Original Article 대한침구의학회지제 30 권제 4 호 (2013 년 9 월 ) : 69-78 The Acupuncture Vol. 30 No. 4 September 2013 : 69-78 pissn 1229-1137 eissn 2287-7797 http://dx.doi.org/10.13045/acupunct.2013023 腎兪 (BL 23 ) 杜仲藥鍼이흰쥐의신경병리성동통모델에미치는영향 이동근, 이욱재, 이주희, 이상현, 이정훈, 신정철, 김재홍 * 동신대학교한의과대학침구의학교실 [Abstract] The Effects of Eucommiae Cortex Pharmacopuncture Injected at Sinsu(BL 23 ) on Neuropathic Pain in Rats Dong Geun Lee, Ook Jae Lee, Ju Hee Lee, Sang Hyun Lee, Jung Hun Lee, Jeong Cheol Shin and Jae Hong Kim* Department of Acupuncture and Moxibustion Medicine, College of Oriental Medicine, Dongshin University Key words : Neuropathic pain; Eucommiae Cortex pharmacopuncture; Sinsu(BL 23) Objectives : The purpose of this study is to examine whether Eucommiae Cortex pharmacopuncture may affect to the neuropathic pain in a rat model. Methods : To produce the model of neuropathic pain, under isoflurane 2.5 % anesthesia, underwent tight ligation by 6.0 silk thread and transection of the tibial and sural nerves, leaving the common peroneal nerve intact. After neuropathic surgery, the author examined if the exhibited the behavioral sign of allodynia. The allodynia was assessed by stimulating the plantar with Dynamic Plantar Aesthesiometer. Three days after the neuropathic surgery, Eucommiae Cortex pharmacopuncture was injected at Sinsu(BL 23) once every week for 6 weeks. After that, the author examined the withdrawal response of neuropathic rats leg by Dynamic Plantar Aesthesiometer. And also the author examined Bax, Bcl-2, Bax/Bcl-2 ratio in the spinal cord of neuropathic rats and the change of WBC, RBC, HGB, HCT count in the blood of neuropathic rats. Results : 1. The Eucommiae Cortex pharmacopuncture decreased the withdrawal response of mechanical allodynia that assessed with Dynamic Plantar Aesthesiometer in EC2-BL 23 group as compared with control group. 2. The Eucommiae Cortex pharmacopuncture decreased Bax/Bcl-2 ratio in EC1-BL 23, EC2-BL 23 group. But The Eucommiae Cortex pharmacopuncture injected at Sinsu(BL 23) didn t change Bax, Bcl-2 expression level in the all group. 3. The Eucommiae Cortex pharmacopuncture decreased WBC count in EC1-BL 23, EC2-BL 23 group. Conclusions : We have noticed that Eucommiae Cortex pharmacopuncture decreased mechanical allodynia in the model of neuropathic pain compared with the control group. Bax/Bcl-2 ratio in spinal cord of that group was also decreased compared with the control group. This study can be used as a basic resource on a study and a treatment of neuropathic pain. Received : 2013. 