6-20 (69-85).hwp

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1 J Korean Med. 2013;34(3):69-85 pissn eissn Original Article 안순선, 허동석 대전대학교한의과대학한방재활의학과교실 Effects of Kyejiinsam-tang in MIA-Induced Osteoarthritis Rats Soon-sun An, K.M.D, Dong-seok Heo, K.M.D Dept. of Oriental Rehabilitation Medicine, College of Oriental Medicine, Dae-Jeon University Objectives: This study investigated the anti-osteoarthritic effects of Kyejiinsam-tang (hereinafter referred to KIT) on the monosodium iodoacetate (MIA)-induced osteoarthritis rats. Methods: <in vitro> Anti-oxidative effects of KIT were measured by scavenging activities of DPPH, reactive oxygen species (ROS) and nitric oxide (NO). Scavenging activities of anti-oxidation in lipopolysaccharide (LPS)-treated RAW cells were also measured for inhibitory effects against the production of inflammatory mediators (tumor necrosis factor-α, interleukin-1β, interleukin-6). <in vivo> Osteoarthritis was induced in rats by injecting MIA in the knee joint. Rats were divided into a total of 4 groups (n=6). The normal group were not treated at all without inducing osteoarthritis whereas the control group were induced for osteoarthritis by MIA and oral medicated physiological saline per day. The positive comparison group was injected with MIA and after 7 days, 2 / of Indomethacin. The experimental group was injected with MIA and after 7 days was medicated with 34 / of KIT. Indomethacin and KIT were orally-medicated for each substance a total of 4 weeks, once per day. Weight-bearing on hind legs was measured every week after MIA injection. At the end of the experiment (5 weeks after MIA injection), micro CT (computed tomography)-arthrography and histopathological examinations on the articular structures of knee joint were performed. The effect on inflammatory cytokines and immunological cells in synovial fluid was measured. Volume of cartilage was measured by micro CT-arthrography. Injury to synovial tissue was measured by H & E (hematoxylin and eosin), Safranin-O immunofluorescence. Results: 1. Cytotoxicity against hfcs was insignificant. 2. KIT showed the potent full term for DPPH. <in vitro> 1. NO was significantly reduced by KIT (at 100, 200 / ) and ROS was also reduced, but not significantly, by KIT (at 200 / ). 2. IL-6 and IL-1β were significantly reduced by KIT (at 100, 200 / ) and TNF-α was also reduced, but not significantly, by KIT (at 200 / ). <in vivo> 1. In hind legs weight-bearing measurement, level of weight increased. 2. Functions of liver and kidney were not affected. 3. IL-1β was significantly reduced and TNF-α, IL-6 were also reduced but not significantly. 4. PGE2 (prostaglandin E2), LTB4 (leukotriene B4) were significantly reduced in the KIT group. 5. MMP-9 (matrix metalloproteinase-9), TIMP-1 (tissue inhibitor of metalloproteinases-1) and Osteocalcin were significantly reduced in the KIT group. 6. Destruction of cartilage on micro CT arthrography was reduced but had no significant differences. 