IL-6, CCL-2, CCL-7 were increased in 10 μm rosiglitazone with TNF-α and then decreased as the concentration of rosiglitazone increased. Rosiglitazone treatment also suppressed FLS proliferation in a dose-dependent manner, and EMSA showed decreased NF-κB expression with rosiglitazone treatment. Conclusion: Rosiglitazone suppressed cellular proliferation and mrna expressions of pro-inflammatory mediators by down-regulating the NF-κB signaling pathway in RA FLS. The outcomes suggest that activation of PPAR-γ can be a novel therapeutic approach in RA. Key Words: Rosiglitazone, Rheumatoid arthritis, Fibroblast-like synoviocytes, Pro-inflammatory mediators, NF-κB 서론류마티스관절염은가장흔한염증성관절염으로약 1% 의유병율을가진다 (1,2). 류마티스관절염의정확한원인이나발병기전은아직완전히밝혀지지는않았으나자가면역현상이주요기전으로알려져있으며대칭성, 다발성말초관절염이특징인만성전신성염증질환이다 (3). 가장먼저염증이시작되는부위는관절을둘러싸고있는활막으로서, 활막세포가비정상적으로과다증식하여연골과뼈의손상을유발하여관절파괴와그에따른기능손실을초래한다 (4,5). 류마티스관절염환자의활막에는다양한면역세포들이침윤되어있으며특히대식세포가많은부분을차지하고있다. 어떠한원인항원에의해염증이시작되면 nuclear factor-kappab (NF-κB), mitogen-activated protein kinases, IκB kinase (IKK) 및 activator protein (AP)-1과같은신호전달체계가관여하여대식세포와단핵구로부터 tumor necrosis factor (TNF)-α, interleukin (IL)-1β 등다양한염증성싸이토카인과 chemokine (C-C motif) ligand (CCL)-2, CCL-7 등의케모카인, 그리고 matrix metalloproteinase (MMP)-9, cyclooxygenase (COX)-2 등이분비되어염증을악화시키게된다 (6-12). 그결과활막의증식이가속화되어 pannus를형성하여연골을파괴하고여러염증매개인자들이 receptor activator of NF-κB lilgand (RANKL) 의활성화를유발하여파골세포를활성화시켜골파괴를일으켜관절손상이진행되는것으로알려져있다 (13,14). 이러한염증을억제하고관절의변형을최소화하 기위해류마티스관절염의치료약제개발에대한연구도꾸준히지속되어왔다. 최근에는류마티스관절염에서활막증식과관절파괴에중심적인역할을할것으로생각되는여러염증성싸이토카인들에대한연구가활발히진행되고있으며, NF-κB의산물인 TNF-α 및 IL-1β의작용을차단하는약제가개발되어류마티스관절염의임상증상의호전뿐아니라방사선소견의진행까지늦출수있음이보고되었다 (15-17). 하지만치료효과가뛰어남에도불구하고일부환자들은여전히치료에반응을보이지않아새로운치료법개발의필요성이강조되고있다. Peroxisome proliferator-activated receptor-γ(ppar-γ) 는유전자전사에관여하는핵수용체 (nuclear receptor family) 의하나로, 지질대사및항상성유지에중요한역할을하는것으로알려져있다 (18,19). 최근들어 PPAR-γ가염증반응에도관여함이밝혀졌는데, PPAR-γ agonist를관절염실험쥐모델에투여하자대식세포에서염증매개물질의생성이감소하는것이관찰되었다 (20,21). 또한 PPAR-γ가 TNF-α 및 IL-1β를비롯한다양한염증성싸이토카인에대한억제작용뿐만아니라 COX-2와 MMP-9을비롯한다양한염증매개물질들의발현을감소시키고이러한항염증작용은주로 NF-κB의억제작용에의한것으로받아들여지고있다 (22,23). 이상의연구결과들을미루어볼때, 대식세포의활성화및 NF-κB 에의해발현이증가된여러염증매개인자들이그병인에있어서중요한류마티스관절염에있어서도 PPAR-γ agonist가항염증효과를나타낼가능성이있을것으로추정된다. 류마티스관절염의병인에있어서관절활막이특 154

