<BBE7BBF3C3BCC1FAC0C7C7D0C8B8C1F6284A53434D D3228C5EBB1C7292E687770>

Transcription

1 Original Article J Sasang Constitut Med 201;26(2): MPTP(1-methyl--phenyl-1,2,3,6-tetrahydropyridine) 로유도된 Parkinson's Disease 동물모델을이용한향사양위탕의신경세포보호효과 고가연 김윤희 안택원 대전대학교한의과대학사상체질의학 Abstract Neuroprotective Effects of Hyangsayangwi-tang in MPTP-induced Mouse Model of Parkinson's Disease Ga-Yeon Go, Yun-Hee Kim, Taek-Won Ahn Dept. of Sasang Constitutional Medicine, College of Oriental Medicine, Daejeon university Objectives To evaluate the neuroprotective effects of Hyangsayangwi-tang (HY), a Korean traditional medicinal prescription in a Parkinson's disease mouse model. Methods Four groups(each of 10 mouse per group) were used in this study. The neuroprotective effect of HY was examined in a Parkinson's disease mouse model. C57BL/6 mouse treated with 1-methyl--phenyl-1,2,3,6-tetrahydropyridine (MPTP, 30mg/kg/day), intraperitoneal (i.p.) for 5 days. Slow behavioral responses and memory disorder is the major clinical symptoms of PD. In order to investigate the effect of HY on recovery of behavioral deficits and memory, we examined the motor function and memory by using Morris water maze and Forced swimming test. Ischemic mouse brain stained with TTC(2,3,5 triphenyl tetrazolium chloride) in the MPTP-induced Parkinson's disease to find out ischemia and tissue damage in mouse. The convenient, simple, and accurate high-performance liquid chromatography (HPLC) method was established for simultaneous determination of neurotransmitters in MPTP-HY group. To measure the amount of dopamine in mice brain, striatum-substantia nigra, was examined by Bradford assay. Immunohistochemistry was examined in the MPTP-induced Parkinson's disease (PD) mouse to evaluate the neuroprotective effects of Hyangsayangwi-tang on hippocampal lesion, ST and SNpc. Results and Conclusions Hyangsayangwi-tang (HY) prevents MPTP-induced loss of serotonin, hippocampus and TH-ir cell. Key Words : Hyangsayangwi-tang, Parkinson s disease, Serotonin, Hippocampus, TH-ir cell Received June 12, 201 Revised June 12, 201 Accepted June 29, 201 Corresponding Author Taek-Won Ahn Dept. of Sasang Constitutional Medicine, Cheonan Oriental Hospital of Daejeon University Doojeong-dong 621, Seobuk-gu, Cheonan city, Chungcheongnamdo, , Korea Tel: Fax: twahn@dju.kr C The Society of Sasang Constitutional Medicine. All rights reserved. This is an open acess article distributed under the terms of the Creative Commons attribution Non-commercial License ( creativecommons.org/licenses/by-nc/3.0/)

2 166 Neuroprotective Effects of Hyangsayangwi-tang in Parkinson's Disease Ⅰ. 緖論 파킨슨병 (Parkinson's disease, PD) 은대뇌기저핵의선조체 (striatum, ST)- 흑질치밀부 (substantia nigra pars compacta, SNpc) 의 dopamine 신경계 (dopamine neuron) 의퇴행으로인한신경질환이다. 주요병인에는도파민뉴런의사멸, 유전자기형 (alpha-synuclein) 의축적으로인한 lewy body 생성으로나타나고이런원인의발병요소로중금속 / 탄화수소노출등의환경적인자, 중추노화관련인자, 산화스트레스 (oxidative stress), 유전인자 (α-synuclein, parkins, PINK1, DJ-1 등 ) 등으로보고되고있다 1-. 각종산화스트레스는활성산소 (reactive oxygen species, ROS) 를생성하고생성된 ROS 에의하여신경퇴행질환인 PD, 알츠하이머병 (Alzheimer's disease) 등의염증및진행과정과관련이있는것으로보고되고있다 5. 지금까지 PD의약물요법은일차적으로 dopamine 효능제또는 dopamine의전구체인 L-DOPA 요법이주로선택되어시행되고있다. 그러나 PD 동물모델에서장기간의 L-DOPA 요법은 ROS 형성에의하여신경독성이발생하고있으며, 약물효능의감소, 이상운동증 (dyskinesia), 운동동요 (motor fluctuation) 및다른합병증을유발한다 6-8. 그러므로기존에쓰이는 L-DOPA 요법외에다른약물치료제및한약처방의개발에대한필요성이제시되고있다. 역학조사에의하면 PD는환경적인요인과밀접한관계가있는것으로알려져있으며생활환경호르몬의하나인 MPTP (1-methyl--phenyl-1,2,3,6-tetrahydropyridine) 는향정신성의약품인 meperidine 유도체로서이에노출될경우평형감감상실, 손떨림, 경직이나자세의불안정등파킨슨병과유사한증상이나타나며파킨슨병모델이필요할때많이사용되고있 다 본연구에사용한처방인少陰人香砂養胃湯은 東醫壽世保元ㆍ辛丑本 12 新定少陰人病應用要藥二十四方의하나로少陰人胃受寒裏寒病의太陰證에사용하는처방이다. 少陰人脾胃病에脾胃의기운을돕고補脾和脾하고健脾直脾하는 효과를기대할수있으며소화기계문제, 특히식욕부진, 흉통, 위염, 위하수, 위무력등을치료하기위해널리응용된다 지금까지파킨슨에관련된한약의효능을알아보는연구에는정등 16 의파킨슨병으로유발된진전이한약치료를통하여호전된치험 1례, 배 17 의파킨슨병병태모델에서自家貪食作用活性化를통한熱多寒少湯加減方의뇌신경보호효과등이있다. 이연구를통해사상체질적접근을통한파킨슨질환환자의운동능력향상에대한효능과 MPTP 가유발한독성으로부터자가탐식을유도하고세포보호및증식의효과를가진열다한소탕의효능을확인할수있었다. 하지만아직태음인열다한소탕처방외에 MPTP 유발파킨슨질환의소음인처방에대한연구는미미한실정이다. PD 환자들은무력이나진전이나타나는부위에체온저하의특징이나타난다. 소음인처방중향사양위탕은胃受寒裏寒病의하복부냉증에다용하는처방으로이에본연구에서는소음인처방인 Hyangsayangwi-tang (HY) 을이용하여이처방의뇌세포및신경세포보호효과를확인하여파킨슨병의치료가능성을알아보기위하여실험을수행하였다. Ⅱ. 硏究材料및方法 1. 材料 1) 약재본실험에사용한약재는대전대학교천안한방병원에서구입한후정선하여사용하였다. HY 는 東醫壽世保元ㆍ辛丑本 12 에근거하였으며, 그구성비는아래와같은방법으로조제되었다 (Table 1). 2) 동물숫컷 6 주령의 g C57bl/6 mouse( 대한바이오링크, 대한민국 ) 를공급받아실험당일까지고형사료 ( 항

