J Dent Hyg Sci Vol. 15, No. 4, 2015, pp.424-429 http://dx.doi.org/10.17135/jdhs.2015.15.4.424 RESEARCH ARTICLE Triptolide 의말초와중추투여에의한흰쥐의안면부통증경감효과 김윤경 1 ㆍ최자형 1 ㆍ이현정 1 ㆍ손유진 2 ㆍ윤소영 2 ㆍ이정화 1,2 ㆍ이민경 1,2 1 동의대학교일반대학원보건의과학과, 2 동의대학교치위생학과 Analgesic Effects of Triptolide via Peripheral and Central Administration in Rat Model of Inflammatory Orofacial Pain Yun-Kyung Kim 1, Ja-Hyeong Choi 1, Hyun-Jung Lee 1, Yoo-Jin Son 2, So-Yeong Yoon 2, Jung-Hwa Lee 1,2, and Min-Kyung Lee 1,2 1 Department of Biomedical Health Science, Dong-Eui University Graduate School, Busan 47340, 2 Department of Dental Hygiene, Dong-Eui University, Busan 47340, Korea The aim of this study was to investigate whether peripheral or central administration of triptolide is involved in pain modulation in inflammatory orofacial pain. The inflammatory orofacial pain was induced by the injection of 5% formalin into right vibrissa pad of rats. The pain behavioral response was measured the number of grooming or scratching on the orofacial area for 9 successive 5 minutes intervals. Triptolide was administrated into the identified vibrissa pad (12.5, 25, 50 g/50 l) or intracisternal space (0.01, 0.1, 1 g/10 l) 10 min before formalin injection. The nociceptive responses were reduced in the 2nd phase (11 45 minutes), particularly 20, 30 minutes after fomalin injection following administration of triptolide into vibrissa pad (25, 50 g/50 l). Intracisternal (1 g/10 l) administration of triptolide alleviated the formalin-induced pain behaviors in the 2nd phase, especially 25 40 minutes after formalin injection. Triptolide could be a promising analgesic agent in the treatment of inflammatory orofacial pain. Key Words: Analgesia, Facial pain, Triptolide 서론 안면부통증은치아, 치조골, 구강연조직및악관절의손상에따른통증뿐아니라, 삼차신경통, 편두통과같이신경병증성통증을포함한다. 이러한통증은조직에손상을주거나잠재적인손상을줄수있는유해자극에의해나타나는일종의경고성감각인통각 (nociceptive pain), 손상된조직과그주변조직으로부터활성화되어분비되는 interleukin- 1 등의염증성매개물들 1) 이유해감수기에작용하여나타나는염증성통증 (inflammatory pain), 그리고말초와중추 신경의자체손상으로인해유해하지않은자극에도지속적으로나타나는신경병증성통증 (neuropathic pain) 으로구별될수있다 2,3). 염증성통증은통증부위의움직임을제한하여더이상의손상을방지하고일정기간후사라지지만때로는해가되는방향으로진행되어신경병증성통증으로발전되는경우도있어염증성통증의조절이중요하다고보인다. 포르말린테스트는염증성통증모델중널리사용되는방법중하나이다. 흰쥐의발바닥 4), 입술 5), 악관절 6), 안면부피하 7) 등으로포르말린을주입하면주입즉시염증반응과조직손상에의한특징적인이상성통증행위반응 (biphasic Received: July 2, 2015, Revised: July 21, 2015, Accepted: July 22, 2015 ISSN 1598-4478 (Print) / ISSN 2233-7679 (Online) Correspondence to: Min-Kyung Lee Department of Biomedical Health Science, Dong-Eui University Graduate School, 176 Eomgwang-ro, Busanjin-gu, Busan 47340, Korea Tel: +82-51-890-4238, Fax: +82-0505-182-6878, E-mail: lmk849@deu.ac.kr Copyright 2015 by the Korean Society of Dental Hygiene Science This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