7. Histopathologically, injury to synovial membrane of the KIT group was decreased and proteoglycan content of KIT group was increased. Conclusions: According to this study, Kyejiinsam-tang has inhibiting effect on the progression of arthritis in MIA-induced osteoarthritis rat. Kyejiinsam-tang has anti-oxidants and anti-inflammation effects, and is related to inhibiting the activity of inflammatory cytokine and injury of volume in cartilage. Key Words : Kyejiinsam-tang, osteoarthritis, anti-inflammation, anti-oxidation, MIA-induced. Received:22 May 2013 Revised:26 June 2013 Accepted:26 June 2013 Correspondence to: 허동석 (Dong-seok Heo) 대전광역시중구대흥동 22-5 대전대학교부속대전한방병원한방재활의학과교실 Tel : , Fax : , hurds@hanmail.net 69

2 (328) 서론골관절염은퇴행성관절염또는퇴행성관절질환이라고도불리며, 주로관절연골의국소적퇴행성변화, 연골하골의비대, 주변골연골부의과잉골형성등을특징으로한다 1). 골관절염의일차적인변화는연골에서시작되고연골, 뼈, 활막세포에서생성된기질단백분해효소 (matrix metalloproteinase, MMP) 와세포질분해효소 (cytokine) 의국소적인생성에의해유도된다 2). 골관절염은의가으로침입하거나관절에응체됨으로써,, 및심하면관절의강직성변형을초래하며, 는하고, 는하며, 등의가있으면하고, 는한다 1). 골관절염연구는,,, 물리적요법등을이용하여혈액학적, 면역학적, 동태학적및조직학적으로접근하고있으며, 연골세포의재생및연골보호, 통증및관절운동개선등의효과가있는것으로보고되고있다 3). 은 4) 에처음등재된,,,, 으로구성된처방으로 6,7), 8), 9), 5), 10) 등은항염증또는항산 화효과와관절염에유효함이알려졌으나, 복합제제인에대해서는골관절염에대한연구를접하지못하였다. 이에저자는이골관절염에미치는영향을살펴보고자, lipopolysaccharide(lps) 로자극된 RAW 세포에대한염증유발인자의변화를관찰하였고, MIA로유발된 rat에대한염증매개인자, 뒷다리체중부하능력, 연골량및병리조직학적변화를관찰한결과, 유의한성적을얻었기에보고하는바이다. 실험 1. 재료 1) 세포및동물실험에사용된 RAW 세포는한국세포주은행 ( 서울, 한국 ) 에서구입하여사용하였다. 동물은수컷 6 주령의 Wistar rat( g)( 오리엔트바이오, 성남, 한국 ) 을사용하였고, 실험당일까지고형사료 (5L79, 오리엔트바이오, 성남, 한국 ) 와물을충분히공급하고, 온도 22±2, 습도 55±15%, 12시간 (light-dark cycle) 의환경에서 1주간적응시켰다. 본연구는대전대학교동물실험윤리위원회의승인을받아동물윤리준칙에의거하여진행하였다 ( 승인번호 : DJUARB ). 2) 약재실험에사용한 (Kyejiinsam-tang, 이하 KIT) 은 4) 과 11) 에근거하여 처방하였고, 구성약물들은대전대학교둔산한방병원에서구입하여정선후사용하였으며, 내용과분량 (1첩) 은다음과같다 (Table 1). 3) 시약본실험에사용된시약은 lipopolysaccharide(lps), dimethyl sulfoxide(dmso), 1,1-diphenyl-2-picrylhydrazyl(DPPH) 는 Sigma-Aldrich Co.(st. Louis, MO, USA) 에서, Dulbecco's Modified Eagle's Medium(DMEM) 배양액, Fetal Bovine Serum(FBS), Penicillin 및 Streptomycin은 Hyclone Co.(Logan, Ut, USA), Cell viability assay kit은 Daeil Lab service Co.( 서울, 한국 ) 에서, Nitric oxide detection kit은 Intron Biotechnology Co.( 수원, 한국 ) 에서구입하였 Pharmacognostic nomenclatures Amount(g) Cinnamomi Ramulus 8.0 Glycyrrhizae Radix 8.0 Atractylodis macrocephalae Rhizoma 6.0 Ginseng Radix 6.0 Zingiberis Rhizoma 6.0 Total

3 안순선외 1 명 : (329) 다. Cytokine Milliplex Map Immunoassay kit은 Millipore Co.(Bellerica, MA, USA) 에서, HPLC 분석에이용된 water와 acetonitrile은 Ducksan Co.( 안산, 한국 ) 에서, Rat Total MMP-9(matrix metalloproteinase-9) kit, LTB4(leukotriene B4) kit, Rat TIMP-1(tissue inhibitor of metalloproteinases-1) kit, Rat IL-6(interleukin-6) kit, Rat TNF-α(tumor necrosis factor-α) kit, PGE2(prostaglandin E2) kit, Rat IL-1β(interleukin-1β) kit은 R&D System Co.(Minneapolis, USA) 에서, 주정추출에이용된주정은대한주정판매 ( 전주, 한국 ) 에서구입하였다. 