3 GY Go et al. 167 Table 1. Prescription of HY * Herbs Scientific name Amount(g) 人蔘白朮白芍藥炙甘草半夏香附子陳皮乾薑山査肉砂仁白荳蔲生薑大棗 Ginseng Radix Atractylodes Macrocephalae Rbizoma Paeonia Radix Alba Glycyrrhiza Radix Pinellia Rbizoma Cyperi Rbizoma Citri Pericarpium Zingiberis Rbizoma Crataegii Fructus Amomi Fructus Alpiniae Katsumadaiv Semen Zingiberis Rbizoma Recenes Jujubae Fructus Total amount 52 * HY:Hyangsayangwi-tang 생제무첨가, 삼양사료 Co.) 와물을충분히공급하고온도 22±2, 습도 55±15%, 12 시간 (light-dark cycle) 의환경에서 1주간적응시킨후실험에사용하였고, 동물실험의윤리적, 과학적타당성검토및효율적인관리를위하여대전대학교동물실험윤리위원회 (IACUC : Institutional Animal Care and Use Committee) 의승인을받았다. 3) 기기機器는열탕추출기 ( 대웅, DWT-1800T, Korea), 감압증류장치 (Rotary vaccum evaporator, BUCHI B-80, Switzerland), 동결건조기 (freeze dryer, EYELA FDU- 50, Japan), CO 2 배양기 (Forma scientific Co., U.S.A.), 원심분리기 ( 한일과학, Korea), plate shaker(lab-line, USA.), spectrophotometer (Shimazue, Japan), Bio-freezer (Sanyo, Japan), Quantitative Real-Time PCR (Applied Biosystems, U.S.A.), ELISA leader (Molecular Devices, U.S.A.), HPLC (Waters Co. Milford, MA, USA) 등을사용하였다. 2. 方法 1) 한약처방약제조본실험에사용한한약처방은 HY로처방약 2첩 분량 (22 g) 에증류수 2,000 ml를가하여열탕추출기에서 3 시간추출하여얻은액을흡입여과하여이를감압증류장치 (rotary vaccum evaporator) 로농축하여, 이를다시동결건조기 (freeze dryer) 를이용하여완전건조한한약처방약추출물을냉동보관 (-8 ) 하면서적당한농도로희석하여사용하였다. Mouse 경구투여를위한 HY의용량은 60kg 성인이하루 3회탕약복용하는것을기준으로삼았다. 성인의 1회복용탕약용량은 120cc 이며 1첩탕약안의약재는 11g 이사용된다. 이를 60kg 성인이하루 3회복용하였을시흡수되는약재의용량을고려하여 Mouse 경구투여량을결정하였으며그결과하루생쥐투여량은 550mg/kg 이다. 2) MPTP에의한파킨슨병 mouse 모델약 25.3g의숫컷 C57bl/6 mouse 에 MPTP (1-methyl --phenyl-1,2,3,6-tetrahydro-pyridine, 30mg/kg, in saline) 1일 1회 5일간복강에투여하여파킨슨병유도 mouse 를제작한후 2일간방치하여격리시켜흡수분해되지않고배출된적출물을제거한후사육실로이동시켜, 1일간 HY와마도파를경구투여하였다. 3) 실험군구성및실험설계실험군은 1주일간적응사육시킨후무게별로균등