김윤경외 : 트립톨라이드의말초와중추투여에의한안면부통증조절 nociceptive response) 을나타내며, 이때주입부위를긁거나문지르는행위또는핥는행위의횟수나시간등을통증행위반응의지표로간주한다. 포르말린주입후유해자극에의한말초감작으로 0 10분간지속되다가사라지는반응을 1차통증행위반응 (first phase) 이라하고, 이후유해자극이사라짐에도불구하고중추감작 (central sensitization) 에의해 11 45분간지속되는반응을 2차통증행위반응 (second phase) 이라고한다 8,9). 이때, 1차통증행위반응은말초감작으로간주하며 2차행위반응은중추감작으로분류할수있다. 말초감작은조직에가해진손상이유해수용기를자극하면서생성된 substance P, histamine, bradykinin 등의여러통증유발물질들이서로상승작용을나타내어구심성 A, C 섬유말단의통각수용기의역치를낮추고작은자극에도강력히활성화됨으로써통증이증강되는현상이다 10). 반면에, 중추감작은 C 섬유를통한고빈도자극과 A 섬유를통한저빈도자극이지속적인경우척수후근에있는광범위영역신경세포 (wide-dynamic range neuron) 나 nociceptive neurons의흥분성이증가하는현상으로, 지속적인 glutamate의분비로인한시냅스후 N-methyl-D-aspartate (NMDA) 수용기가활성화되고이로인해세포내유입된 Ca 2+ 가증가되면서중추감작이일어나게된다 11). 안면부통증조절을위해임상에서는 acetaminophen, 근육이완제, 항우울제, 비스테로이드성항염증제 (nonsteroidal anti-inflammatory drugs), opioid계약물등다양한합성화학약물들이처방되고있으나 12), 일부는의존성, 내성, 변비, 오심, 구토등의부작용을야기하므로최근연구는상대적으로부작용이적은천연물유래의통증조절약물에대한관심이증가하고있는상황이다 13-15). Triptolide는천연약초인미역순나무 (Tripterygium wilfordii Hook F) 에서추출한주요활성성분의하나이며, 다양한실험동물의통증모델에서 triptolide의통증경감효과가보고되고있다. 이와관련한연구결과로, 유선암종세포를실험동물의좌측경골골수강에주입하여유도된골암통증모델에서 triptolide의척수강내투여는실험동물의뒷발바닥에증가된기계적통각과민반응을완화시켰으며 16), 신경병증성통증모델에서반복투여한 triptolide의복강투여는제5번요수신경결찰에의해증가된기계적이질통을감소시켰다 17). 또한 complete Freund s adjuvant (CFA) 를실험동물의발바닥에주입하여유도한염증성통증모델에서 triptolide의복강투여는증가된열및기계적통증반응을완화시켰다고보고된다 18). In vivo 실험을통해확인된 triptolide의적용에따른통증및염증신호와관련된경로인 substance P 19), NMDA receptor 2 20), mitogen-activated protein kinase 및염증성매개물 1,17) 등의감소는이러한 triptolide의통증경감효과를뒷받침하고있다. 그러나아직까지 triptolide의통증경감효과를안면부의염증성통증모델에서연구한보고는미미한실정이다. 따라서본연구는실험동물의안면부피하에포르말린을주입하여염증성통증을유발하였고, triptolide의중추및말초로의투여가안면부통증조절에미치는영향을평가하고자하였다. 연구대상및방법 1. 실험동물본연구는의식이있는동물의실험에관한통증연구학회의윤리적규정에따라수행하였으며, 동의대학교동물실험윤리위원회의연구승인을얻은후실시하였다 (R2015-002). 실험동물은 240 280 g의 Sprague-Dawley 계수컷흰쥐를효창사이언스 (Daegu, Korea) 에서공급받아사용하였으며, 실험기간동안실험동물용투명플라스틱통에서사료와물을자유로이공급하며사육하였다. 12시간주 / 야순환주기및 23 o C 25 o C의일정한환경을유지하였다. 2. 포르말린으로유도한안면부통증실험동물의스트레스를최소화하기위해플라스틱통에 10분이상적응시키고테스트동안물과음식의섭취를금하였다. 인슐린주사기 (0.25 8 mm) 를이용하여실험동물 (n=6) 의오른쪽수염부피하에 5% 포르말린 (30 l) 을주입하였고, 약물이주입된안면부위를문지르거나긁는행위를통증지표로간주하였다. 