08. 06. Revised : 2013. 08. 15. Accepted : 2013. 08. 26. On-line : 2013. 09. 20. Corresponding author : Department of Acupuncture & Moxibustion Medicine, Gwangju Oriental Medicine Hospital of Dongshin University, 377-12, Wolsan-dong, Nam-gu, Gwangju, 503-850, Republic of Korea Tel : +82-62-350-7209 E-mail : nahonga@hanmail.net This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The Acupuncture is the Journal of Korean Acupuncture & Moxibustion Medicine Society. (http://www.theacupuncture.or.kr) Copyright 2013 KAMMS. Korean Acupuncture & Moxibustion Medicine Society. All rights reserved. http://dx.doi.org/10.13045/acupunct.2013023 69
The Acupuncture Vol. 30 No. 4 September 2013 Ⅰ. 서론 Ⅱ. 재료및방법 신경병리성동통 (neuropathic pain) 은말초신경이나조직의손상으로인해발생되는과통증 (hyperalgesia), 이질통 (allodynia), 자발적인통증 (spontaneous pain) 등을수반하는만성통증의하나로 1), 1872 년 Mitchell 이처음으로외상으로인한작열통을보고한이래 2) 많은환자에게서발견되었으며, 현재우리나라에서도이와유사한통증을호소하는환자가많다. 한의학에서신경병리성통증은痺證의범주로인식되어왔는데, 黃帝內經 素問 痺論 에서 痺者閉也, 氣血凝滯不行也, 有風寒濕三氣之痺, 有皮氣脈筋骨五臟外合之痺 라하여風寒혹은風濕의邪氣에의해氣血의흐름이막혀격렬한통증이발생하는것으로보았다 3). 약침요법은다양한방법에의해제조된약침액을질환과연관된양성반응점 ( 압통점, 아시혈 ) 및혈관에약침주입용주사기를사용하여시술하는방법으로서혈위의치료작용과약물의약리작용을이용하여생리적인기능을조정하고병리적인상태를개선시켜질병을치료하는신침요법이다 3,4). 杜仲은杜仲科에속한낙엽교목인두충 (Eucommiae ulmoides O LIV.) 의樹皮를건조한것으로味는甘微辛하고性은溫하며, 補肝腎强筋骨의효능으로腰膝酸痛과足膝痿弱에사용된다고하였으며, 肝腎不足으로인한腰膝痠痛과筋骨無力을치료하는要藥이라고하였다 5). 두충에대한실험적연구로 Hong et al 6) 은혈압강하효과를, Kim 7) 은소염, 진통작용을, Kim et al 8) 은면역증강작용을, Han 9) 은지질대사작용을보고한바있으나아직杜仲藥鍼이신경병리성통증에미치는영향에대한연구는전무한실정이다. 이에저자는소염, 진통작용이있으며補肝腎, 强筋骨효능이있는杜仲藥鍼이신경병리성동통에미치는영향을관찰하기위하여, 흰쥐의 tibial nerve 와 sural nerve 절제모델의腎兪 (BL 23 ) 에杜仲藥鍼을주입한후 dynamic plantar aesthesiometer 를이용하여이질통에대한회피반응시간및자극강도의변화, bax, bcl-2 발현과혈액내 WBC, RBC, HGB, HCT 변화를관찰하여유의한결과를얻었기에보고하는바이다. 1. 재료 1) 동물 체중이약 180 200 g의 Sprague Dawley 계의白鼠를사육장 ( 실내온도, 24 26 ) 내에서물과사료를충분히섭취하게하면서적응사육후실험에사용하였다. 실험동물을관리하거나실험할때인도적이고윤리적인관점을견지하였으며, 동신대학교동물실험윤리위원회의규정을준수하였다. 2) 약침조제한국산杜仲 (Eucommiae Cortex) 200 g을증류수 1,000 ml 와함께 3시간동안끓인다음원심분리기 (Centricon T-42K, Italy) 로 5,000 rpm 에서 30 분간원심분리하여상등액을취하였다. 상등액은 rotary evaporator(buchi, Netheland) 로수분을증발시켜 100 ml 로감압농축하였으며, 농축된약침액을동결건조기 ( 삼원주식회사 ) 로 -70 에서동결건조시켜최종적으로얻어진약침액의양은 15.87 g이었다. 이를소분하여생리식염수로희석하였으며, ph meter(orion, USA) 로각각 ph 7 산도를조절하여조제한후냉장보관하였다가시술에사용하였다. 