3 권용진외 : PPAR-γ Agonist 의류마티스관절염치료효과 이적면역반응이일어나는주요부위임은분명히밝혀져있는바, 류마티스관절염의관절활막에서발생되는병태생리를연구하고활막염의발생을억제혹은감소시키는인자를발견하는것은류마티스관절염의발생기전을이해하며새로운치료를도모할수있는매우중요한임상적의미를갖는다고생각된다. 본연구에서는류마티스관절염환자의관절활막에대하여 synthetic PPAR-γ agonist인 rosiglitazone을투여하고투여용량과비례하여활막에서발생하는염증반응이감소되는지, 그리고활막세포의증식이억제되는지의여부를확인하고자하였다. 대상및방법 1. 임상시료수집 1987년미국류마티스학회가제정한류마티스관절염의진단기준 (24) 에의거하여류마티스관절염으로진단된환자를대상으로하였으며, 류마티스관절염의진단은 1 1시간이상의조조강직을가지며, 2 손과손목의관절에염증이증명되고, 3 좌우대칭적으로관절의염증이증명되고, 4 3부위이상의다발성관절염이증명되며, 5 혈액검사상류마티스인자가양성이며, 6 피부에류마티스결절이증명되고 7 방사선소견상특징적인골파괴소견을보이는경우로, 상기사항중 4가지이상을만족할경우류마티스관절염으로진단하였고다른결체조직질환혹은관절질환및관절외상의과거력이있는경우는배제하였다. 류마티스관절염환자에서진단적관절경수술혹은인공관절치환술을통하여환자동의하에관절활막을채취하였다. 2. 섬유모세포양활막세포 (Fibroblast-like synoviocyte) 의분리및배양류마티스관절염환자의활막조직을인산완충액 (cell culture phosphate-buffered saline, ccpbs) 이담겨있는 100 mm dish에담아지방조직, 인대, 연골등을제거한다음 Dulbeco's Modified Eagle Medium (DMEM: GIBCO BRL, Grand Island, NY, USA) 으로세척하였다. 여기에 250배희석된 2.5% collagenase (Sigma, St. Louis, MO, USA) 를섞어 37 o C에서 4시간동안반응시켰다. 분리된섬유모세포양활막세포의세포 용액을 1,200 rpm으로 4 o C에서 10분간원심분리한후 20% 우태아혈청 (fetal bovine serum: FBS, GIBCO BRL) 이함유된 DMEM에서 7일간배양하였다. 배양된활막세포는 5 8회계대배양한후실험에사용하였다. Rosiglitazone (Cayman, Ann arbor, MI, USA) 은 dimethylsulphoxide (DMSO) 에각각 (10 μm, 50 μm 및 150 μm) 의농도로용해시켜사용하였다. 3. MTT assay를통한세포증식도평가활막세포의증식정도는 method of transcriptional and translational (MTT) assay를통해정량분석하였다. EZ-CyTox Kit를이용하여 rosiglitazone이활막세포의증식억제를유발하는적정농도를측정하였는데 96 well에배양한활막세포를 10 μm, 50 μm 및 150 μm의 rosiglitazone으로처리한뒤 24시간, 48시간및 72시간후에활막세포의증식억제여부를확인하기위해각 well 마다 kit reagent를넣고 6시간동안 incubation후 microtitre plate reader로 wave length 에서흡광도를측정하였다. 4. RNA 분리및정량적 real-time PCR 활막세포에 rosiglitazone을각각 10 μm, 50 μm 및 150 μm의농도로처리한뒤 TNF-α (10 ng/ml) 를함께투여한군과투여하지않은군으로나누어 48 시간동안반응시킨후 total RNA를 Trizol-reagent (Molecular Research Center Inc., Cincinnati, OH, USA) 를이용하여활막세포로부터분리하였다. RNA의농도는분광광도계를이용하여 260 nm에서측정하고 RNA의순도는 260 nm와 280 nm에서측정한흡광도의비율을이용하여평가하였다. 역전사반응은 TaKaRa RNA PCR Kit (AMV) 를사용하였으며 MgCl 2, RT buffer, dnt, RNase Inhibitor, AMV, Oligo dt, RNase Free water를이용하여 cdna를합성하였다. 염증성싸이토카인 (IL-1β, IL-6), 케모카인 (CCL-2, CCL-7), COX-2와 MMP-9의 mrna 발현감소정도를확인하기위해각각의염증매개물질들에대한특이적인 primer를사용하여증폭하였으며 PCR buffer, D.W., Ex Taq과 20 pmol sense와 anti-sense primer를각각첨가하여 95 o C, 58 o C, 72 o C에서 30 cycle PCR 반응을수행한후 ethidium bromide를사용하여 2% agarose gel상에서 band를분석하였다. 155