주입직후부터 5분단위로누적하여총 45분간기록하였으며, 1차통증행위반응 (0 10분) 과 2차통증행위반응 (11 45분) 으로구분하여평가하였다 21). 3. Triptolide의말초및중추투여 Triptolide (Sigma, St. Louis, MO, USA) 는 5% dimethylsulfoxide에 1 g/ l의농도로희석시켜 70 o C에서보관하였고, 실험직전사용농도로희석하여사용하였다. Triptolide 의말초효과를평가하기위해 12.5, 25, 50 g/50 l의농도로포르말린주입 10분전동일한부위에각각투여하였다. 중추효과를평가하기위해서 0.01, 0.1, 1 g/10 l 의농도로 Hamilton 시린지 (Hamilton Co., Reno, NV, USA) 를사용하여포르말린주입 10분전소뇌연수조내로투여하였다. Triptolide의투여농도는 Hu 등 22) 의연구를참고하였다. 포르말린은 5% 의농도로생리식염수에희석시켜사용하였다. 425
J Dent Hyg Sci Vol. 15, No. 4, 2015 4. 중추로의약물투여를위한카테터삽입실험동물은 zoletil (1 ml/kg) 과 xylazine (0.25 ml/kg) 혼합액을근육주사하여마취하였다. 마취된쥐의뒤통수부위를면도하여 stereotaxic frame (Model 1404; David Kopf Instruments, Tujunga, CA, USA) 에고정시키고정수리점부터뒤통수뼈아래까지피부를절개하였다. 후두근을포함한주변근육을젖히고소뇌연수조 (intracisternal space) 에 26 gauge의주사바늘로작은구멍을내어뇌척수액의미세유출을확인후폴리에틸렌카테터 (8 cm, PE10; Caly Adams, Parsippany, NJ, USA) 를약 1 2 mm 삽입하였다. 카테터는마루뼈에미리삽입된 mini implant screw에회전시켜치과용자가중합레진 (Dentsply, York, PA, USA) 으로두개골에고정하였으며, 카테타끝은뇌척수액의흐름을방지하기위하여 stainless steel wire (0.32 mm) 로막아두었다. 절개부위는 4-0 나일론봉합사로봉합하였고, gentamycin (0.05 ml/kg) 을근육주사하였다. 술후실험동물은각각별도의플라스틱통에두었고, 72시간이상회복후실험에사용되었다. 5. 통계분석실험결과의통계분석에사용된프로그램은 IBM SPSS Statistics ver. 19.0 (IBM Co., Armonk, NY, USA) 과 sigmaplot 2001을사용했으며, 반복측정자료는 LSD 사후분석을이용한일원배치분산분석을하였다. 통계적인비교를위해통계적유의성의표준값은 p<0.05로설정하였으며, 결과는평균 ± 표준오차 (standard error of the mean) 로표시하였다. 결과 1. 말초 triptolide의투여에의한통증행위반응조절효과실험동물의안면부에포르말린으로유도된통증행위반응에대한 triptolide의말초투여의영향에대한결과는 Fig. 1 과같다. 본실험에앞선예비실험에서그래프에는나타나지않았으나포르말린주입군은아무것도처치하지않은 naive군에비해통증행위반응이증가됨을확인하였다. 1차통증행위반응의결과, 안면부피하로 triptolide (12.5, 25, 50 g/50 l) 투여후포르말린을주입한군은각농도에따라통증행위반응수치가 36.67±9.84회, 24.17±10.76회, 15.50±2.21회로나타났으며, 36.67±13.44회로나타난포르말린주입군과비교시유의한차이를나타내지않았다. 하지만 2차통증행위반응의결과, 포르말린군에서 299.00± 28.14회, triptolide 투여후포르말린을주입한군에서각농도에따라 226.00±35.44회, 179.17±15.36회, 114.33± 16.65회로나타나, 안면부피하로주입한 25, 50 g/50 l의 triptolide는포르말린으로유도된통증행위반응을 2차통증행위반응시기에서유의하게감소시켰음을확인하였다 (Fig. 1). 통증행위반응의변화를시간의경과에따라보면, 안면부피하로 triptolide 투여후포르말린을주입한군 (50 g/50 l) 의통증행위반응은포르말린주입군과비교시 20분, 30 분에유의하게감소되었음을확인하였다 (Fig. 2). 2. 중추 triptolide 의투여에의한통증행위반응조절효과실험동물의안면부에포르말린으로유도된통증행위반 Fig. 1. Peripheral effects of triptolide on nociceptive behavior. The nociceptive responses were reduced in 2nd phase (11 45 minutes), following administration of triptolid into rat s vibrissa pad 10 min before formalin injection (n=6). *p<0.05, F vs. triptolide 25, 50 g/50 l+f. F: formalin. Fig. 2. Changes in nociceptive responses following peripheral administration of triptolide. Administration of triptolide into rat s vibrissa pad significantly reduced the nociceptive responses 20, 30 minutes after induction of pain. *p<0.05, F vs. triptolide 50 g/50 l+f. F: formalin. 426