4 168 Neuroprotective Effects of Hyangsayangwi-tang in Parkinson's Disease 하게 10마리씩 group으로나누고, 각 group 별로다음과같이처치하였다. Control group : mouse received saline only. MPTP group : mouse received intraperitoneal(i.p.) injections of MPTP-HCL (30mg/kg of free base; Sigma, Saint Louis, Missouri, USA) in saline at 2h intervals for 5 days. 마도파 + MPTP group : MPTP 복강주입한 Mouse 에 1일간마도파 ( 염산벤세라짓 ) 2mg/kg을경구투여하였다. HY + MPTP group : MPTP 복강주입한 Mouse 에 1일간 HY 550mg/kg을경구투여하였다. ) Morris water maze 연구방법 Morris water maze 란직경이 90cm 이고높이가약 30cm 인수조로수온이 28 인수돗물을 2/3정도채우고, 그안에 mouse 가올라갈수있는직경이 10cm 인원통형 platform 을설치한것이다. 원형수조의중심을지나면서직교하는두개의가상선을그어수조를 개의사분면으로나누었다. 그중하나의사분면중앙에아크릴로만든도피대, 즉 platform 을설치하였다. 수조에 0cm 깊이로물을담고도피대가보이지않게하기위해서 1kg 의탈지분유를풀었다. 수조의위천장에는비디오카메라를설치하여학습이이루어지는동안동물의움직임을촬영하였다. 학습은 1일 1회 30초이내에 pool에서 platform 으로올라가는연습을반복하여실시하였다. 선별된 mouse 10 마리를한군으로하여정상군 (C57bl/6-nr), 대조군 (MPTP control), 양성대조군 (MPTP-MD 2mg/kg) 과 HY(MPTP-HY 550 mg/kg) 으로분류하였고, 1일간 1일 1회약물투여와 platform 에오르는반복훈련을실시하였다. 5) Forced swimming test 방법강제수영검사는다른말로절망행동검사라고불리며약물개발시의항우울효과를검색하는기본적인실험으로알려져있다. 이실험은총 2번에걸쳐 진행된다. 윗면이개방된원통모양의아크릴실린더 ( 지름 20cm, 높이 0cm) 에깨끗한수돗물 ( 수온 25± 2 ) 을실린더바닥에서부터 15cm 까지붓고우울증생쥐모델을 15분동안강제로수영시킨다. 그후물에서건져올려마른수건으로닦고사육상자로돌려보낸다 (pretest session). 2시간후에동물을동일한장비에 5분동안빠뜨려쥐가하는행동을측정한다. (test session). 처음실험시수분간은이를벗어나기위해심한저항을보이나시간이흐를수록점점부동자세를보이는시간이늘어난다. 두번째날검사시에는 5분간같은환경에서강제수영을시키고기어오르기. 수영, 부동행동등의자세를측정한다. 전형적인부동행동이란동물이얼굴을포함한상체의일부분만수면뒤로드러낸채몸의균형을유지하기위하여약간의움직임만을나타낼뿐물위에단순떠있는상태이다. 사람에의한오차를줄이기위해캠코더 (Figure 3) 등의기록장치를이용하여기록하고, 기록된영상을통하여 test session (5분 ) 동안동물의행동이나타나는시간을측정하였다. 이번검사에서는동물이발버둥치지않고물위에가만히떠있는행동상태를부동성의증가로보고절망행동의지표로삼았다. (Immobility behavior). (Figure ) 그외실린더주위를수평방향으로움직이는행동으로 Immobility behavior 보다도더적극적으로움직이는행동인수영과 (Swimming behavior) 과벽을향해앞발을물밖으로차올리는다소격렬하게움직이는행동인기어오르기는 (Climbing behavior) 는측정하지않았다. 6) 허혈의크기측정 Mouse 를마취한후후두부에서전두부방향으로두개를열어그안에있는뇌를꺼낸다. 꺼낸뇌를생리식염수에씻은후 brain matrix 를이용하여 2mm의두께로자르고 2% TTC 용액을가하여 20분간염색한다. TTC 용액에의하여정상조직은적색으로염색이되고, 허혈된부분, 즉혈액공급이되지않아뇌세포가파괴된곳은콜라겐으로채워지므로하얗게염색된다. 허혈크기의측정은 Brian 의방법으로수행하였

5 GY Go et al. 169 다. 허혈의크기 (AT) 는 [(A1 / ST1) + (A2 / ST2) + (A3 / ST3) + (A / ST)] 이고, A는사진상 (Figure 5) 에나타난허혈면적, ST는각각 section(2 mm ) 의전체면적. BH(brain Hipocampus) 는뇌의 hipocampus부분의면적이며, risk 에대한허혈크기는전체면적의 percent로표현하였다. 즉 BH 부위의허혈면적 (LV) 은 (AT of area at risk/st of BH) x 100 로분석하였다 ) HPLC를이용한표준성분분석생쥐의뇌 ( 총마리 ) 를적출하여호모겐나이즈 (homogenizer) 로균질화한후 5ml 에탄올 (Ethanol, Merck) 을가하여 6시간이상 shaking water bath 에서배양한다. 그후 3M filter로모두거른다음 0.2ml 로가압농축한다. 농축된메탄올추출액을 HPLC 분석을통해도파민 (Dopamine), 세로토닌 (Serotonine), 멜라토닌 (Melatonin), 에피네프린 (epinephrine), 노르에피네프린 (norepinephrine) 의함량을분석하였다. 뇌에탄올추출물분석을위한 HPLC (Waters Co. USA) 는 Agilent 1200 series 로서 autosampler, column oven, binary pump, DAD detector (Agilent Technologies, Waldbronn, Germany), degasser (Agilent Technologies, Tokyo, Japan) 를사용하였으며, software는 Agilent 사의 Chemstation software (Agilent Technologies, Avondale, CA, USA) 를사용하였다. HPLC 조건으로컬럼오븐의온도는 2 5, 분석파장은 220nm, 유속은 1.0ml/min 이였고, 컬럼은 Luna C18 column (250mm ⅹ.6mm; particle size 5μm, Phenomenex, Torrance, CA, USA) 를사용하였다. 표준성분시료는 Sigma USA 사의 Serotonin 과 Melatonin 을이용하였다. 이동상은 A 용매로 100% 3 차증류수 (0.1% TFA 함유 ), B 용매로 100% 아세토니트릴에각각 0.1% 포름산을첨가하여사용하였고용매계는용매기울기용리로서아래표 (Table 2) 에나타내었다. 8) 도파민양측정방법 Mouse 의뇌 ( 총2마리 ) 를적출하여 -20 에서얼린후뇌시료 ( 선조체 -흑질부위 ) 부분을분리하였다. 뇌시료 ( 선조체 -흑질부위, 0.25g) 부분에 200~500μl 의 extraction buffer (0.3M sucrose, 0.15mM spermine, 0.5mM spermidine, 10 mm HEPES (ph 7.9), 1.5 mm MgCl2, 10mM KCl, 0.5mM DTT, 0.2mM PMSF, 0.1% protease inhibitor, 0.1% phosphatase inhibitor, 0.5% NP 0) 를넣어호모겐나이즈 (homogenizer) 로균질화한후최종부피가 0.2ml 이되도록 extraction buffer 를가해잘혼합한뒤 2시간동안 에서용해시켰다. 여기에반응이끝나면시료를 1.5ml tube에담아 3분씩 5번동안 vortex하고, 15,000rpm 에서 15분간원심분리한후상등액 (100 μl ) 을취하였다. 단백질양은 Bio-Rad 단백질분석키트에기술된 Bradford 방법 (Bio-Rad Laboratories, Hercules, CA, USA) 에따라측정하였다. Mouse/Rat Dopamine ELISA Assay Kit (DOU39-K01, Eagle Biosciences, Inc) 를사용하여제조사의지시에따라각 well 을 wash buffer 로세척하고 Assay diluent 를 100μl씩넣어서 1시간동안 well 을막은후실온에서배양하였다. 표준품을희석하고상층액을 20배희석한후 microplate 를세척하고각표준품과상층액을 Table 2. Solvent Gradient Condition for HPLC-DAD Final time (min) Flow rate (ml/min) A a B b A a Water with 0.1% formic acid. B b Acetonitrile with 0.1% formic acid.