4 대한류마티스학회지제 17 권제 2 호 2010 정량적 real-time PCR은 Taqman master mix (Applied Biosystems, Foster City, CA), sense와 anti-sense primer (Applied Biosystems, Foster City, CA) 를사용하여 gene Amp 7300 sequence detector를통해 threshold cycle (Ct) 를측정하였다. 5. Electrophoretic mobility shift assays (EMSA) 100 mm 배양접시에활막세포와 rosiglitazone 100 μm을처리하고 48시간뒤에인산완충액 (phosphate buffered saline: PBS) 으로두번세척하고세포를긁어낸후, 저장성용해완충액 (hypotonic lysis buffer : 10 mm HEPES, ph 8.0, 0.5% Nonidet P-40, 1.5 mm MgCl 2, 10 mm KCl, 0.5 mm DTT, and 200 mm sucrose) 조성의 buffer A 1 ml를첨가하여 vortex한후 10분간얼음위에놓아두었다. 다시 4 o C에서 5분간 3,000 rpm으로원심분리한후상층액을걷어내고 20 mm HEPES-KOH, ph 7.9, 0.42 M NaCl, 1.2 mm MgCl, 0.5 mm DTT, 0.2 mm EDTA, 25% glycerol, 0.5 mm PMSF, 5 μg/ml aprotinin, 5 μg/ml leupeptin 조성의 buffer B 100 μl를첨가한후 20분간얼음위에놓아두었다. 이후 4 o C에서 15분간 1,500 rpm으로원심분리한후상층액을걷어새로운 tube에옮긴후 BCA protein assay를이용하여 protein의농도를측정한후 20 o C에서보관하였다. Double-stranded NF-κB oligonucleotide를 biotin 부착된것을이용하여 LightShift R Chemiluminescent EMSA Kit (Pierce Biotechnology, Rockford, IL, USA) 를이용하여 DNA-protein interction을확인하였는데먼저 4 6% polyacrylamide gel에 pre-run 이후 nylon membrane 을이용하여 transfer시킨후 chemiluminescence으로 biotin-labeled DNA를 detect하였다. Membrane은 cassette에 expose시킨후 X-ray film에 2 3분후 delope 하여 shift된정도를 film 상에서확인하였다. Band의크기가커진만큼 negative acrylamide gel에서 mobility가 shift된것을확인하였다. 결과 1. Rosiglitazone 처치에따른섬유모세포양활막세포의증식억제 PPAR-γ가류마티스관절염의병인에관여하는지 Fig. 1. The effects of rosiglitazone on the viability of rheumatoid FLS. Rheumatoid FLS were treated with varying concentrations of rosiglitazone (10 μm, 50 μm, and 150 μm) for 24 hours, 48 hours and 72 hours and the extent of FLS viability was measured by MTT assay. Results were expressed as % of control and data was the mean of five separate experiments. Rosiglitazone inhibited cellular proliferation of rheumatoid FLS dose and time dependently. *p-value<0.05, p-value<0.01. 여부를알아보기위해류마티스관절염환자의활막으로부터섬유모세포양활막세포를분리배양하여 rosiglitazone을각각 10 μm, 50 μm 및 150 μm의농도로처리한뒤 24시간, 48시간및 72시간후에활막세포의증식억제여부를 MTT assay를통해측정한결과, 그림 1과같이 rosiglitazone을투여한군에서섬유모세포양활막세포의증식억제가유도되었으며이러한억제효과는농도와시간에비례하여증가하였다. 2. Rosiglitazone 처치에따른 IL-1β, IL-6, CCL-2, CCL-7, COX-2와 MMP-9의 mrna 발현감소 Rosiglitazone의항염증효과를확인하기위하여류마티스관절염의활막에서염증을유발하고악화시켜병태생리에가장중요한역할을하는 IL-1β, IL-6, CCL-2, CCL-7, MMP-9 그리고 COX-2의 mrna 발현이 rosiglitazone 처치후감소하는지관찰하였다. 류마티스관절염환자의활막으로부터분리배양된섬유모세포양활막세포에 rosiglitazone을각각 10 μm, 50 μm 및 150 μm의농도로처리한뒤 TNF-α (10 ng/ml) 를함께투여한군과투여하지않은군으 156