2. 방법 1) 신경병리성동통유발 10) 백서를 isoflurane 2.5 % 로도입, 유지마취후고관절후측의피부를절개한후실체줌현미경을사용하여 biceps femoris muscle 사이의 sciatic nerve 를찾아 tibial nerve와 common peroneal nerve 및 sural nerve 를확인한후 microforcep 으로주변조직및혈관으로부터분리시킨다음, common peroneal nerve 는남겨두고, tibial nerve 와 sural nerve 는현미경하에서 6.0 silk thread 로결찰한후미세수술가위로절단하였다. 절개된부위는 kanamycin 을점적하고피부를봉합하였다. 2) 군분리 실험군은동통을유발시킨후처치를하지않은대조군 (control, n=5), saline 를시술한 saline 군 (saline, n=5), 70 http://dx.doi.org/10.13045/acupunct.2013023
腎兪 (BL 23) 杜仲藥鍼이흰쥐의신경병리성동통모델에미치는영향 9.1 μg/kg/μl 두충약침을처치한약침1군 (EC-1, n=5), 45.5 μg/kg/μl 두충약침을처치한약침2군 (EC-2, n=5) 으로총 4개군으로분류하였다. 3) 취혈및약침시술 11) 약침시술은足太陽膀胱經의腎兪 (BL 23) 에시행하였으며, 위치는뒤정중선의第 2 腰椎下에서兩旁 1.5 寸에서인체와상응하게정하였다. 약침시술은 insulin syringe(31 G 8 mm, BD, USA) 를이용하여 1주일에 1회씩총 6회를양측腎兪 (BL 23) 부위에 20 μl 씩각각시행하였다. 4) Dynamic plantar aesthesiometer 측정 수술후 3일째부터 6주간 1주에 1회씩 dynamic plantar aesthesiometer(ugo Basile 37450, Italy) 를이용하여, 각각이에대한철회반응 (withdrawal response) 을망으로된 cage 에옮겨 5분간안정시킨후관찰하였다. Stimulator 자극에의한이질통측정방법은 0 g에서부터 50 g 강도까지환측발바닥부위에자극하여발의철회반응의 react time(stimulator 가발바닥에 contact 하여다시떨어질때까지의시간 : second) 과, force intensity( 실험체가 stimulator 를피한순간의힘 : gram) 를얻었다. 5) 혈액검사 채혈에의하여얻은혈액중약 100 μl 를 EDTA-bottle에넣은후곧바로 multispecies hematology analyser(950, Hemavet, USA) 에주입하여 WBC, RBC, HGB, HCT 를측정하였다. 6) 혈청검사 분리된혈청은측정하기전까지는 4 에보관하였다. GOT 는 GOT/AST -PⅢ slide(fujifilm Corporation, Japan) 로, GPT 는 GPT/ALT-PⅢ slide(fuji film Corporation, Japan) 로 Fuji dri-chem clinical chemistry analyzer (DRI-Chem 4000ie, Fuji film Corporation, Japan) 를이용하여측정하였다. 7) Total RNA 분리및 reverse transcription polymerase chain reaction (RT-PCR) 1 Total RNA 분리 Total RNA 의분리는 spinal cord 부위의조직 (20 mg) 을 500 ul TRIZOL reagent(gibco-brl, USA) 로균질화 하고균질액에대해 200 μl의 chloroform(sigma, USA) 을가하여 15초동안흔들어잘혼합한후, 실온상태에서 5 분방치하고난다음세포유잔물을제거하기위하여 4, 14,000 rpm 에서 5분동안원심분리하였다. 원심분리로얻은상층액과 500 μl의 isopropanol(sigma, USA) 을첨가하여실온상태에서 5분동안방치한후 RNA pellet 을얻기위하여 4, 14,000 rpm 에서 8분간원심분리하고, 원심분리로생긴 pellet 에냉장보관된 70 % ethanol 과함께 DEPC 를넣고 4, 7,500 rpm 에서 5분간원심분리후 pellet 만남기고모두제거하고, 남은 ethanol 은실온에서 5분간방치시켜건조시킨다음 DEPC-treated water 에녹여 Spectrophotometer(Eppendorf, Germany) 에서 OD260 값을읽어 RNA 의순도및농도를정량하였다. 이들 spinal cord 조직의 total RNA 는사용시까지 -70 에서보관하였다. 2 RT-PCR 분리된 total RNA 3 μg 과 2.5 μl oligo(dt), DEPC-treated water를 RT premix(bioneer, Korea) 에넣어 Mastercycler gradient(eppendorf, Germany) 를이용하여 20 μl cdna 를합성하여 PCR 증폭을위한 template 로사용하였다. 