6 170 Neuroprotective Effects of Hyangsayangwi-tang in Parkinson's Disease 100 μl씩넣었다. 그후 2시간동안 well 을막은후실온에서배양하였다. microplate를세척하고 warking detector를만들어서각 well 에 100μl씩넣고 1시간동안 well 을막은후실온에서배양하였다. microplate 를세척하고 substrate solution 을만들어서각 well 에 200μl씩넣고 30분동안어두운곳에서실온으로배양하였다. stop solution 을각 well 에 50μl씩넣고 Microplate spectrophotometer에서흡광도 50nm로측정하였다. 9) 뇌조직검사생쥐의뇌를 10% formaldehyde 용액에고정한후세절하여흐르는물에 8시간수세한다음파라필린물질을삼투시켜고화시킨다. 이것을 microtome으로절편을만들어 Hematoxylin & Eosin 염색을실시하고광학현미경상에서관찰하였다. 저하됨을알수있다. MPTP 와 HY을병용주입한 MPTP-HY group 은 MPTP group 에비해 platform 까지찾아가는데걸린시간과거리모두줄어들었지만통계학적으로유의성있는단축을보이진못하였다 (Figure 1,2). 2. 행동관찰실험 (Forced swimming test) PD의또다른증상으로운동능력저하를들수있다. 이에대한변화를알아보기위하여강제수영검사 (Forced swimming test) 를실험하였다. 그결과 MPTP group은무동의시간이 212초, MPTP-HY group 은무동시간이 189.7로 HY를처리한군에서약 22초가량 3. 통계 모든실험결과는평균 ± 표준편차 (mean±s.d.) 로나타내었고, 비모수검정방법인 KW방법 (Kruskal-Wallis test) 과 MW(Mann-Whitney U test) 방법을사용하였다. p values 가 5% 미만일때를 유의적인차이가있음 으로판정하였다. Ⅲ. 硏究結果 1. 기억력측정실험 PD의대표증상인기억력저하, 인지장애를연구하기위하여수중미로테스트 (Morris water maze) 를이용하였다. 연구에사용된 mouse 들을마도파와 HY 경구투여하면서 Morris water maze에서 1주 1회반복학습훈련을실시하여선별된 mouse 의군을나누어실험을진행하였다. 그결과 MPTP group 은정상군에비해 platform 까지찾아가는데걸리는시간은 6배, 찾아가면서헤맨거리는 8배에달해기억력이현저히 Figure 1. Effects of Hyangsayangwi-tang on the impairment of learning and memory of the stop-through type Morris water maze test in the MPTP -induced Parkinson's disease (PD) mouse. C57BL/6 mice were orally administered of Hyangsayangwi -tang or MD and the acquisition taining trial continued once a day for 21 days. Lardist this is the total distance (in cm) was initiated at 3 weeks after orally administered of HY extract (Hyangsayangwi-tang, 550mg/kg,p.o) the retention trial was carried out after day 7 after the acquisition training trial and injected MPTP induced PD mice. Control, MPTPinduced PD mice; MD ( 마도파, 2mg/kg,p.o); HY extract (Hyangsayangwi-tang, 550mg/kg,p.o). Lardist this is the total distance (in cm) covered By the animal in large movement, and smldist this is total distance covered by the animal in small movement. Data (Lardist plus Smldist) represent means±s.e (N=10). Data represent means±s.e (N=10). Statistically significant value compared with MPTPinduced PD mice group (control) data by T test ( *** p<0.001).

7 GY Go et al. 171 Figure 2. Effects of Hyangsayangwi-tang on the spatial working memory deficits of the distance movement-through type Morris water maze test in the MPTP-induced Parkinson's disease (PD) mouse. C57BL/6 mice were orally administered of Hyangsayangwi-tang or MD and the acquisition taining trial continued once a day for 21 days. Lardist this is the total distance (in cm) was initiated at 3 weeks after orally administered of HY extract (550mg/kg,p.o) the retention trial was carried out after day 7 after the acquisition training trial and injected MPTP induced PD mice. Control, MPTP-induced PD mice; MD ( 마도파, 2mg/kg, p.o); HY extract (550mg/kg,p.o). o). Lardist this is the total distance (in cm) covered By the animal in large movement, and smldist this is total distance covered by the animal in small movement. Data (Lardist plus Smldist) represent means±.e (N=10). Data represent means.±e (N=10). Statistically significant value compared with MPTP-induced PD mice group (control) data by T test ( ** p<0.01) 무동의시간이단축된것을확인할수있다. 그러나 Figure 에서확인할수있듯이시간단축외에통계학적인의미를가지지는못하였다. 3. MPTP 처리 Mouse 에서의허혈및조직손상 Mouse 의뇌를 TTC 용액처리하여허혈된면적을살펴보았다. 그결과 MPTP group 에서는뇌혈관의 50% 에해당되는부분이허혈상태로변할것을확인할수있다. 그에반해마도파는뇌혈관의 20%, MPTP-HY group 은뇌혈관의약 25% 정도가허혈상태로변한것을확인할수있었다. MPTP-HY group 은 MPTP group 에비해콜라겐으로채워진허혈부분이 Figure 3. Forced swimming test The forced swim test is a rodent behavioral test used for evaluation of antidepressant drugs, antidepressant efficacy of new compounds, and experimental manipulations that are aimed at rendering or preventing depressive-like states. Mouse are placed in an inescapable transparent tank that is filled with water and their escape related mobility behavior is measured. Figure. Effects of Hyangsayangwi-tang on the immobility time during the forced swim test in the MPTP -induced Parkinson's disease (PD) mouse. C57BL/6 mice were orally administered of Hyangsayangwi-tang or MD and the acquisition taining trial continued once a day for 21 days. ). The FST behaviour was videotaped for later analysis and the periods of immobility were scored from videotapes. Control, MPTP-induced PD mouse; MD ( 마도파, 2mg/kg,p.o); Hyangsayangwi-tang extract (550mg/kg,p.o). Data represent means±.e (N=10). Statistically significant value compared with MPTPinduced PD mice group (control) data by T test ( ** p<0.01.). 줄어들었으나통계학적으로유의성을가지지는못하