양성대조군에는 Indomethacin(Sigma Co., USA), 골관절염유발주사제로는 Monosodium iodoacetate (MIA)(Sigma. Chemical Co. Ltd, Cat. No. I2512, USA), 마취제로는 Zoletil(Virbae Co., France) 과 Rompun( 바이엘코리아, 서울, 한국 ) 을사용하였다. Hematoxylin and eosin 염색과 Safranin-O 염색에는 Hematoxylin(Merck Co., Germany), Eosin Y(Wako Co., Japan), Safranin-O(Merck Co., Germany) 를, Micro CT arthrography의조영제는 Hexabrics 320(Guerbet Co., France) 을사용하였다. 4) 기기본실험에사용된기기는 Rotary vacuum evaporator(eyela, Japan), Freeze dryer( 일신바이오베이스, 한국 ), ELISA reader(molecular Devices, USA), Luminex(Millipore Co., USA), HPLC (Shimadzu LC-20AD, Japan), HPLC column(ace 5 C18, , 5 ) 를, Flow cytometry system (BD Biosciences Immunocytometry Systems, USA), Incapacitance Meter Tester(IITC Life Science, california, USA), BD Ultra-Fine ll(becton, Dickinson & Company, USA), Light Microscope(Carl ZEISS AXIOSKOP-40, Germany), micro CT arthrography (SkyScan, Optoscan, Belgium) 를이용하였다. 2. 방법 1) 시료추출 KIT 6첩을분말한후, 70% 주정과 30% 증류수를혼합한 800 에 24시간침지후여과하여, 그여액 을 rotary evaporator 를이용하여 50 로감압, 추출, 동결건조하여사용하였다. 추출수율에따른한첩분량은 4.08 g이었다. in vitro 1) 세포독성측정 RAW 세포는 96well plates에 cells/well로분주하여 24시간동안배양한후, KIT을각각 50, 100, 200 / 의농도로처리하여 24시간동안배양하였다. 배양후 10 의 WST solution을첨가하여 CO 2 배양기 (37, 5% CO 2 ) 에서 30분간반응시킨후, 450 에서흡광도의변화를측정하여, only cell을대조군으로하여대조군에대한세포생존율을백분율로표시하였다. 2) 항산화활성측정 (1) 1,1-diphenyl-2-picryl-hydrazyl(DPPH) 소거능자유라디칼소거활성시험은안정한자유라디칼 DPPH를사용하는방법으로주정에용해시킨 0.2 mm의 DPPH 용액 150 와 KIT(25, 50, 100, 200, 400, 800 / ) 100 를각각혼합하고, 37 에서 30분간반응시킨후, 517 에서흡광도를측정하였다. 대조군은시료액대신증류수를넣고, DPPH 용액대신주정을넣어보정값을얻었다. 자유라디칼소거율은아래의식에따라계산하였다. 소거율 (%) = ( (2) 세포내 ROS 생성 대조군의흡광도 - 시료첨가군의흡광도 ) 100 대조군의흡광도 Lipopolysaccharide(LPS) 로자극된 RAW 세포내에서 reactive oxygen species(ros) 를측정하기위하여 2,7 -dichlorofluorescin diacetate(dcf-da) 를이용하였다. 48 well plate에 RAW 세포를 cells/well이되게분주하였다. 24시간동안배양한후여기에 LPS(1 / ) 및 KIT(50, 100, 200 / ) 를각각의 well에첨가한후 24시간동안 37, 5% CO 2 배양기에서배양한후 1,200 rpm에서 5분간원심분리하였다. 원심분리후모은세포를차가운 PBS로 2회세척한후, DCF-DA 10 이되도록첨가하여 15분동안빛이차단된상온에서염색하였다. 71

4 (330) 염색후차가운 PBS를넣어 1,200 rpm에서 5분간원심분리한다음상층액을제거하고, 다시 PBS 400 를부유시켜유세포분석기 (Flow cytometer, Becton, Dickinson & Company, Franklin Lakes, NJ, USA) 를이용하여형광강도의세기에따른변화를분석하였다. (3) 세포내 NO 생성 Total nitric oxide(no) 의농도는배양액내의 nitrite 농도를 Griess Reagent 방법으로측정하였다. RAW 세포는 96 well plates에 cells/well 로분주하여 24시간동안배양한후 KIT(50, 100, 200 / ) 을처리하고, LPS(1 / ) 로처리하여다시 24시간동안배양하였다. N1 buffer 50 를각 well에처리한후 10분간상온에서암소반응후, N1 buffer 50 를처리한것에 N2 buffer 50 를각 well에처리하고 10분간반응시킨후, 540 에서흡광도를측정하였다. Nitrite standard의농도별표준곡선을이용하여배양액의 NO 농도를결정하였다. 2) 군분리및약물투여 MIA 주사 7일후에관절염유발유무를확인하여관절염이유발된동물만을대조군 (Control), 양성대조군 (Indomethacin), KIT 투여군 (KIT) 으로나누었고, MIA를주사하지않은정상군 (Normal) 을포함하여동물수는각군당 6마리로하였다. Wistar rats에 MIA를주사하여골관절염을유발시킨후일주일이경과된이후부터 1일 1회매일오전 10시에대조군에는생리식염수 2 / 을, 양성대조군에는 Indomethacin 2 / 을, KIT 투여군에는 KIT 34 / 을 4주동안경구투여하였다. 모든군에서자유음수와식이를실시하였고, 총 5주간실시하였다. 3) 뒷다리체중부하검사뒷다리체중부하는 Incapacitance Meter Tester를이용하여플라스틱방에 60도기울기로세운후, 각뒷다리에가해진세기를 10초에걸쳐평균을산출하였다. 처치된동측의뒷다리에분포된체중의백분율은다음과같은방정식을이용하여계산하였다. 3) 염증관련 cytokine 분석 RAW 세포를 6 well plates에 cells/ 가되도록분주하고, 24시간동안배양한후, KIT(50, 100, 200 / ) 를처리하고, LPS(1 / ) 로처리하였다. 