5 권용진외 : PPAR-γ Agonist 의류마티스관절염치료효과 Fig. 2. Inhibition of cytokine production from rheumatoid FLS by rosiglitazone. Rheumatoid FLS were treated with varying concentrations of rosiglitazone (10 μm, 50 μm, and 150 μm) with or without TNF-α (10 ng/ml) stimulation for 48 h. The mrna expressions of (A) IL-1β, (B) IL-6, (C) CCL-2, (D) CCL-7, (E) COX-2, (F) MMP-9 were dose-dependently suppressed by rosiglitazone without TNF-α. When TNF-α were treated with rosiglitazone, rosiglitazone treatment induced a dose-dependent reduction in mrna expressions of (E) COX-2, (F) MMP-9. But mrna expressions of (A) IL-1β, (B) IL-6, (C) CCL-2, (D) CCL-7 were increased with 10 μm rosiglitazone and TNF-α, and then decreased as the concentration of rosiglitazone was increased by 50 μm and 150 μm. The results shown in this figure are the mean of five independent experiments, *p-value <0.05 compared to control, p-value<0.05 compared to rosiglitazone 10 μm. 157

대한류마티스학회지제 17 권제 2 호 2010 로나누어 48시간동안반응시킨후 IL-1β, IL-6, CCL-2, CCL-7, COX-2와 MMP-9의 mrna 감소정도를 RT-PCR 및정량적 real-time PCR을통해확인한결과, 그림 2와같이 TNF-α를투여하지않은경우 rosiglitazone의농도가증가함에비례하여 IL-1β, IL-6, CCL-2,

6 대한류마티스학회지제 17 권제 2 호 2010 로나누어 48시간동안반응시킨후 IL-1β, IL-6, CCL-2, CCL-7, COX-2와 MMP-9의 mrna 감소정도를 RT-PCR 및정량적 real-time PCR을통해확인한결과, 그림 2와같이 TNF-α를투여하지않은경우 rosiglitazone의농도가증가함에비례하여 IL-1β, IL-6, CCL-2, CCL-7, COX-2와 MMP-9의 mrna 발현이현저하게감소하였으며 COX-2와 MMP-9의경우 TNF-α를투여한군에서도 rosiglitazone의농도에비례하여 mrna의발현이감소되었다. 그러나 IL- 1β, IL-6, CCL-2와 CCL-7의경우 TNF-α를투여한군에서는 10 μm의 rosiglitazone을처리하였을때오히려 rosiglitazone을처리하지않은대조군에비해 mrna의발현이증가하였으며 rosiglitazone의농도가증가함에따라점차발현이감소하였으나대조군에비해서는여전히증가되어있는것을관찰할수있었다. 3. Rosiglitazone 에의한 NF-κB 의 down-regulation Rosiglitazone의항염증작용을매개하는신호전달체계를확인하기위해 nuclear extracts로부터 electrophoretic mobility shift assays (EMSA) 를시행하여 NFκB의 억제여부를확인한결과, 그림 3과같이섬유모세포양활막세포에서 TNF-α에의해활성화된 NF-κB는 rosiglitazone을처리한후 down-regulation되 Fig. 3. Inhibition of NF-κB by rosiglitazone in rheumatoid FLS. The effects of rosiglitazone on the DNA-binding activity of NF-κB was tested by EMSA. (A) Treated with vehicle only, (B) treated with rosiglitazone 100 μm for 48 h. NF-κB was significantly inhibited by rosiglitazone. The results are representative of five independent experiments. 는것을관찰하였다. 