8 172 Neuroprotective Effects of Hyangsayangwi-tang in Parkinson's Disease C57bl/6-Nr MPTP_MD 2mg/kg MPTP_control MPTP_HY 550mg/kg Figure 5. Infarction and brain edema coronal sections from ischemic rat brain stained with TTC in the MPTP -induced Parkinson's disease (PD) mouse. C57BL/6 mice were orally administered of HY or MD and the acquisition taining trial continued once a day for 21 days. Brain cerebral infarction is visualized by 2,3,5-triphenyl-2H tetrazolium chloride. Control, MPTP-induced PD mouse; MD( 마도파, 2mg/kg,p.o); HY extract (550mg/kg,p.o). Data represent means±.e (N=10). Statistically significant value compared with MPTP-induced PD mice group (control) data by T test 였다.. HPLC를이용한표준성분및신경전달물질분석 Figure 6. Effects of Hyangsayangwi-tang on the coronal section in the MPTP-induced Parkinson's disease (PD) mouse. C57BL/6 mice were orally administered of HY or MD and the acquisition taining trial continued once a day for 21 days). Brain cerebral infarction is visualized by 2,3,5-triphenyl-2H tetrazolium chloride, and % LV using various BH(brain Hipocampus) imaging measurements. Control, MPTP-induced PD mice; MD ( 마도파 2mg/kg,p.o); HY extract (550 mg/kg,p.o. Data represent means±.e (N=10). Statistically significant value compared with MPTPinduced PD mice group (control) data by T test (*p<0.001). HPLC 를이용한표준분석및신경전달물질분석결과도파민, 세로토닌, 멜라토닌, 에피네프린, 노르에피네프린중에서세로토닌만 HPLC 분석에서확인되는것을볼수있었다. 세로토닌은저하시우울증, 불안장애, 공격성, 자살등과관련이있으며 PD 환자들에게서세로토닌작동성신경의장애와퇴행이나타난다. PD 환자의우울증상과관련하여각종신경전달물질의변화를알아보기위하여정상쥐의표준물질동정외우울증생쥐의뇌성분에함유된 serotonin, melatonin 의패턴을확인하여표준물질을동정하였다. (Figure 7). 이자료를토대로정상군과 MPTP group, 마도파, MPTP-HY group 의뇌의세로토닌함량을비교해보았을때정상군에비하여 MPTP group 의세로

9 GY Go et al. 173 토닌생성량이감소를나타내었다. 그리고양성대조군인마도파 (p<0.001) 와 MPTP-HY group (p<0.01) 을 투여한실험군은대조군에비하여세로토닌생성량이통계학적으로유의성있게증가를나타내었다 (Figure 8). Standard patern C57bl/6-Nr Depression model Figure 7. Using HPLC analysis of standard pattern and the neurotransmitter Figure 8. Effects of Hyangsayangwi-tang on the levels of serotonin in the brain tissue in the MPTPinduced Parkinson's disease (PD) mouse. C57BL/6 mice were orally administered of HY or MD and the acquisition taining trial continued once a day for 21 days. The serotonin level was initiated at 3 weeks after orally administered of HY extract (550 mg/kg,p.o) and injected MPTP induced PD mice. Data represent HY extract (550mg/kg,p.o). The brains were removed and the levels of serotonin were determined by an HPLC method. Statistically significant value compared with MPTP-induced PD mouse group (control) data by T test (*p<0.001). Figure 9. Effects of Hyangsayangwi-tang on the levels of dopamine in the brain tissue in the MPTP -induced Parkinson's disease (PD) mouse. C57BL/6 mice were orally administered of HY or MD and the acquisition taining trial continued once a day for 21 days. The dopamine level was initiated at 3 weeks after orally administered of HY extract (550mg/kg,p.o) and injected MPTP induced PD mice. Data represent means±e (N=10). Control, MPTP-induced PD mouse; MD ( 마도파 2mg/kg, p.o); HY extract (550mg/kg,p.o). The brains were removed and the levels of dopamine were determined by an ELISA method. Statistically significant value compared with MPTP-induced PD mice group (control) data by T test (*p<0.001).

10 17 Neuroprotective Effects of Hyangsayangwi-tang in Parkinson's Disease 5. Mouse 뇌 ( 선조체 -흑질) 혼탁액에서도파민양측정 Mouse 2마리의뇌선조체- 흑질부위를이용하여직접적으로도파민의양을측정하였다. 그결과 MPTP group 의도파민양에비해 MPTP-HY group 의도파민양이약 2배이상증가한것을볼수있다. 양성대조군인마도파처리군은 배넘게증가한것을확인할수있다 (Figure 9). 그러나마도파와는달리 MPTP-HY group은통계적으로유의성을가지지는못하였다. 6. 뇌조직손상분석이번연구에서는해마의조직검사를위해총 마리의 Mouse 가실험에이용되었으며정상군보다 MPTP group 에서눈에띄게해마부분이줄어든것을확인할수있었다. 양성대조군인 MPTP-MD group 과 MPTP-HY group 은 MPTP group 에비하여사라졌던해마뉴런등이많이회복된것을볼수있었다 (Figure 10). 또 Mouse 의선조체 (ST) 와흑질 (SN) 에서 TH 면역반응세포의분포와범위를면역염색을통하여알아 보았다 (Figure 11,12). 그림을참고해보면 MPTP group 에비해 MPTP-HY group 에서 TH 면역반응세포의갯수와밀집도가더높은것을확인할수있다. Ⅳ. 考察및結論 파킨슨병은뇌의흑색질의파괴로인하여신경전달물질인도파민 (dopamin) 이부족해져생기는질환으로대표적인퇴행성뇌질환이다 20. 파킨슨병의원인으로다양한기전이알려져있지만최근에와서는환경호르몬에의한질병유발이주된원인으로알려지고있다. 파킨슨병을유발하는것1 로알려진화학물질로는유기농약제인 paraquat, rotenone, amphetamine, MPTP 등이있다 MPTP 는생체내로유입되면뇌에서몇단계를거친후 MPP + 로변환되어중뇌의흑질부위를비가역적으로파괴한다. 현재까지 MPTP 의신경독성을완화시킨다고알려진것은 Vitamine E, allopurinol, selenium, centrophenoxine 등이있으며, 주로활성산소를제거하여도파민성신경세포의파괴를 C57bl/6-Nr MPTP_control MPTP_MD 2mg/kg MPTP_HY 550mg/kg Figure 10. Effects of Hyangsayangwi-tang on the histological analysis of hippocampal lesion in the MPTP-induced Parkinson's disease (PD) mouse. C57BL/6 mice were orally administered of HY or MD and the acquisition taining trial continued once a day for 21 days. The hippocampal lesion level was initiated at 3 weeks after orally administered of HY extract (550mg/kg,p.o) and injected MPTP induced PD mice. Data represent means±e (N=10). Control, MPTP-induced PD mice; MD( 마도파 2mg/kg,p.o); HY extract (550mg/kg,p.o). They were sacrificed, brain was fixed in 10% buffered formalin and embedded in paraffin. Paraffin sections were stained with hematoxylin/eosin. The panels are representative photomicrographs of each of these risk at bright microscope (Nikon, 0).