24시간동안배양한후세포배양액을수거하여배양액에함유된 TNF-α, IL-1β, IL-6를 Milliplex map cytokine kit(millipore Co., Bellerica, MA, USA) 을이용하여측정하였다. 분석은 Milliplex analyst software( 고마바이오텍, 서울, 한국 ) 를통해이루어졌다. in vivo 1) MIA 주사에의한골관절염유발 Zoletil 0.5 와 Rompun 0.1 로 rat을마취시킨후왼쪽무릎주변을깨끗이제모하고골관절염유발물질인 Monosodium iodoacetate(mia) 를 31G의 1 Insulin Syringe(0.25 8, BD Medical-Diabetes Care, USA) 를사용하여왼쪽무릎관절내에 50 (60 / ) 씩투여하였다. MIA 희석시에는 0.9% saline을사용하였다. 유발된하지의무게백분율 (%) = ( ) 100 정상하지의무게 4) 혈액분석최종실험종료후튜브형주사기를이용하여심장에서채혈하였다. 전혈을바이오톡스텍 ( 주 )( 청주, 한국 ) 에의뢰하여 AST, ALT, BUN, Creatine 을측정하였다. 5) 혈청내염증 cytokine 및염증매개인자측정최종실험종료후혈액을채취하여혈청을분리한뒤, 염증성지표로서 TNF-α, IL-1β, IL-6 등을측정하고, Prostaglandin E 2 (PGE 2 ), Leukotriene B 4 (LTB 4 ), Matrix metalloproteinase-9(mmp-9), Tissue inhibitor of metalloproteinase-1(timp-1), Osteocalcin 등의염증매개인자들을 ELISA assay kit로측정하였다. 6) 연골량측정조영제인 Hexabrics 320을왼쪽무릎에투여하고, micro CT arthrography를사용하여 cartilage volume 72

5 안순선외 1 명 : (331) 을측정하여서, 무릎관절연골의파괴정도를분석하였다. 7) 병리조직학적검사 Micro CT arthrography가끝난후무릎부위를절단하여, 10% EDTA가포함된 10% 포르말린용액에넣어관절조직을탈칼슘화시켰다. Radiographic technique를이용하여 decalcification 유무를확인한후파라핀왁스에관절조직을넣고고정한다음 coronal section을실시하였다. Decalcification 과정을거쳐파라핀으로고정된조직을 7 의크기로자른뒤, hematoxylin and eosin(h & E) 및 Safranin-O 염색을실시하여 100배의비율로조직상태를관찰하였다. 염증반응발생유무나활막세포의증식, 염증세포의조직침윤여부는 H & E 염색결과에서확인할수있으며, proteoglycan 층을염색하는 Safranin- O 염색결과에서는연골조직의손상여부를확인하였다. 3. 통계처리실험결과는 SPSS 11.0의 unpaired student's t-test 를이용하여통계처리하였으며, p< 0.05에서유의성을검정하였고, 그값은평균 (mean) ± 표준편차 (standard deviation) 로표기하였다. 결과 1. 세포독성에미치는영향세포독성을관찰한결과 RAW 세포에서대조군 (KIT 0 / ) 의생존율은 100.0±0.4% 로나타난반면, KIT의 50, 100, 200( / ) 농도에서는각각 112.1±11.7%, 95.9±13.8%, 91.7±13.8% 로나타났다 (Fig. 1). in vitro 1) 항산화활성에미치는영향 (1) DPPH 소거능 RAW cells were treated with 50, 100, 200 μg / ml of KIT for 24 hours. Cell viability was determined using the WST assay. Extracts were incubated with DPPH solution at 37 for 30 mins. Activities were determined by measurement of absorbance at 517 nm. The ROS production was analysed following incubation with DCFH-DA by flow cytometry. Significant value was calculated by compared with control group by student's t-test(***p<0.001). 73

6 (332) DPPH 소거능은 KIT 25, 50, 100, 200, 400, 800( / ) 에서각각 4.9±0.5%, 10.8±2.4%, 15.3± 1.9%, 17.2±3.0%, 30.2±3.0%, 51.3±3.5% 로나타났다 (Fig. 1). (2) ROS 와 NO 생성 Reactive oxygen species(ros) 생성이대조군에서 100.0±0.0% 였을때정상군은 51.0±6.7%, KIT 50, 100, 200( / ) 에서는각각 102.7±5.2%, 93.9±5.3%, 92.3±12.9% 로나타났다. Nitric oxide (NO) 생성이대조군에서 100.0±2.7% 였을때정상군은 38.0±4.0%, KIT 50, 100, 200( / ) 에서는각각 93.6±1.7%, 68.9±0.9%, 60.7±4.0% 로, KIT 100, 200( / ) 에서유의성있는감소 (p<0.001) 가나타났다 (Fig. 1). 