이때 housekeeping 유전자인 glyceraldehyde-3-phosphate dehydrogenase(gapdh) (sense primer: 5'-ACTCCATCACCATCTTCCAG-3', antisense primer: 5'-CCTGCTTTCACCACCTCCTTG-3') 를 internal control로사용하였다. Reverse transcription temperature cycle은 42 C에서 1시간동안 cdna synthesis, 94 에서 5 분동안 denature 그리고 4 C에서 5분동안 cooling 시키는단계를거쳤다. Bax, bcl2, mglur5 유전자에대한白鼠의특이 primer 는 PCRpremix kit(bioneer, Korea) 를사용하였다. Polymerase chain reaction 은 cdna 2 μl, sense primer 1 μl, antisense primer 1 μl, DEPC-treated water를 PCR premix(bioneer, Korea) 에넣었다. PCR temperature cycle 은 cdna 의증폭을위하여 95 C에서 300 초동안 pre-denaturation, 94 C 에서 40 초동안 melting, 55 C 에서 40 초동안 annealing, 72 C에서 90초동안 extension 하는과정을 30 회반복수행하고마지막 cycle 에서 72 C에서 600 초동안 extension 단계를거쳐 bax 유전자증폭을 primer(sense primer: 5'-CATCTTCTTCCA GATGGTGA-3', antisense primer: 5'-GTTTCATCCAG GATCGAGCAG-3'), bcl2 유전자증폭을 primer(sense primer: 5'-CGGTTCAGGTACTCAGTCAT-3', antisense primer: 5'-ACTTTGCACAGATGTCCAGT-3'), mglur5 유전자증 http://dx.doi.org/10.13045/acupunct.2013023 71
The Acupuncture Vol. 30 No. 4 September 2013 폭을 primer(sense primer: 5'-TCCAATCTGCTCCTCC TACC-3', antisense primer: 5'-CAACGATGAAGAACTCT GCG-3') 룰이용하여 mastercycler gradient(eppendorf, Germany) 에서시행하였다. 이렇게증폭된 bax, bcl2, mglur5 의 DNA 를 ethidium bromide(etbr, 10 mg/ml) 를포함한 1.5 % agarose gel 상에서 0.5x TBE buffer(80 mm Tris-HCL, 80 mm boric acid, 2 mm EDTA, ph 8.3) 로 70 V에서전기영동시켜관찰한후 image station (Kodak, USA) 을이용하여촬영하였으며, Kodak MI software(kodak, USA) 를이용하여측정하였다. 이에비하여, EC-2 군은 2주째 66.3±15.47 %(p<0.05), 3주째 54.8±9.05 % (p<0.05), 4주째 34.8±7.00 %(p<0.001), 5주째 64.9±13.68 %(p<0.05), 6주째 65.5±10.02 %(p<0.05) 로 2주째부터 6주째까지모두유의한감소를보였다 (Table 1, Fig. 1). 3. 통계처리 실험성적은평균값과표준오차 (mean±se) 로표시하였으며, Window 용 SPSS(version 10.05, SPSS) 를이용하여, 비모수적방법중 Mann-Whitney U test 를시행하여실험군사이의통계적유의성을관찰하였다. 전체실험의통계적인유의성은신뢰구간 p<0.05 에서의미를부여하였다. Ⅲ. 결과 Fig. 1. Effects of Pharmacopuncuture(Eucommiae Cortex) at BL 23 Acupoint on the Withdrawal React Time in Neuropathic Pain Rats * : p <0.05, ** : p <0.001, as compared with the corresponding data of control group. 1. Plantar 철회반응의 react time 변화 신경병리성동통을유발한백서에대한두충약침이철회반응의 react time 에미치는영향을비교관찰한결과, control 군은 1주째 100.0±0.00 %, 2주째 122.6±21.20 %, 3주째 128.3±24.81 %, 4주째 102.1±10.94 %, 5주째 173.7±21.46 %, 6주째 176.3±47.93 % 를나타내었다. 