김윤경외 : 트립톨라이드의말초와중추투여에의한안면부통증조절 응에대한 triptolide의중추투여의영향에대한결과는 Fig. 3과같다. 1차통증행위반응의결과, 소뇌연수조내로 triptolide (0.01, 0.1, 1 g/10 l) 투여후포르말린을주입한군은각농도에따라 31.00±10.23회, 27.50±8.49회, 25.33± 8.95회로통증행위반응이나타나, 42.00±12.27회로나타난포르말린주입군과차이를보이지않았다. 2차통증행위반응의결과로, 소뇌연수조내로 triptolide 투여후포르말린을주입한군에서각농도에따라 216.50±35.61회, 190.50± 30.72회, 134.00±23.03회로나타났으며, 포르말린주입군에서 289.50±44.30회로나타나 triptolide 1 g/10 l의투여군에서포르말린으로유도된통증행위반응을감소시킴으로서통증조절효과를나타내었다 (Fig. 3). 그래프에는나타나지있지않으나, triptolide 10 g/10 l의투여군도 triptolide 1 g/10 l의투여군과유사한수준의통증조절효과를나타내었다. 시간의경과에따른통증행위반응의변화를보면, 소뇌연수조내로 triptolide (1 g/10 l) 투여후포르말린을주입한군의통증행위반응은포르말린주입후 25 40분에포르말린군과비교하여유의하게감소되었음을확인하였다 (Fig. 4). 고찰 구강및턱관절을포함하는안면부는삼차신경 (trigeminal nerve) 에의해지배를받으며, 여러가지외부의자극에의해염증성혹은신경병증성통증이유발되면감정적혹은정신적인부분에서도불편감을나타내는인체에서가장민감한부위중하나이다 23). 안면부에서발생한염증성급성 통증이해소되지못할경우신경병증성통증으로발전될수있어통증조절의중요성이강조되며, 최근연구는합성성분의약물로인한부작용을최소화하기위해천연물을이용한통증조절에관한연구로이어지고있다 15,24). 따라서본연구는포르말린으로유도된염증성안면통증모델에서천연약초인 triptolide의말초조직의피하또는소뇌연수조내로투여가안면부통증조절에미치는영향을확인하고자하였다. 약물의진통효과를평가하기위해실험동물을대상으로하는약물의투여경로에는복강투여, 국소부위의말초침윤투여, 경구투여, 중추투여등다양한방법으로적용될수있다. 약물의효과는약물의투여방법에따라서도크게좌우되므로어떠한방법으로약물을투여하여야유효한양의약물이병적부위에신속히도달할수있는가를고려하여투여방법을선택하여야한다. 예를들어, Cho 등 25) 은 ketamine 의말초제통효과확인을위한포르말린테스트에서 ketamine 의복강내투여보다말초침윤이더효과적이었고, 제통효과를얻기위해약물의말초침윤투여가복강투여보다더적은양으로전신부작용을최소화하면서말초통증관리가가능한장점이있다고보고하였다. 또한모르핀의척수강내 (intrathecal) 투여는운동기능및자율신경기능장애등의부작용을최소화하면서경구투여용량의약 1/300로동일한진통효과를나타낼수있다고하였다 26,27). 척수강내투여법은실험동물의통증모델에서약물의효과를검증하기위해널리사용되는투여법중하나로 17,22), 제4 5번요수사이의지주막하로약물을투여하여뇌혈관장벽의투과성이낮은약물의최대치료효과를유도할수있다고하였고, 약물주입을위한카테터삽입후마비, 운동성감소, 배변배 Fig. 3. Central effects of triptolide on nociceptive behavior. The nociceptive responses were reduced in 2nd phase (11 45 minutes), following Intracisternal administration of triptolide 10 min before formalin injection (n=6). *p<0.05, F vs. triptolide 1 g/10 l+f. F: formalin. Fig. 4. Changes in nociceptive responses following central administration of triptolide. Intracisternal administration of triptolide significantly reduced the nociceptive responses 25 40 minutes after induction of pain. *p<0.05, F vs. triptolide 1 g/10 l+f. F: formalin. 427