고찰류마티스관절염은다발성으로관절활막에염증이발생하는만성염증성자가면역질환이다. 대부분수년에걸쳐관절파괴를유발하며특히연골과뼈의비가역적손상을유발하여관절의기능저하를초래한다 (4). 염증이있는활막에는수많은단핵구와대식세포가침윤되어있으며특히 T 세포로부터의신호와면역복합체에의해활성화된대식세포가 TNF-α, IL-1β, IL-6와같은염증성싸이토카인을형성하게되고이로인해세포접착분자들 (cell-adhesion molecules) 과 CCL-2, CCL-7와같은케모카인, COX-2, MMP-9 등의발현을증가시켜관절염증및섬유모세포양활막세포의과다증식을유발하여연골파괴를일으키고 RANKL 및파골세포 (osteocalst) 를활성화시켜골파괴를유발하게된다 (25-27). 또한염증성싸이토카인을비롯한여러염증매개인자들은염증세포의침윤을가속화시키고신생혈관을생성하며 collagenase 등의 MMP분비를촉진하여섬유모세포양활막세포의과다증식및연골파괴를일으킨다. 즉류마티스관절염의병인에있어가장핵심적인역할을하는것이대식세포에서분비되는여러염증매개물질이므로대식세포의활성을억제하는것이류마티스관절염의치료에매우중요할것으로생각된다 (28). Peroxisome은진핵세포내에존재하는미세기관으로, 지방산의산화에관여하며, peroxisome의증식을일으키는약물 (peroxisome proliferators) 중지질대사에중요한영향을나타내는 fibrates 계통의 clofibrates, fenofibrates 등은임상에서널리사용되고있다. 이러한 peroxisome proliferator의수용체는 1990년대이후밝혀져 peroxisome proliferator-activated receptor로총칭되고있다. PPAR는핵내에위치하는핵수용체로서부신피질호르몬수용체나활성형지용성비타민수용체와유사한구조를가지며유전자전사활성화를매개하는 DNA 결합성전사인자이다. PPAR는갑상선호르몬수용체나비타민 D 수용체와비슷하게 9-cis retinoic acid 수용체인 retinoid X receptor (RXR) 와 heterodimer를형성하지만 peroxisome 158

7 권용진외 : PPAR-γ Agonist 의류마티스관절염치료효과 proliferator responsive element와결합하여표적유전자에특이적으로작용한다 (29). PPAR는 α, β, γ 의 3가지아형이존재하는데그중 PPAR-γ는지방세포에발현하여지방세포분화와지질대사를조절한다. 지방산대사산물이염증반응의신호전달과정에중요한역할을하므로 PPAR-γ가이러한과정에관여할것이라는가능성이제시되어왔으며최근들어 PPAR-γ가대식세포에서발현되어염증성싸이토카인의생성을억제하여염증반응을억제하는것이밝혀졌다 (30,31). PPAR-γ agonist는두가지종류로분류될수있는데 fatty acid와 15-deoxy-Δ 12,14 - prostaglandin J 2 (15d-PGJ 2) 등의 endogenous agonist (32-34) 와 thiazolinedions (rosiglitazone, troglitazone), fibrates 등의 synthetic agonist가있다 (35). 류마티스관절염에서도이들 PPAR-γ agonist의항염증작용에대한연구가진행되었는데 Yutaka 등이발표한연구에의하면 15d-PGJ 2 와 troglitazone이류마티스관절염환자의활막세포의증식억제와 apoptosis를유도하고관절염실험쥐모델에서활막조직에단핵구의침윤과 pannus 형성을감소시켰다 (36). 그러나 Ji 등이발표한연구에의하면류마티스관절염환자의활막세포에서 15d-PGJ 2 가 TNF-α, IL-1β 의발현을억제하였고 troglitazone이 NF-κB의활성을감소시키기는하였으나활막세포의증식억제및 apoptosis를유도하지는못하였다 (37). 이처럼 synthetic PPAR-γ agonist의항염증효과에대한관심이높아지고있음에도불구하고만성염증성질환의대표라고할수있는류마티스관절염을대상으로한연구들마다그결과에차이를보이고있으며대표적인 synthetic PPAR-γ agonist인 rosiglitazone의류마티스관절염에있어서의작용을조사한연구는아직까지충분히진행되지않았다. 본연구에서는류마티스관절염환자의섬유모세포양활막세포에대표적인 synthetic PPAR-γ agonist인 rosiglitazone을다양한농도로처리하였을때투여농도가증가함에비례하여 IL-1β, IL-6, CCL-2, CCL-7, COX-2와 MMP-9의 mrna 발현이감소됨을관찰하였다. 섬유모세포양활막세포에염증을더욱증가시키기위해 TNF-α를투여하였는데 COX-2와 MMP- 9의경우 TNF-α를투여한군에서도 rosiglitazone의농도에비례하여 mrna의발현이감소되었 다. 