2. Plantar 철회반응의 force intensity 변화 신경병리성동통을유발한백서에대한두충약침이철회반응의 force intensity 에미치는영향을비교관찰한결과, control 군은 1주째 100.0±0.00 %, 2주째 122.0± 20.69 %, 3주째 127.5±24.27 %, 4주째 102.0±10.83 %, Table 1. Effects of Pharmacopuncuture(Eucommiae Cortex) at BL 23 Acupoint on the Withdrawal React Time in Neuropathic Pain Rats (%) Weeks Group 1 2 3 4 5 6 Control 100.±0.00 122.6±21.20 128.3±24.81 102.1±10.94 173.7±21.46 176.3±47.93 Saline 100.±0.00 141.9±47.06 76.6±15.33 77.4±13.91 202.7±51.36 144.7±58.31 EC-1 100.±0.00 95.6±24.42 110.2±42.84 69.5±14.32 139.6±40.45 131.4±45.71 EC-2 100.±0.00 66.3±15.47 54.8±9.05* 34.8±7.00** 64.9±13.68* 65.5±10.02* Pharmacopuncture was treated on the rats for 6 weeks. Results are shown as means±se. * : p <0.05, ** : p <0.001, as compared with the corresponding data of control group. 72 http://dx.doi.org/10.13045/acupunct.2013023
腎兪 (BL 23) 杜仲藥鍼이흰쥐의신경병리성동통모델에미치는영향 Table 2. Effects of Pharmacopuncuture(Eucommiae Cortex) at BL 23 Acupoint on the Withdrawal Force Intensity in Neuropathic Pain Rats (%) Weeks Group 1 2 3 4 5 6 Control 100.±0.00 122.0±20.69 127.5±24.27 102.0±10.83 160.0±22.94 169.6±43.80 Saline 100.±0.00 130.9±36.52 77.1±15.08 77.8±13.69 169.8±29.53 122.5±34.39 EC-1 100.±0.00 95.5±23.87 109.5±41.67 70.0±14.21 138.4±39.51 130.3±44.53 EC-2 100.±0.00 66.9±15.14* 55.5±8.93* 35.9±6.86** 65.5±13.34* 66.1±9.80* Pharmacopuncture was treated on the rats for 6 weeks. Results are shown as means±se. * : p <0.05, ** : p <0.001 as compared with the corresponding data of control group. 5 주째 160.0±22.94 %, 6주째 169.6±43.80 % 를나타내었다. 이에비하여, EC-2 군은 2주째 66.9±15.14 %(p<0.05), 3주째 55.5±8.93 %(p<0.05), 4주째 35.9±6.86 %(p<0.001), 5 주째 65.5±13.34 %(p<0.05), 6주째 66.1± 9.80 %(p<0.05) 로 2주째부터 6주째까지모두유의한감소를보였다 (Table 2, Fig. 2). Control 군에비하여모든실험군에서유의한변화를보이지않았다 (Fig. 3). GAPDH 579 bp Bax 498 bp Fig. 2. Effects of pharmacopuncuture(eucommiae Cortex) at BL 23 acupoint on the withdrawal force intensity in neuropathic pain rats * : p<0.05, ** : p<0.001, as compared with the corresponding data of control group. Fig. 3. Effects of pharmacopuncture(eucommiae Cortex) at BL 23 acupoint on the intensity of bax mrna in neuropathic pain rats 4. Bcl-2 발현변화 3. Bax 발현변화 신경병리성동통을유발한백서에대한두충약침이 bax 발현정도에미치는영향을비교관찰한결과, control 군은 113.2±3.20( 1,000 OD), saline 군은 115.4±4.20( 1,000 OD), EC-1 군은 110.1±2.34( 1,000 OD), EC-2 군은 108.1±1.66( 1,000 OD) 을나타내었다. 신경병리성동통을유발한백서에대한두충약침이 bcl-2 발현정도에미치는영향을비교관찰한결과, control 군은 106.1±2.66( 1,000 OD), saline 군은 109.8± 3.14( 1,000 OD), EC-1 군은 111.9±3.33( 1,000 OD), EC-2 군은 113.0±5.09( 1,000 OD) 를나타내었다. Control 군에비하여모든실험군에서유의한변화를보이지않았다 (Fig. 4). http://dx.doi.org/10.13045/acupunct.2013023 73