J Dent Hyg Sci Vol. 15, No. 4, 2015 뇨등의이상소견이없었으며, 기계적이질통에대한실험에서도정상군과비교하여차이가나지않았다고보고 26) 하여실험동물에서약물의약효를검증하는데유용한투여법으로제시되어진다. 실험동물의소뇌연수조 (intracistenal) 투여법은척수강내투여법과유사한방법으로안면부통증유발모델에서중추약물투여방법으로널리사용된다 7,21). 소뇌연수조는소뇌의아래쪽과연수의뒤쪽사이에지주막밑공간이커져있는부위로뇌척수액을함유하고있으며, 소뇌연수조내로의약물투여시안면부의감각정보를투사받는삼차신경핵군에대한약물의직접적인중추효과를확인할수있기때문이다. 따라서본연구에서는 triptolide를통증이발생한해당말초부위의피하에투여했을때의효과와적은용량으로약물의효과를유도할수있는소뇌연수조내로 triptolide을투여했을때의진통효과를평가하였다. 그결과, 두약물투여법모두유의한통증조절효과를나타내었다. 말초조직에직접투여한 triptolide는 25, 50 g/50 l를투여한실험군에서효과적인통증조절효과를나타내었으며, 소뇌연수로조투여한 triptolide는 1 g/10 l의적은용량에서통증행위반응에유의한감소효과를나타내었다. Triptolide의통증조절효과에대한연구로, 척수신경결찰에의한신경병증성통증모델에서복강투여한 triptolide 는증가된척수후각의미세아교세포 (microglia) 와성상교세포 (astrocyte) 의발현및염증성매개물인 interlukin (IL)-6, IL-1beta, monocyte chemotactic protein, tumor necrosis factor- (TNF- ) 의 messenger RNA의발현을억제함으로써기계적이질통을완화시켰고 17), triptolide의척수강내투여는신경결찰에따라증가된 TNF-, IL-2를억제함으로써기계적이질통을완화시켰다 22) 는보고가있다. 또한 CFA로실험동물의발바닥에유도된지속성염증성통증모델에서복강으로반복투여한 triptolide는척수후각의미세아교세포와성상교세포의발현을억제하여열및기계적과민반응을완화시켰다 18) 는보고가있다. 본연구에서 triptolide는안면부염증성통증행위반응경감효과를나타내었으며, 이는이전연구결과들을근거로하는 triptolide의생리약리학적특성이포르말린으로유도된염증및통증신호경로를조절함으로나타난것으로생각된다. 본연구의결과, 염증및통증신호의조절효과를가지는 triptolide는말초와중추투여를통해포르말린으로유도한염증성안면부통증모델에서실험동물의행위반응을감소시킴으로써유의한통증경감효과를나타내었다. 따라서 triptolide는안면부통증의예방및치료의연구에있어활용가능한천연대체약물로제시될수있으나, 이를위해서 는염증및통증신호의전달과정에서 triptolide의생리약리학적역할에대한추가적인연구가필요할것으로생각된다. 요약 본연구는 trtiptolide의안면부통증조절효과를평가하기위하여포르말린으로유도된실험동물의안면부통증모델에서말초와중추로투여한 trtiptolide의농도별차이를확인하였고, 다음과같은결과를얻었다. 먼저, 안면부피하로 triptolide 투여후포르말린주입군에서포르말린주입후 11 45분에발생하는 2차통증행위반응은 25, 50 g/50 l 의각농도에따라 179.17±15.36회, 114.33±16.65회로나타났고 299.00±28.14회로나타난포르말린주입군과비교시통증행위반응이유의하게경감되었다. 시간의경과에따른변화에서 50 g/50 l의투여군에서포르말린주입후 20분, 30분에통증행위반응이포르말린군과비교하여유의하게감소되었음을확인하였다. 안면부피하로투여한 triptolide는포르말린으로유도된통증행위반응을감소시킴으로써안면부통증조절효과를나타내었다. 다음으로, 소뇌연수조내로 triptolide 투여후포르말린주군에서 2차통증행위반응은 1 g/10 l의 134.00±23.03회로나타났고, 289.50±44.30회로나타난포르말린군과비교시통증행위반응이경감되었다. 시간의경과에따른변화에서, 1 g/10 l의 triptolide 투여군에서포르말린주입후 25 40분에통증행위반응이감소되었음을확인하였다. 소뇌연수조로투여한 triptolide는포르말린으로유도된통증행위반응을감소시킴으로써안면부통증조절효과를나타내었다. 이상의결과는, triptolide는구강및턱관절등을포함하는안면부통증조절의예방및치료에있어활용가능한천연대체약물로제시될수있을것으로생각된다. 감사의글 이논문은 2012년도정부 ( 교과부 ) 의재원으로한국연구재단바이오. 의료기술개발사업의지원을받아수행된연구임 (No. 2012M3A9B6055414). References 1. Kim JH, Kim KR, Jin HJ, Im SU, Song KB, Choi YH: The effect of polycan-calcium gluconate complex on inflammatory mediators from periodontitis patients. J Dent Hyg Sci 14: 223-229, 2014. 428
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