그러나 IL-1β, IL-6, CCL-2와 CCL-7의경우 TNF-α를투여한군에서는 10 μm의 rosiglitazone을처리하였을때오히려 rosiglitazone을처리하지않은대조군에비해 mrna 발현이증가하였고 rosiglitazone의농도가증가함에따라점차발현이감소하였으나대조군에비해서는증가되어있는상태임을관찰할수있었다. 이러한현상의원인으로는 TNF-α 처리와같이염증이더욱조장된경우에는저농도의 rosiglitazone이오히려 pro-inflammatory action을할가능성이있을수있겠다. 하지만 50 μm과 150 μ M의 rosiglitazone을투여한경우 TNF-α만투여한경우보다 pro-inflammatory gene의발현이증가되어있기는하나, rosiglitazone의투여용량을늘릴수록그증가정도가감소되는양상으로보아본연구에서사용한 rosiglitazone의용량보다더욱고용량을투여하였을경우의 pro-inflammatory gene의발현 pattern의변화는추후조사해볼필요가있겠다. 일반적으로알려진 rosiglitazone의염증반응에서의작용기전은본연구에서도확인된바와같이 NFκB의 down-regulation을통한 anti-inflammatory action 이주를이룰것으로생각되나 TNF-α와 rosiglitazone을같이투여한경우에본연구에서관찰된 IL-1β, IL-6, CCL-2와 CCL-7의증가현상이어떠한기전을매개로하는것인지에대해서는추후추가적인연구가필요하겠다. 본연구에서는 rosiglitazone이섬유모세포양활막세포의증식에미치는영향을알아보기위해 MTT assay를시행했고그결과, rosiglitazone의투여농도에비례하여섬유모세포양활막세포의증식이억제됨을확인할수있었다. PPAR-γ의세포증식억제효과는싸이토카인의발현을조절하는 NF-κB, AP- 1 등의전사인자를억제하여초래되는것으로알려져있는데 (38), 특히 NF-κB는류마티스관절염에서중요한전사인자로서염증작용을조절하는데섬유모세포양활막세포에서발현이증가되어염증성싸이토카인의생성을증가시킴으로서관절염을악화시키는것으로알려져있다. PPAR-γ agonists가류마티스관절염에서항염증작용을나타내는대표적인신호전달체계역시 NF-κB의억제에의한것으로생각되어지는데이를확인하기위해본연구에서는 rosiglitazone이대식세포의대표적인염증반응신호전달 159

8 대한류마티스학회지제 17 권제 2 호 2010 체계인 NF-κB를억제시킬수있는지여부를 EMSA 를통해알아보았고그결과활성화된 NF-κB가 rosiglitazone의투여에의해억제되는것을관찰할수있었다. 이러한결과를바탕으로 TNF-α를처리하지않은상태에서단독으로투여된 rosiglitazone이대식세포의핵수용체인 PPAR-γ를활성화시켜류마티스관절염의병인에중요한여러가지염증매개물질들의생성을감소시키고관절파괴를유발하는섬유모세포양활막세포의과다증식을억제하며이러한반응은 NF-κB 활성억제에의해유발되는것으로생각되어진다. 본연구를통하여 PPAR-γ가류마티스관절염의병인에관여함을확인하고 synthetic PPAR-γ agonist 를투여했을때관절활막에서일어나는면역반응을감소시키고활막의증식을억제함을규명하였으며이는임상적으로는질병의진행을늦추어기능적손실을감소시키는등류마티스관절염의치료에있어서새로운치료방침을제공하는것이며경제적으로는새로운표적물질의증명으로인한신약개발및치료전략수립에크게이바지할것이다. 결 본연구에서는 PPAR-γ가류마티스관절염의병인에관여하는지여부를알아보기위해류마티스관절염환자의활막으로부터섬유모세포양활막세포를분리배양하여다양한농도의 rosiglitazone을단독으로처리한뒤농도에비례하여 TNF-α, IL-1β, CCL-2, CCL-7, COX-2와 MMP-9의 mrna 발현이감소되는것을확인하였다. 또한 rosiglitazone 처치에따른활막세포의세포증식이억제되고대식세포의대표적인염증반응신호전달체계인 NF-κB down-regulation을확인하였다. 본연구를통하여 PPAR-γ가류마티스관절염의병인에관여하여항염증작용이있음을확인하였으며류마티스관절염의치료에있어서새로운치료방침을제공할수있을것으로생각한다. 론 참고문헌 1) Alamanos Y, Voulgari PV, Drosos AA. Incidence and prevalence of rheumatoid arthritis, based on the 1987 American College of Rheumatology criteria: a systematic review. Semin Arthritis Rheum 2006;36: ) Gabriel SE, Michaud K. Epidemiological studies in incidence, prevalence, mortality, and comorbidity of the rheumatic diseases. Arthritis Res Ther 2009;11: ) Lee DM, Weinblatt ME. Rheumatoid