The Acupuncture Vol. 30 No. 4 September 2013 GAPDH 579 bp Bcl-2 186 bp Fig. 4. Effects of pharmacopuncture(eucommiae Cortex) at BL 23 acupoint on the intensity of bcl-2 mrna in neuropathic pain rats Fig. 5. Effects of pharmacopuncture(eucommiae Cortex) at BL 23 acupoint on the bax/bcl-2 ratio in neuropathic pain rats * : p<0.05, ** : p<0.001, as compared with the corresponding data of control group. 5. Bax/Bcl-2 ratio 변화 신경병리성동통을유발한백서에대한두충약침이 Bax/Bcl-2 ratio 변화에미치는영향을비교관찰한결과, control군은 1.07±0.005, saline 군은 1.05±0.056, EC-1 군은 0.98±0.010, EC-2 군은 0.96±0.039 를나타내었다. Control 군에비하여 EC-1 군 (p<0.001) 과 EC-2 군 (p<0.05) 에서유의한감소를보였다 (Fig. 5). 6. WBC, RBC, HGB, HCT 변화에미치는영향 신경병리성동통을유발한백서에대한두충약침이 WBC, RBC, HGB, HCT 의변화에미치는영향을비교관찰한결과, control 군에비하여 RBC, HGB, HCT 는유의 Fig. 6. Effects of pharmacopuncture(eucommiae Cortex) at BL 23 acupoint on the WBC, RBC, HGB, HCT in hematology of neuropathic pain rats * : p<0.05, as compared with the corresponding data of control group. Table 3. Effects of Pharmacopuncture(Eucommiae Cortex) at BL 23 Acupoint on the WBC, RBC, HGB, HCT in Hematology of Neuropathic Pain Rats Group Group Control Saline EC-1 EC-2 WBC(K/ul) 6.5±0.52 5.9±0.41 4.7±0.48* 4.6±0.22* RBC(M/ul) 8.5±0.13 8.6±0.19 8.7±0.15 8.7±0.17 HGB(g/dl) 15.4±0.33 14.8±0.45 16.0±0.35 14.9±0.22 HCT(%) 55.1±1.04 53.9±2.31 55.5±1.62 53.2±0.46 * : p <0.05 as compared with the corresponding data of control group. 74 http://dx.doi.org/10.13045/acupunct.2013023
腎兪 (BL 23) 杜仲藥鍼이흰쥐의신경병리성동통모델에미치는영향 한변화가없었으나, WBC 의경우 control 군이 6.5±0.52 K/ul, saline 군이 5.9±0.41 K/ul, EC-1군이 4.7±0.48 K/ul, EC-2 군이 4.6±0.22 K/ul 로 EC-1 군과 EC-2 군에서유의한감소를보였다 (p<0.05)(table 3, Fig. 6). Ⅳ. 고찰 통증이란실질적인또는잠재적인조직손상이나, 이러한손상과관련하여표현되는감각적이고정서적인불유쾌한경험으로, 크게침해성통증 (nociceptive pain), 염증성통증 (inflammatory pain) 및신경병리성통증 (neuropathic pain) 으로분류된다. 이러한통증은감각적인면과정서적인면을포함하고있으며, 같은자극에대해서도개개인에따라그반응이다르게나타난다 12). 일반적으로손상부위가치유되면서통증은자연히소실되지만신경계손상에의한통증은손상부위가치유된후에도수개월또는수년이상지속되기도한다. 이러한신경계손상의결과발생한통증을신경병리성통증이라고한다 13). 신경병리성통증은신경계의일차적병변이나기능이상에의해시작되거나발생한통증으로정의되는데, 신경병리성통증은손상부위에따라중추성과말초성으로나눌수있고유발자극의유무에따라자발통증 (spontaneous pain) 과유발통증 (evoked pain) 으로나눌수있다. 자발통증은지속성통증과발작성통증으로나타나며, 유발통증은역치이하의비유해자극에의한통증인이질통 (allodynia) 과유해자극에의한통증인통각과민 (hyperalgesia) 으로분류할수있다 14,15). 