2) 염증 cytokine 생성에미치는영향 TNF-α 생성량이대조군에서 100.0±2.0% 였을때정상군은 10.0±1.0%, KIT 50, 100, 200( / ) 에서는각각 104.9±3.4%, 103.0±9.4, 96.4±7.3% 로나타났다. IL-1β 생성량이대조군에서 100.0± 2.0% 였을때, 정상군은 4.3±4.7%, KIT 50, 100, 200( / ) 에서는각각 84.8±16.0%, 45.1±8.8%, 34.4±2.9% 로, KIT 100, 200( / ) 에서유의성있는감소 (p<0.001) 가나타났다. IL-6 생성량이대조군에서 100.0±2.0% 였을때, 정상군은 2.0±1.0%, KIT 50, 100, 200( / ) 에서는각각 89.3±6.3%, 50.0±7.9%, 41.4±14.1% 로, KIT 100, 200( / ) 에서유의성있는감소 (p<0.01, p<0.001) 가나타났다 (Fig. 2). in vivo 1) 뒷다리체중부하측정뒷다리체중부하를측정한결과대조군이 100±11.2% 였을때, 정상군 166.9±2.3%, 양성대조군 164.1±4.7%, KIT 투여군 142.0±16.4% 로, KIT 투여군에서유의성있는증가 (p<0.05) 가나타났다 (Fig. 3). α β TNF-α: tumor necrosis factor-α, IL-1β: interleukin-1β, IL-6: interleukin-6. Cells were treated with LPS(1 μg / ml ) and control(0 μg / ml ), 50, 100, 200 μg / ml of KIT for 24 hours. The results are expressed as mean ± SD from three independent experiments. Significant value was calculated by compared with control group by student's t-test(**p<0.01, ***p<0.001). 74

7 안순선외 1 명 : (333) KIT 투여군 43.0±8.2 로나타났다 (Fig. 4). 3) 신장기능에미치는영향혈청중의 BUN 농도는정상군이 15.30±0.89, 대조군 18.10±1.45, 양성대조군 18.90±2.03, KIT 투여군 17.50±3.04로나타났고, 혈청중의 creatinine 농도는정상군이 0.53±0.02, 대조군 0.55±0.02, 양성대조군 0.58±0.01, KIT 투여군 0.59±0.02로나타났다 (Fig. 4). Normal; Normal Wister rat group, Control; MIA-induced osteoarthritis group treated with normal saline, Indomethacin; MIA-induced osteoarthritis group treated with Indomethacin, KIT; MIA-induced osteoarthritis group treated with KIT. The results are expressed as mean ± SD from three independent experiments. Statistically significant value compared with control by t-test(*p<0.05, **p<0.01). 2) 간기능에미치는영향혈청 AST는정상군, 대조군, 양성대조군, KIT 투여군에서각각 130.0±8.7, 162.7±7.1, 160.0±15.1, 172.0±28.2로, 혈청 ALT는정상군이 32.0±1.7, 대조군 33.7±1.2, 양성대조군 37.7±4.2, 4) 혈청내염증 cytokine 및염증매개인자생성에미치는영향 (1) TNF-α TNF-α 생성량이대조군에서 100.0±10.2% 였을때, 정상군은 82.8±18.5%, 양성대조군 85.0±9.0%, KIT 투여군이 94.9±7.2% 로나타나, KIT 투여군에서감소하였으나유의성은없었다 (Fig. 5). (2) IL-1β IL-1β 생성량이대조군에서 100.0±1.3% 였을때, 정상군은 67.8±3.6%, 양성대조군 80.2±4.1%, 250 AST ALT AST & ALT level in serum (U/L) Normal Control Indomethacin KIT Normal; Normal Wister rat group, Control; MIA-induced osteoarthritis group treated with normal saline, Indomethacin; MIA-induced osteoarthritis group treated with Indomethacin, KIT; MIA-induced osteoarthritis group treated with KIT. AST: aspatrate aminotransferase, ALT: alanine aminotransterase, BUN: blood urea nitrogen. Values represent the means ± SD. 75

8 (334) α β Normal; Normal Wister rat group, Control; MIA-induced osteoarthritis group treated with normal saline, Indomethacin; MIA-induced osteoarthritis group treated with Indomethacin, KIT; MIA-induced osteoarthritis group treated with KIT, TNF-α: tumor necrosis factor-α, IL-1β: interleukin-1β, IL-6: interleukin-6, PGE 2: Prostaglandin E 2. Values represent the means ± SD. Statistically significant value compared with control by t-test(*p<0.05, ** p<0.01, ***p<0.001). KIT 투여군 87.3±2.5% 로나타나, KIT 투여군에서유의성있는감소 (p<0.001) 가나타났다 (Fig. 5). KIT 투여군에서유의성있는감소 (p<0.05) 가나타났다 (Fig. 6). (3) IL-6 IL-6 생성량이대조군에서 100.0±14.0% 였을때, 정상군은 77.2±11.4%, 양성대조군 78.5±11.1 %, KIT 투여군 83.1±15.5% 로나타나, KIT 투여군에서감소하였으나유의성은없었다 (Fig. 5). (4) Prostaglandin E2 Prostaglandin E2 생성량이대조군에서 ±13.2% 였을때, 정상군이 24.7±4.9%, 양성대조군 42.2±9.5%, KIT 투여군 40.0±20.9% 로나타나, KIT 투여군에서유의성있는감소 (p<0.01) 가나타났다 (Fig. 5). (5) Leukotriene B4 Leukotriene B4 생성량이대조군에서 100.0± 12.3% 였을때, 정상군 67.4±10.2%, 