11 GY Go et al. 175 완화하거나, 생체내에서의 MAO-B inhibitor 로작용하여 MPTP 대사를저해시키는기전으로파킨슨병의치료에활용되고있다 23,2. 급증하는노인으로인해 퇴행성뇌질환에대한연구의필요성이대두되고있는가운데퇴행성뇌질환의병태생리적원인과밀접한관련이있는산화스트레스의치료에관심이쏠리 C57bl/6-Nr MPTP_control MPTP_MD 2mg/kg MPTP_HY 550mg/kg Figure 11. Effects of Hyangsayangwi-tang on the immunohistochemistry of TH-immunoresponsive (TH-IR) neurons in the striatum (ST) in the MPTP-induced Parkinson's disease (PD) mice C57BL/6 mice were orally administered of HY or MD and the acquisition taining trial continued once a day for 21 days. The hippocampal lesion level was initiated at 3 weeks after orally administered of HY extract (550mg/kg,p.o) and injected MPTP induced PD mice. Data represent means±.e (N=10). Control, MPTP-induced PD mice; MD( 마도파 2mg/kg,p.o); HY extract (550mg/kg,p.oE). TH-IR cells in the ST of mouse were determined by an immunostaining assay with rabbit anti-th antibody. Representative photomicrographs are shown of the ST from each group. The panels are representative photomicrographs of each of these risk at bright microscope (Nikon, 0). C57bl/6-Nr MPTP_control MPTP_MD 2mg/kg MPTP_HY 550mg/kg Figure 12. TH-IR cells in the SNpc of mice were determined by an immunostaining assay with rabbit anti-th antibody in a subacute MPTP mouse model of Parkinson's disease. C57BL/6 mice were orally administered of HY or MD and the acquisition taining trial continued once a day for 21 days. The hippocampal lesion level was initiated at 3 weeks after orally administered of HY extract (550mg/kg,p.o) and injected MPTP induced PD mice. Data represent means±.e (N=10). Control, MPTP-induced PD mouse; MD( 마도파 2mg/kg,p.o); HY extract (550mg/kg,p.oE). TH-IR cells in the SNpc of mice were determined by an immunostaining assay with rabbit anti-th antibody. Representative photomicrographs are shown of the SNpc from each group. The panels are representative photomicrographs of each of these risk at bright microscope (Nikon, 0).

12 176 Neuroprotective Effects of Hyangsayangwi-tang in Parkinson's Disease 고있다. 이에본저자는 MPTP 을이용하여파킨슨병을유발한동물모델을이용하여향사양위탕 (HY) 의파킨슨치료제로서의가능성을실험을통해규명하고자하였다. 지금까지파킨슨병의한약처방에관한연구로는증례논문이거나배 17 의파킨슨병병태모델에서自家貪食作用活性化를통한熱多寒少湯加減方의뇌신경보호효과에관한연구가있다. 배 17 의연구에서언급된自家貪食은에너지결핍시 ATP 합성을위해오래된단백질을분해또는제거하는일종의세포내 house-keeping 작용을하는것으로세포사멸이라는의미를넘어서세포가끊임없이죽고재생되는과정을조절하는세포항상성에관여하는중요기작으로주목받고있다. 배 17 의연구는열다한소탕가감방을복용하였을시어떤기전으로자가탐식작용이일어나는지밝혀낸연구이다. 그에반해열다한소탕외의다른사상체질방을이용한 PD 연구는거의전무한상태이다. 따라서이번연구를통해 PD 환자치료에소음인처방이효과를나타낼수있을지알아보고자하였다. 또한본연구에서는뇌조직분석, 도파민양측정, 표준성분분석과허혈상태인뇌의면적측정과같은실험외에 Mouse 의행동상태와우울정도를측정하는행동실험도같이진행하였다. 그동안 Morris water maze 와 Forced swimming test 를이용한 Mouse 의실험은다양하게있었으나파킨슨행동양상을측정하는데이용한연구는없었다. 따라서이번연구를통해지금까지언급된적없는소음인 HY 처방의 PD 치료효과에대한근거를마련하고자하였다. 본연구에서는먼저파킨슨유발 Mouse (MPTP group) 군과양성대조군인 MPTP MD group, MPTP 후 HY 경구투여한군 (MPTP HY group) 으로나눴으며각 group마다실험대상쥐는 10마리로책정하였다. PD 발생시가장보편적으로사용하는양약은마도파정125mg 으로 Benserazide hydrochloride 28.5mg (benserazide 로서 25mg), Levodopa 100mg 을성분으로한다. 이약은말그대로도파민이부족한 PD 환자들에게직접적으로도파민의작용을할수있는성분을 흡수시켜주는약이다. 먼저 PD 질환의주요증상중하나인인지장애, 기억력저하에대한 HY의효과를알아보기위해수중미로테스트 (Morris water maze) 를시행하였다. 수중미로테스트는일정기간동안 Mouse 를 Morris water maze내 platform 에찾아갈수있도록훈련을시킨다음, 얼마간의시간과거리가걸려서 platform 까지찾아가는지헤매는시간과거리를측정하는실험이다. 실험결과확연한시간, 거리단축을보인양성대조군에반해 MPTP-HY group 은시간, 거리모두다소감소되는경향을보였으나통계적인유의성은없었다. 파킨슨병에걸리게되면정신과적증상이흔히동반된다는것은오래전부터알려져있다. 특히우울증상이대표적인증상인데대부분의연구결과에따르면파킨슨병에서우울증상의동반빈도는약 0~70% 에달한다. 그동안발표된연구들에의하면, 파킨슨병의운동기능장애가발생하기전에우울증이흔히발견된다는연구들이있으며파킨슨병환자군과신체장애의정도가비슷한다른질환의환자군을비교해보면파킨슨병환자군에서우울증의빈도가더높다고보고되고있다 25,26,27. 그렇지만이렇게많은수의환자군이우울증상을겪고있으나겉으로드러난신체강직이나진전등의재활치료에밀려우울증상에대한관심이상대적으로적은것이사실이다. 이번실험에서는 PD로인한무운동성과우울경향에대해알아보기위해강제수영검사 (Forced swimming test) 를시행하였다. 강제수영검사는윗면이개방된원통모양의아크릴실린더에물을붓고 Mouse 를 15분간강제로수영을시킨뒤 2시간후똑같은아크릴실린더에 5분동안다시떨어드려놓는실험이다. 그럴때 Mouse 의행동을캠코더로촬영하며측정값은 Mouse 가움직이고자하는의지없이무동증상을보이고있는시간이다. 그결과 MPTP 투여로파킨슨병유발된군에비해 MPTP-HY group 의무동시간은약 30초가량줄어든것을볼수있다. 시간상으로는소폭감소하였으나통계적인유의성을가지지못해 HY의우울증상감소역할은미미한것으로나타났다.