arthritis. Lancet 2001;358: ) Feldmann M, Brennan FM, Maini RN. Rheumatoid arthritis. Cell 1996;85: ) Arend WP, Dayer JM. Inhibition of the production and effects of interleukin-1 and tumor necrosis factor alpha in rheumatoid arthritis. Arthritis Rheum 1995; 38: ) Dayer JM. The pivotal role of interleukin-1 in the clinical manifestations of rheumatoid arthritis. Rheumatology (Oxford) 2003;42 Suppl 2:3-10S. 7) Arend WP, Dayer JM. Cytokines and cytokine inhibitors or antagonists in rheumatoid arthritis. Arthritis Rheum 1990;33: ) Morand EF, Leech M. Macrophage migration inhibitory factor in rheumatoid arthritis. Front Biosci 2005;10: ) Sweeney SE, Firestein GS. Rheumatoid arthritis: regulation of synovial inflammation. Int J Biochem Cell Biol 2004;36: ) Okamoto H, Hoshi D, Kiire A, Yamanaka H, Kamatani N. Molecular targets of rheumatoid arthritis. Inflamm Allergy Drug Targets 2008;7: ) Simmonds RE, Foxwell BM. Signalling, inflammation and arthritis: NF-kappaB and its relevance to arthritis and inflammation. Rheumatology (Oxford) 2008;47: ) Brown KD, Claudio E, Siebenlist U. The roles of the classical and alternative nuclear factor-kappab pathways: potential implications for autoimmunity and rheumatoid arthritis. Arthritis Res Ther 2008;10: ) Pettit AR, Walsh NC, Manning C, Goldring SR, Gravallese EM. RANKL protein is expressed at the pannus-bone interface at sites of articular bone erosion in rheumatoid arthritis. Rheumatology (Oxford) 2006; 45: ) Ainola MM, Mandelin JA, Liljestrom MP, Li TF, Hukkanen MV, Konttinen YT. Pannus invasion and cartilage degradation in rheumatoid arthritis: involvement of MMP-3 and interleukin-1beta. Clin Exp Rheumatol 2005;23: ) Gartlehner G, Hansen RA, Jonas BL, Thieda P, Lohr 160

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Pharmacotherapeutics Application of New Pathogenesis on the Drug Treatment of Diabetes Young Seol Kim, M.D. Department of Endocrinology Kyung Hee Univ

Pharmacotherapeutics Application of New Pathogenesis on the Drug Treatment of Diabetes Young Seol Kim, M.D. Department of Endocrinology Kyung Hee Univ Application of New Pathogenesis on the Drug Treatment of Diabetes Young Seol Kim, M.D. Department of Endocrinology Kyung Hee University College of Medicine & Hospital E mail : ycell2@yahoo.co.kr Abstract