양성대조군 69.8±6.5%, KIT 투여군 74.1±12.3% 로나타나, (6) Matrix metalloproteinase-9 Matrix metalloproteinase-9 생성량이대조군에서 100.0±1.8% 였을때, 정상군 50.0±4.1%, 양성대조군 64.6±1.0%, KIT 투여군 81.9±8.1% 로나타나, KIT 투여군에서유의성있는감소 (p<0.01) 가나타났다 (Fig. 6). (7) Tissue inhibitor of metalloproteinase-1 Tissue inhibitor of metalloproteinase-1 생성량이대조군에서 100.0±1.6% 였을때, 정상군 54.4±3.9 %, 양성대조군 72.0±4.2%, KIT 투여군 89.4±7.1 % 로나타나, KIT 투여군에서유의성있는감소 (p<0.05) 가나타났다 (Fig. 6). (8) Osteocalcin Osteocalcin 생성량이대조군에서 100.0±8.5% 였을때, 정상군 41.2±3.0%, 양성대조군 60.1±4.1 %, 76

9 안순선외 1 명 : (335) Normal; Normal Wister rat group, Control; MIA-induced osteoarthritis group treated with normal saline, Indomethacin; MIA-induced osteoarthritis group treated with Indomethacin, KIT; MIA-induced osteoarthritis group treated with KIT, LTB 4: Leukotriene B 4, MMP-9: Matrix metalloproteinase-9, TIMP-1: Tissue inhibitor of metalloproteinase-1. Values represent the means ± SD. Statistically significant value compared with control by t-test(*p<0.05, **p<0.01, ***p<0.001). Normal; Normal Wister rat group, Control; MIA-induced osteoarthritis group treated with normal saline, Indomethacin; MIA-induced osteoarthritis group treated with Indomethacin KIT; MIA-induced osteoarthritis group treated with KIT KIT 투여군 70.4±8.6% 로나타나, KIT 투여군에서유의성있는감소 (p<0.01) 가나타났다 (Fig. 6). 5) 연골량에미치는영향 연골량을측정한결과정상군 4.64, 대조군 1.15, 양성대조군 3.70, KIT 투여군이 3.29 로나타나, 대조군에비해 KIT 투여군에서연골이덜파괴되었다 (Fig. 7). 77

10 (336) 6) 병리조직학적변화 (1) Hematoxylin & Eosin 염색 (H & E) 조직염색후 100배비율로관찰한결과, 정상군의관절은활막조직이정상적으로위치한반면, 대조군은관절주변에활막세포의과다침투로인해연골과뼈의침윤으로활막조직의손실이나타났다. 양성대조군은대조군에서비해서연골과뼈의침윤등이상대적으로감소하였고, KIT 투여군도연골과뼈의침윤이많이억제되는것으로나타났다 (Fig. 8). 이보였다 (Fig. 9). Normal; Normal Wister rat group, Control; MIA-induced osteoarthritis group treated with normal saline, Indomethacin; MIA-induced osteoarthritis group treated with Indomethacin, KIT; MIA-induced osteoarthritis group treated with KIT. Stain intensity of red color increased in proportion to proteoglycan content in normal. The red color disappeared in almost articular cartilage and red color remained in periphery of necrotic area in control. Compare the stain intensity and areas of red color in Indomethacin and KIT with the normal and control. 고찰 Normal; Normal Wister rat group, Control; MIA-induced osteoarthritis group treated with normal saline, Indomethacin; MIA-induced osteoarthritis group treated with Indomethacin, KIT; MIA-induced osteoarthritis group treated with KIT. In normal, articular cartilages are sell developed. In control, most of articular cartilage was degenerated. In Indomethacin and KIT, most of articular cartilages are degenerated and appeared homogeneous, but proteoglycan in periphery of degenerated area are remained. (2) Safranin-O 염색조직염색후 100배비율로관찰한결과, 정상군의관절은연골조직이정상적으로적으로위치하였고, 대조군은관절주변에염증세포의침투가일어나붉게염색된정상연골조직이 MIA에의해파괴되어, proteoglycan 조직이사라진것을관찰할수있었다. 양성대조군은관절면에대조군에서비해연골과뼈의침윤등이적었으며, 붉게염색된 proteoglycan 조직이활막주변에많이분포하였다. KIT 투여군은대조군에비해붉게염색된강도가높아 proteoglycan 조직의분포가많 골관절염은관절연골의퇴행성질환으로, 기질의감소와구성요소의합성억제로인한 13) 연골의파괴를특징으로하며, 골극형성, 연골하골경화및미세골절, 경증의활막염증등과같은병리적소견을보인다. 연골의항상성유지에중요한연골세포의변화와사멸로인하여 type II collagen, proteoglycan, aggrecan 등세포외기질의생산이감소하고분해가촉진되면서, 연골의기본구조가파괴되어관절부하를견디지못한다 14). 이러한연골파괴과정에 IL-1β, TNF-α, TGF-β 등과같은 cytokines이중요한역할을수행한다 15). 고령화사회와비만인구의증가로인한유병율은지속적으로증가하고있으며 2008년국민건강영양조사에따르면 2008년성인중 10.7 % 가골관절염을앓고있으며연령에따라비율이증가하여 80대에는 34.5% 에이르는것으로추정된다 16). 골관절염은의범주로주요한임상증상은 78