사람이나동물에서의신경병리성동통의유발기전에대한다양한연구가이루어지고있으나정확한유발기전이나조절기전은아직밝혀지지않고있으며, 약물및비약물에의한치료가있지만만족할만한통증제어방법이없어새로운치료법이절대적으로요구된다 16). 신경병리성통증은사람에서는주관적인호소에의존해야하고, 통증양상및통증범위도불분명하며, 정서적원인에의해다양하고복합적인양상으로나타나기에효과적인치료방법모색에많은어려움이있다 15). 이러한어려움때문에신경병리성통증의발생기전을연구하기위한동물모델의필요성으로신경절제모델 17) 개발을통한연구가오래전부터이루어지고있는데, 이번연구에사용한 Lee et al 10) 의좌골신경절제모델은총비골신경 (common peroneal nerve) 은남겨두고, 경골신경 (tibial nerve) 과비복신경 (sural nerve) 만손상시켜기존신경절 제모델에비해만들기쉽고, 손상시킨신경섬유의재생가능성이없다 1). 좌골신경절제모델에서의신경병리성통증은좌골신경통과근접한데, 좌골신경통은한의학적으로腰脚痛, 腰脚痙攣, 股幹痛, 脚筋痛등의범주에속하며, 風寒濕邪, 肝腎兩虛, 氣血瘀滯로변증되는데 18), 諸病源候論 腰背病諸候 腰脚疼痛候에서는 腎氣不足, 受風邪之所爲也. 勞傷則腎虛, 虛則受于風冷, 風冷與眞氣交爭, 故腰脚疼痛 이라하여腰脚疼痛이腎虛한뒤에風邪가침범하여발생되는것으로보았다 19). 腎兪는족태양방광경의경혈로滋補腎陰, 振氣化, 祛水濕, 强腰脊, 益水壯火의穴性 20) 으로비뇨생식기질환, 소화기질환, 부인과질환, 전신성만성소모성질환에다용되며, 耳聾, 遺精, 腰痛, 月經不調, 身腫如水등의主治症을갖는다 21). 약침요법은침구요법과약물요법이결합된新鍼療法의일종으로경혈과약물의효과를동시에얻는장점이있다. 임상에서진정, 소염, 해열, 구급효과가뛰어나동통질환에서속효성을보이며, 약을복용할수없는환자에게도사용할수있다는장점이있다 22). 杜仲은補肝腎, 强筋骨의효능으로腰膝酸痛과足膝痿弱을치료하는要藥으로최근의연구에따르면 Hong et al 6) 은혈압강하효과를, Kim 7) 은소염, 진통작용을, Kim et al 8) 은면역증강작용을, Han 9) 은지질대사작용을보고하였고, Oh et al 23) 과 Lee et al 24) 은골흡수및골무기질의손실을방지하고골신생에유의한효과가있으므로골다공증유발억제에응용이가능하다고보고하였다. 또한 Cho et al 25) 은동물실험을통해경구투여시신경지배차단에따른골격근위축에대해근세포의 apoptosis 억제효과가있음을밝힌바있다. 이와같이杜仲에대한활발한연구가진행되고있으나신경병리성동통에대한진통효과를다룬실험보고는없는상태로두충의補肝腎, 强筋骨의효능으로主治하는腰膝酸痛과足膝痿弱이허리와다리의통증과함께화끈거리거나저린느낌이나거나감각이둔해지고다리에힘이빠지는등의좌골신경통증상과같음에착안하여신경병리성동통모델白鼠의신수 (BL 23) 에杜仲약침을시술한후 dynamic plantar aesthesiometer 를이용하여이질통에대한회피반응시간및자극강도의변화, bax, bcl-2 발현과혈액내 WBC, RBC, HGB, HCT 변화를관찰하여유의한결과를얻었다. 본실험에서시행한행동학적검사중물리적자극에의한회피반응에사용한 dynamic plantar aesthesiometer 는 0 g에서 50 g 강도까지 20초동안 2.5 g/s 로일정하게증가하는자극을가할수있는데이때물리적이질통이발 http://dx.doi.org/10.13045/acupunct.2013023 75
The Acupuncture Vol. 30 No. 4 September 2013 현된다. 이러한물리적이질통은회피반응으로나타나고회피반응시자극은자동적으로멈추는데이때의자극량 (force intensity) 은 0.1 g단위까지나타낼수있다 26). Dynamic plantar aesthesiometer 를이용하여물리적으로이질통을발생시킨후회피반응의 react time 과 force intensity 를관찰한결과 react time 의변화는대조군에비해 EC-2 군은 2주째 (p<0.05) 부터, 3주째 (p<0.05), 4주째 (p<0.001), 5주째 (p<0.05), 6주째 (p<0.05) 까지모두유의한감소를보였다. Force intensity 의변화에서도 EC-2 군은대조군에비해 2주째 (p<0.05) 부터, 3주째 (p<0.05), 4주째 (p<0.001), 5주째 (p<0.05), 6주째 (p<0.05) 까지모두유의한감소를보였다. 이는杜仲藥鍼이신경절제로손상된흰쥐의낮아진역치를높여줌으로써회피반응의감소를보여주는것으로말초신경손상에의한통증을억제시킬수있는가능성을제시한다. 