13 GY Go et al. 177 Mouse 의뇌를후두부에서전두부방향으로두개를열어 2mm 두께로자른후 TTC 용액을가하게되면정상조직은적색, 허혈상태로인한뇌세포사멸조직은염색이되지않아하얗게표시가된다. 이염색법은조직학적으로뇌신경세포파괴를단기간에알수있는방법으로서허혈부분을육안으로쉽게비교할수있는장점이있다. 앞서첨부한그림을살펴보면 (Figure 5) MPTT group은뇌세포사멸부분이많아군데군데하얗게표시된곳이많고, MPTP-HY group 은상대적으로허혈로인한하얀부분의면적이좁았다. 그러나육안으로확인한것과는달리허혈면적의비중을이용하여통계처리하였을때는유의성을가지지못했다. 모든물질은고유의파장을가지고있으며이미연구를통해몇몇물질에대한고유파장을알고있다. 대표적인뇌신경전달물질인도파민, 세로토닌, 노르에피네프린, 에피네프린, 멜라토닌의표준품을이용하여실험에사용된 Mouse 의뇌에탄올추출물을 HPLC 를사용하여분석해보았다. 위에서언급한뇌신경전달물질 5가지종류모두추출될것으로기대하였으나 MPTP 처리한뇌시료에서세로토닌외에다른물질은분석이되지않았다. 세로토닌은신경세포의발달, 운동, 기분, 공격성, 식욕, 수면, 인지기능등고위대뇌기능에중요한역할을하는신경전달물질로서인체의생리기능에중요한역할을한다. HPLC 를이용하여검출된세로토닌양을 Nr group, MPTT group, MPTP-MD group, MPTP-HY group으로나누어살펴보았다. 그결과 Nr group 에비해 MPTP-HY group 의뇌무게당세로토닌양이유의성있게증가한것을확인할수있었다. 앞서설명하였듯이직접적으로도파민을채워넣어주는마도파와비교했을때도크게차이나지않는유의성을보여줬는데, 이는한약이양약못지않게 PD 치료에큰효과를낼수있음을보여주는예이다. 또다른실험은파킨슨병유발의중요한원인이되는도파민양을측정하는실험이다. 파킨슨병은여러가지원인에의해중뇌선조체- 흑질부위의도파 민이사멸하여발생하는질환이다. 따라서 Mouse 뇌의선조체- 흑질혼탁액에서도파민양을측정한다면직접적이면서도중요한뇌의변화를알수있는방법이될것이다. 이실험역시 MPTT group 에비해 MPTP- HY group 에서도파민양이늘어난것을볼수있었다. 그러나향사양위탕이파킨슨환자의도파민양을직접적으로늘리게한다는통계적유의성을가지지는못하였다. 마지막으로 Mouse 의뇌를조직검사하여면역기능의변화와기억력을담당하는해마의손상정도를살펴보았다. 파킨슨병에걸리게되면해마의위축이일어나게되고그로인해인지장애및기억력저하더나아가서치매로발전하는경우가많이있다. 따라서조직검사를통해 MPTP 로파킨슨병유발한 Mouse 의해마위축정도와함께향사양위탕투여군에서는어느정도해마의위축이회복되는지확인하였다. 또한 TH IR CELL 의증가, 감소까지함께알아보았다. Figure 10-12를보면해마의위축정도가 MPTT group 에비해 MPTP-HY group 에서많이회복된것을볼수있다. TH-IR CELL 역시 MPTT group 보다 MPTP-HY group 에서더많이분포하였다. 이는 HY가파킨슨병으로인해인지장애를겪고면역기능이저하된환자의증상개선에도움을줄수있다는것을뜻한다. 이상의연구결과는 HY가 MPTP 로유도된 Parkinson s disease 동물모델에대해일정부분신경보호효과가있음을나타내고있다. 뇌의기억력담당기관인해마와면역양성세포, 그리고몸의고급생리기능을담당하는신경전달물질인세로토닌의증가에도움을줄뿐만아니라기억력이증대하여행동이빨라지고우울증상의경감이나타나는것을확인하였다. 이를바탕으로 HY가파킨슨병을비롯한퇴행성뇌신경질환의치료와예방에유의한효과가있을것으로예상할수있다. 그러나실험을진행한 Mouse 의수가많지않았다는한계가있다. 따라서 in vitro 실험결과를바탕으로하는임상시험으로의연계가추가적으로이루어져야할것으로사료된다.