11 안순선외 1 명 : (337) 이며 이공통된 이다. 는 한약으로 를고조시켜 를축출하고, 는 시키며, 한사람의 는 시키거나 시키도록한다 1). 치료법으로는약물치료, 물리적요법, 수술및 주사치료등이있으나 17), 골관절염으로인한사 회경제적비용이증가되면서효과적인치료법을 개발하기위한연구가진행되고있다. 발병기전에 대한연구, 연골파괴를억제하고진행을막기위 한약물에대한연구 14), 및 의항산화및 항염증작용 18-20) 과이것을토대로골관절염과결 합시키는노력 21) 도이루어지고있다. 본연구에서사용한 (KIT) 은 4) 에처음등재된,,,, 으로구성된처방으로 이라하여, 과 을 겸한경우사용하는처방 22) 이다. 이 한 23) 에서 는. 이 라하여진통작용이언급되었고, 김 6) 은 collagen 유발관절염모델에서관절염억제에유효함을 보고하였다. 은 23) 에서. 라하여 및 의진통작용이언급되 었고, 추출액이관절염유도생쥐모델의 TNF-α, IL-1β 등의 cytokine 감소와항산화효과 가밝혀졌다 6,7). 은 23) 에서 라하여 에효과적이라하였고, 김 8) 은 이 collagen유도관절염생쥐모델에서 류마티스관절염의중등도감소와 TNF-α 증가를 억제하는효과가있다고하였다. 는 23) 에 라하여진통효과 가언급되었고, 의쥐의대식세포에 대한항염증효과가보고 9) 되었다. 은 23) 에서. 라하여진통작용이언 급되었으며, 쥐의면역세포에대한항염증효과 10) 와 이들어간처방의골관절염유발쥐에대 한항골관절염효과와항산화및항염증작용이보고 18,20) 되었다. 통증에유효하다는문헌적근거와항염증항산화작용이있는본초의구성, 과을겸한와으로구성되어의효능 을가지고있음에착안하여이초기이후의골관절염에적합하리라는가설을세우고, 염증유발인자및 MIA로유발된병태동물모델의관절내염증상태변화를관찰하였다. (KIT) 의주정추출물을시료로하여 RAW 세포에서항산화활성과염증 cytokine에미치는영향을분석하였고, rat의무릎내로 monosodium iodoacetate(mia) 를주입하여골관절염을유발한후 KIT를경구투여하여, 뒷다리체중부하와염증 cytokine 및염증매개인자에미치는영향, 병리조직학적소견을관찰하였다. 본연구에서이용한 RAW 세포는대식세포주로서, cytokine의자극만으로도스스로 nitric oxide(no) 를생성하여세포독성을갖는물질의독성검정을위해유용하게사용된다 24). RAW 세포활성에이용한 lipopolysaccharide (LPS) 는동물실험에서의자가면역질환유발물질로, 대식세포를자극하여 IL-1을분비시켜 TNF-α 분비를유발하고 PGE2 생성을증가시킨다 25). 골관절염유발물질로사용된 MIA는관절연골세포의해당과정의 glyceraldehyde-3-phosphate dehydrogenase 활성을억제하여변성을일으켜골관절염을유발시키는물질로, 쥐를대상으로한골관절염유발실험에서관절연골의손상, 관절의기능장애, 통증등이인체의골관절염과매우유사하다고알려져있다 26). MIA 주입 7일째에는윤활막과주위조직내에서변형이발생된다 27). Proteoglycan의소실 28) 과연골및연골하골의기질붕괴가일어나며, 금속효소및 MMP, 아교질분해효소, gelatinase의활성이증가한다 29). 또한파골세포와골아세포의활동이증가하면서관절연골의소실로인해연골하골에부하가증가하게되어 30), 연구에적합한병변상태라생각하여 MIA 주사 7일이후부터 KIT를경구투여하였다. 양성대조군에경구투여한 Indomethacin은 COX-1 을억제하여소염, 진통작용을갖는비스테 79

12 (338) 로이드성소염진통제로급성관절염에유효하다 31). 약재의안정성검증을위한세포독성검사에서는 KIT 50, 100, 200( / ) 농도에서세포생존율이모두 90% 이상으로안정성이검증되었다 (Fig. 1). 항산화물질의가장특징적인것은유리기와반응하는것으로, 활성 radical에전자를공여하는 radical 소거작용은항산화효과와인체에서의노화억제척도로이용된다 32). DPPH는 hydrazyl의질소원자가항산화성물질과의반응을통해수소원자를받아들여고유의정색성을잃는것을이용한방법으로, 항산화효능의정도를알수있다 33). KIT의 DPPH 소거활성을측정한결과, 농도가증가함에따라소거활성역시증가하여 KIT의항산화효능이확인되었다 (Fig. 1). 염증반응을조절하는 NO는두개의원자로이루어진매우불안정한 free radical로혈관확장, 신경전달체제, 항균물질, 면역조절등에관여하여 34,35), 과량생성되면 TNF-α, IL-1 및 IL-6 뿐만아니라 COX-2 같은염증매개물질을과생산하여, 과도한면역반응을일으킨다 36). 