또한고농도에서만회피반응의감소가나타남을통해치료시농도에따른효과의차이는앞으로더많은실험적연구가필요하리라사료된다. Bax 는주로세포질에존재하는데 DNA 손상등스트레스가전해지면사립체외막으로이동하여이의투과를유발하여 cytochrome-c 를유리시켜 apoptosis 를유도한다. Bax 가이러한현상을초래하는정확한기전은아직확실하게밝혀지지는않았으나사립체외막에서 bax 단백질들의소중합체협성 (oligomerization) 에의해초래되는것으로알려져있다. Bcl-2 는 bax 의작용을억제하는대표적인인자로사립체막에서 bax 의소중합체형성을억제하여 bax 의작용을방해하고궁극적으로 apoptosis 를억제하여세포생존을촉진하게한다. 따라서 apoptosis 를유도할때 bcl-2 는감소하고, bax 는증가하므로 bax/bcl-2 ratio 는 apoptosis 에있어중요한의의가있다고알려졌다 27). Bcl-2 는신경손상후발생한신경병리성통증상태에서증가하는데, 이는신경손상에따른신경퇴행과정의지속을막기위한생체의적응변화로가정된다 28,29). 본연구에서 bax, bcl-2, bax/bcl-2 ratio 발현정도에미치는영향을비교관찰한결과 bax, bcl-2 는대조군에비하여모든실험군에서유의한변화를보이지않았으나 bax/bcl-2 ratio 는 EC-1 군 (p<0.001) 과 EC-2 군 (p<0.05) 에서모두유의한감소를보였다. 이는두충약침이신경병리성동통에서신경손상에따른신경퇴행과정에서 apoptosis 를막아 neuron 의손실을줄일수있음을보여주는것으로농도에따른효과차이와 bax, bcl-2 각각의발현정도에대한연구는추후필요할것으로사료된다. 또한본연구에서두충약침이실험과정중 WBC, RBC, HGB, HCT 수치의변화에미치는영향을관찰한결과대조 군에비하여 WBC 의경우만 EC-1 군과 EC-2 군에서유의한감소를보였다 (p<0.05). Kim 7) 은두충추출물이 bradykinin 과 prostaglandin 의합성을억제하여염증작용을억제한다고보고했는데이를볼때신경병리성통증이염증으로인한신경손상과동반하였을경우두충약침이소염효과를나타내어동통억제에관여할수있을것으로사료된다. 이상의결과를종합해보면신경병리성동통흰쥐모델에있어腎兪 (BL 23) 杜仲藥鍼은 EC-1 군은 bax/bcl-2 ratio 와 WBC 감소에서만유의한효과를보였고, 5배약침처치한 EC-2 군은물리적이질통, bax/bcl-2 ratio, WBC 감소에서유의한효과를보였다. 이를통해두충약침이신경병리성동통억제에유의성이있었으며, 향후동통억제기전및농도와투여기간에대한상세한연구의필요와함께실험적모델에대한임상적연관성에대해서도심도있는연구가필요할것으로사료된다. Ⅴ. 결론 신경병리성동통이유발된흰쥐에대하여腎兪 (BL 23) 杜仲藥鍼시술이물리적이질통, bax 와 bcl-2 발현, 혈액학적변화에미치는영향을측정한결과다음과같은결론을얻었다. 1. 물리적이질통에대한반응은 EC-2 군은 2주부터, 3주, 4주, 5주째까지모두유의하게감소하였다. 2. Bax 와 bcl-2 발현은모든실험군에서유의한변화를보이지않았다. 3. Bax/bcl-2 ratio 변화는 EC-1 군과 EC-2 군에서모두유의한감소를보였다. 4. WBC 에대한변화는 EC-1 군과 EC-2 군에서유의한감소를보였다. Ⅵ. References 1. Lee BH, Sohn JH, Park YG. Effectiveness of Opioids in the Management of Neuropathic Pain. The Journal of Korean Brain Society. 2001 ; 1(1) : 53-64. 2. Mitchell SW. Injuries of Nerve and Their Consequences. Philadelphia : JB Lippincott. 1872 : 252. 76 http://dx.doi.org/10.13045/acupunct.2013023
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The Acupuncture Vol. 30 No. 4 September 2013 overload in the rat. Circulation. 1999 ; 99(23) : 3071-8. 28. De Novellis V, Siniscalco D, Galderisi U et al. Blockade of glutamate mglu5 receptors in a rat model of neuropathic pain prevents early overexpression of pro-apoptic genes and morphological change in dorsal horn lamina Ⅱ. Neuropharmacology. 2004 ; 46(4) : 468-79. 29. Kim YY. Differential expression of Bcl-2, Bax and heat shock proteins in the spinal cord of rat with neuropathic pain [dissertation]. Seoul : Seoul National Univ. 2003. English. 78 http://dx.doi.org/10.13045/acupunct.2013023