14 178 Neuroprotective Effects of Hyangsayangwi-tang in Parkinson's Disease Ⅴ. Acknowledgement 본연구는한국연구재단의이공분야기초연구사업의일환으로수행되었음 (NRF ) Ⅵ. References 1. Imaki T, Katsumata H, Miyata M, Naruse M, Imaki J, Minami S. Expression of corticotropin-releasing hormone type 1 receptor in paraventricular nucleus after acute stress. Neuroendocrinology. 2001;73(5): Bae NY, Yang HO, Ahn TW. Protection effect of New-Yeolda-Hanso tang againstβ-amyloid Induced Cytotoxicity in NGF-differentiated PC12 Cells. J Sasang Constitut Med. 2009;21: (Korean) 3. Bae NY, Ahn TK, Chung SK, Oh MS, Ko HS, Oh HG et al. The neuroprotective effect of modified Yeoldahanso-tang via autophagy enhancement in modelsof Parkinson s disease. Journal of Ethnopharmacology. 2011;13: (Korean). Dauer W, Przedborski S. Parkinson s disease: mechanisms and models. Neuron. 2003;39: Seo JH, Shin MS, Kim CJ, Lee KS, lim BB. The influence of treadmill exercise in hot environments about serotonin expression in the rat brain, generating neurons and short-term memory. Korean Journal of Sport Science. 2009;20();7. (Korean) 6. Smith GW, Aubry JM, Dellu F, Contarino A, Bilezikjian LM, Gold LH, et al. Corticotropin releasing factor receptor 1-deficient micedisplay decreased anxiety, impaired stress response, and aberrant neuroendocrine development. Neuron. 1998;20(6): Porsolt RD, Anton G, Blavet N et al. Behavioral despair in rats : A new model sensitive to antidepressant treatments. Eur J Pharmacol 1978;7: Porsolt RD. Animal model of depression. Biomedicine 1979;30: Langston, J.W., Irwin, I., Langston, E.B., Forno, L.S. Pargyline prevents MPTP-induced parkinsonism in primates. Science 225(669): , Arai N, Isaji M, Kojima M, Mizuta E, Kuno S. Combined effects of cabergoline and L-dopa on parkinsonism in MPTP-treated cynomolgus monkeys. J Neural Transmission 103(11): , Lau, Y.S., Meredith, G.E. From drugs of abuse to parkinsonism: The MPTP Mouse Model of Parkinson s Disease. Methods in Molecular Medicine 79(1): , Lee JM. Dongeuisoosebowonsasangbonchokwon. Seoul: Eulyumoonhwasa. 2002: (Korean) 13. The National College of Oriental Medicine Sasangeuihakclassroom. Sasangeuihak. Seoul: Jipmundang. 200: 260,265,276,39-5,2-80. (Korean) 1. Song IB et all. Sasangeuihak. Seoul:Jipmundang. 200:33,366. (Korean) 15. Lee JC. Sinsasangeuihakron Ⅱ. Seoul:Mokkwato. 2001: (Korean) 16. Jeong BJ, Kim JW, Kim BC, Woo SH, Na YJ, Shin HJ et al. A case of tremor in Parkinson's disease treated with Korean medicine. Korean J. Orient.int.Med. 2006:27() (Korean) 17. Bae NY. Neuroprotective effect of modified Yeoldahanso-tang via autophagy enhancement on Parkinsonian model. Dae-jeon National University. 2013;25(3): (Korean) 18. Brian R. Perri, Douglas H. Smith, Hisayuki Murai, Grant Sinson, Kathryn E. Saatman, Ramesh Raghupathl, Raymond T. Bartus, Tracy K. Mcintosh. Metabolic Quantification of Lesion Volume following Experimental Traumatic Brain Injury in the Rat. Journal of Neurotrauma. 1997;1: Vosselera M., N. Abegunewardenea, N. Hoffmannb,

15 GY Go et al. 179 S.E. Petersene, D. Beckera, D. Cleppienb, P. Kunzc, K.-F. Kreitnerc, M. Lauterbacha, B. Bierbachd, C. Düberc, T. Goria, T. Münzela, L.M. Schreiberb, G. Horsticka. Area at Risk and Viability after Myocardial Ischemia and Reperfusion Can Be Determined by Contrast-Enhanced Cardiac Magnetic Resonance Imaging. Eur Surg Res. 2009;3: Kasper, Braunwald, Fauci, Hauser, Longo, Jameson. Harrison`s Principles of Internal medicine. 16th edition. Seoul: MIP. 2006: Widdowson, P.S., Farnworth, M.J., Upton, R., Simpson, M.G. No changes in behavior, nigro-striatal system neurochemistry or neuronal cell death following toxic multiple oral paraquat administration to rat. Human and Experomental Toxicology. 1996; 15(7): Ferger, B., Teismann, P., Earl, C,D., Kuschinsky, K., Oertel, W.H. The propective effects of PBN against MPTP toxocity are independent of hydroxyl radical trapping. Phamaco Biochem Behav. 2000;65(3): Westm B.D., Sheghrue, P.J., Vanko, A.E.H,, Ransorn, R.W. Kinney, G.G. Amphetamine-induced locomotor activity is reduced in mice following MPTP treatment but not following selegiline/mptp treatment. Pharmacol. Biochm. Behav 2006;8(1): Sun, L., Xu, S., Zhou, M., Wang, C., Wu, Y., Chan, P. Effects of cysteamine on MPTP-induced dopaminergic neurodegeneration in mice. Brain Research. 2010;1335: Cummings JL. Depression in Parkinson s disease:a review. Am J Psychiatry 1992;19: Fetoni V, Soliveri P, Monza D, Testa D, Girotti F. Affective symptoms in multiple system atropy and Parkinson's disease:response to levodopa therapy. J Neurol Neurosurg Psychiatry. 1999;66: Burn DJ. Depression in Parkinson s disease. Eur J Neurol :-5.