또한 NO가다량생성되면 reactive oxygen species(ros) 도같이생성된다 37,38). In vitro에서 RAW 세포에서 ROS와 NO 생성량을측정한결과, 대조군에비해 KIT 군에서감소하였으며, 특히 NO 생성량이 KIT 100, 200( / ) 에서유의성있게감소하여, KIT의항산화활성이 in vitro에서유효함을알수있다 (Fig. 1). Cytokine인 TNF-α, IL-1β 및 IL-6은염증성활막에서다량생산되어기질의합성을저해하고분해를촉진하며, PGE2는활막섬유아세포에작용하여파골세포의생성을유도해서골관절염을일으키는것으로, cytokine의억제가관절염치료에서중요한역할을한다 39,40). TNF-α는면역반응초기에분비되어 IL-1, IL-6, IL-8 및 MMP 등의생성을유발하여염증을증폭시키며 41), 관절연골기질의 proteoglycan 소실증가및재합성억제하여골관절염을진행시킨다 42). IL-1β는골관절염에서 MMP의생성및분비를촉진하여연골기질파괴를증가시키고, PGE2의생성을촉진하여 proteoglycan을소실시키고재합성을억제한다 43). IL-6은 IL-1β과 TNF-α에의해분비가촉진되어파골세포분화를증가시켜, 골흡수와골생성을방해한다 44). Proteoglycan은관절표면에 proteoglycan membrane을형성하여관절면을보호하고, 관절의운동으로발생되는삼투압을통해연골의영양공급을한다 45). TNF-α, IL-1β, IL-6 생성에대한 KIT의억제효과를측정한결과, IL-1β와 IL-6 생성량은 KIT 100, 200( / ) 에서유의성있게감소하였고, TNF-α 생성량은 KIT 200( / ) 에서감소하였으나유의성은없었다 (Fig. 2). KIT 100, 200( / ) 에서의유의성있는 IL-1β와 IL-6 생성감소로 KIT의항염증작용을알수있다. In vivo에서 rat의뒷다리체중부하는대조군이정상군에비해감소하였고, KIT 군은대조군에비해유의성있게증가하였다 (Fig. 3). 이는 KIT의투여가골관절염의통증을완화시켜체중부하를증가시킨것으로생각된다. 간기능을반영하는혈청 AST와 ALT 농도, 신장기능을반영하는 BUN과사구체의여과율을반영하는혈청 creatinine 농도는모두대조군에비해유의한증감을보이지않아 KIT가간과신장기능에관해안전한약물임을확인하였다 (Fig. 4). 혈청내 cytokine의변화를확인하기위해 TNF-α, IL-1β 및 IL-6 생성량을측정한결과, KIT 군에서감소하였으며그중 IL-1β 생성량은유의성있게감소하였다 (Fig. 5). KIT 투여가관절내의염증반응을억제하여골관절염의진행을막는다는것을알수있다. Arachidonic acid는 cytokine이나호르몬등에의해분비되어 prostaglandin, thromboxane, leukotriene과같은 eicosanoids로전환되는불포화지방산이다 46). PGE2는 COX의활성화의주요산 물로 47) 염증시주변조직을파괴하고 48), 통증과 연관있다 49,50). LTB4는과도하게생산되면염증성질환을악화시키고, 류머티스관절염의관절액에서백혈구수, 면역복합체의농도등과상관관계를나타내는인자로알려져있으며 51), LTB4의생산저해제투여가류머티스관절염에억제효과가있다고보고되었다 52). PGE2와 LTB4의생성량은 KIT 군에서유의성 80

13 안순선외 1 명 : (339) 있게감소하여 (Fig. 5, 6), KIT가 COX 반응을억제하는항염증작용과더불어통증감소및연골파괴방지에도효과적임을알수있다. 염증성관절에서연골및골파괴에중요한역할을하는것이 Matrix metalloproteinase(mmp) 같은단백분해효소이며, 그작용은 Tissue inhibitor of metalloproteinases(timp) system에의해길항된다 53). MMP는생체내에서 extracellular matrix protein으로작용하며, 골관절염에서는 proteoglycan의분해와연관있어 54), MMP 활성조절이관절염치료에서중요하다 55). MMP의분비를제어하고활성도를조절하는 TIMP는교원질및단백다당과같은세포외기질성분을분해하는효소로, 골관절염의병인에중요한역할을한다 56). Osteocalcin은골아세포의활동을반영하는골조직생성표지자로, 파골세포에서형성되어파골세포의분화와골재형성에중대한작용을한다 57). 본연구에서 MMP-9, TIMP-1 및 Osteocalcin 생성량은 KIT 군에서유의성있게감소하여 (Fig. 6), KIT 투여가 MMP-9, TIMP-1, Osteocalcin의생성을억제하여 proteoglycan 분해와파골세포의분화를감소시켜연골및골을보호하는효과가있음을알수있다. Micro CT arthrography를사용하여연골량을측정한결과, KIT 군이대조군에비해연골량파괴가감소하였으며 (Fig. 7), H & E 및 Safranin-O 염색을통한병리조직학적검사에서도 KIT 군이증가된 proteoglycan량과감소된염증세포소견을보여활막의손상정도를감소시켰다 (Fig. 8, 9). 이는 KIT가항염증및진통작용뿐만아니라뼈와연골의침윤을감소시켜골관절염의진행을억제함을의미한다. 이상의연구결과를종합하면, (KIT) 의투여가 LPS로자극된 RAW 세포에서항산화및항염증효과를확인할수있었고, MIA 유발골관절염 rat에서뒷다리의체중부하증가, 혈액내의염증 cytokine과염증매개인자발현을억제하는항염증효과, 연골의변성및파괴를방지에유효하였다. 따라서임상적범주로확장하는추가연구를통하여향후골관절염약물 로써치료에이용할수있으리라사료된다. 결론 (KIT) 이골관절염에미치는영향을살펴보고자, lipopolysaccharide(lps) 로자극된 RAW 세포에대한염증유발인자의변화를관찰하였고, MIA로유발된 rat에대한염증매개인자, 뒷다리체중부하능력, 연골량및병리조직학적변화를관찰하여다음과같은결론을얻었다. 1. 세포독성은없었다. 2. 농도증가에따라 DPPH 소거활성은증가하였다. <in vitro> 1. KIT 100, 200( / ) 에서 ROS 생성량은감소하였으나유의성은없었으며, NO의생성량은유의성있게감소하였다. 2. TNF-α 생성량은 KIT 200( / ) 에서감소하였고, IL-1β와 IL-6 생성량은 KIT 100, 200( / ) 에서유의성있게감소하였다. <in vivo> 1. 뒷다리체중부하는유의성있게증가하였다. 2. 간기능및신장기능에영향을미치지않았다. 3. TNF-α, IL-6 및 IL-1β는감소하였으며, 그중 IL-1β가유의성있게감소하였다. 4. PGE2와 LTB4는유의성있게감소하였다. 5. MMP-9, TIMP-1 및 Osteocalcin은유의성있게감소하였다. 6. Micro CT arthrography에서연골의파괴가감소하였다. 7. 병리조직학적소견에서활막의손상정도가감소하였고 proteoglycan의생성은증가하였다. 이상과같이 (KIT) 은항산화및항염증효과가있으며, 골관절염병리과정에서혈액내의염증 cytokine과염증매개인자발현및연골변성 81

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