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1 ORIGINAL ARTICLE pissn / eissn Korean Journal of Acupuncture Vol.30, No.1, pp.37-46, 2013 白鼠의經穴 (LU8, SP5, HT4, KI7, PC5, LR4) 針刺가 Nitric Oxide Synthase 및 Norepinephrine 의변화에미치는影響 김영선 1 ㆍ최동희 1 ㆍ장호선 1 ㆍ나창수 1 ㆍ신헌태 1 ㆍ조주현 2 ㆍ이경인 2 ㆍ김선민 2 ㆍ표병식 2 ㆍ윤대환 1 1 동신대학교한의과대학, 2 동신대학교생물자원산업화지원센터 Effects of Acupuncture at the River Points(LU8, HT4, PC5, SP5, KI7, LR4) on the Changes of Nitric Oxide Synthase and Norepinephrine in Rats Youngsun Kim 1, Donghee Choi 1, Hosun Jang 1, Changsu Na 1, Heontae Shin 1, Joohyun Cho 2, Kyoungin Lee 2, Sunmin Kim 2, Byoungsik Pyo 2, Daehwan Youn 1 1 College of Oriental Medicine, Dongshin University, 2 Biotechnology Industrialization Center, Dongshin University Objectives : The Meridians and acupuncture points are the fundamental theories for acupuncture therapy. They have been associated with nervous system, but It is not well defined. We investigated that the effects of acupuncture at the river points(lu8, HT4, PC5, SP5, KI7, LR4) on the changes in the expression of nnos, inos, enos, and NE in rats. Methods : The Male Sprague-Dawley rats were divided into 6 groups each non-acupuncture and acupuncture group. We inserted needle and retained for 5 minutes on both left and right sides of LU8, HT4, PC5, SP5, KI7, LR4 which were the river points of five transport points for 6 yin meridian vessels. After that, blood was drawn via cardiac puncture, and tissues for each point near meridian vessels were extracted to examine the changes in the changes of nnos, enos, inos and NE. Results : The LU8 and HT4 group showed a significant decrease on nnos. In terms of enos and inos, the LU8 group decreased significantly while the KI7 group increased significantly. However, the experimental groups didn t show any significant changes on the plasma and tissue norepinephrine without plama NE in SP5 group. Conclusions : The effect on the nnos, inos, enos of acupuncture at LU8 and KI7 could be observed, and it is considered that the effect of acupuncture related with on nervous system could be studied by additional researches based on this one. Key words : five transport points, river point, nnos, inos, enos, norepinephrine(ne) Received December 26, 2012; Revised February 28, 2013, Accepted February 28, 2013 Corresponding author: Daehwan Youn College of Oriental Medicine, Dongshin University, 185, Geonjae-ro, Naju , Korea Tel: , Fax: , human22@dsu.kr This research was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education, Science and Technology(No ). CC This is an open access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Copyright 2013, SOCIETY FOR MERIDIAN AND ACUPOINT 37

2 김영선ㆍ최동희ㆍ장호선ㆍ나창수ㆍ신헌태ㆍ조주현ㆍ이경인ㆍ김선민ㆍ표병식ㆍ윤대환 서론 경락은經脈과絡脈을포함하고있으며, 내부로는五臟六腑를, 외부로는皮膚와肢節을연결하는氣血運行하는통로가되며, 유기적정체의기능활동들을실현시키고있는기능적체계및구조적체계이다 1,2). 五輸穴은十二經의인체의팔꿈치와무릎아래에분포하는 5개의특정혈자리를말하며, 경락중의經氣운행을자연계의흐르는물에비유한특정혈위로, 오행의상생과상극의원리에맞추어虛實을補瀉하여경락의기능을조절하고, 소속장부및연계장부의기능조절을담당한다. 五輸穴중經穴은경기의흐름이강물이흐르는듯하여經이라고칭하였으며, 대부분이손목과발목관절부근과그사이에위치하고있다 3). Nitric oxide synthase(nos) 는체내에서생성되어중추신경계와말초신경계의신경신호전달물질역할을하는 nitric oxide (NO) 를생성하여혈관확장과같은생리학적과정에서중요한작용을하며, 신경퇴행성질환의진행에도영향을미치는것으로알려져있다 4-7). NOS는 neuronal NOS(nNOS), inducible NOS (inos), endothelial NOS(eNOS) 로세개의 isotype으로알려져있는데, nnos 는신경조직에서 NO를생성하여세포간의정보전달을담당하는기능으로, 말초신경계, 중추신경계, 혈관계에작용한다고알려져있으며 5-7), enos 는혈관내피세포및세포질에가장풍부하게존재하고있다고알려져있고 8), inos 는백혈구나상피세포등에서발현되어 NO를생성하며염증반응시 NO의생성은대부분이 inos에의하여생성된것이라할수있다고알려져있다 9,10). Norepinephrine(NE) 또한신경전달물질이며, 집중력, 섭식행동, 불면증, 수면등에영향을준다고알려져있다 11). 이에관련된최근의침연구로는 Jeong 12) 과 Lee 등 13) 이足三里배합침자가중추신경의 NO에영향을미친다고보고하였으며, Kang 과 Lee 14) 는足厥陰肝經의行間과曲泉에補瀉를시행한침자가자율신경의흥분상태와안정상태를유발하였다고보고하였고, Chang 등 15) 은足陽明胃經의足三里자침이 atropine 으로유발된교감신경항진에영향을미친다고보고하였고, Kim 등 16) 은商陽과足竅陰에瀉法을, 足通谷과俠谿에補法을시행한자침이야간간호사의자율신경불균형을조절하였다고보고하였으며, Knardahl 등 17) 은手陽明大腸經의曲池등에시행한전침이 muscle sympathetic activity 측정을통한교감신경의활성도에미치는영향을보고하였으나, 침자와관련된경혈국소부를비롯한생체에서의신경전달물질의변화에대한연구및신경활성기전에대한영향연구보고가미흡한실정이다. 본연구에서는해당혈위에침자했을때혈위내국소적인작용변화에관찰하고자하여흰쥐를대상으로五輸穴의經穴에해당하는 LU8, HT4, PC5, SP5, KI7, LR4의鍼刺를시행하였으며, 그결과신경전달관여물질중의 nnos, inos, enos 및 NE의발현변화를다음과같이관찰하였기에보고하고자한다. 재료및방법 1. 동물실험동물은 Sprague Dawley 계 16주령 ( g, Samtaco, Korea) 수컷흰쥐들을준비하여, 동신대학교실험윤리에따라사육하고처치하였다. 사육되는동물은 12h light dark cycle 의명암조절이되었고, 항온항습 (21±2 o C, 40 60%) 이유지되는사육실에서사육되면서물과사료는자유롭게섭취하도록하였다. 2. 군분리실험군들은手太陰肺經의經渠 (LU8, n=3), 手少陰心經의靈道 (HT4, n=3), 手厥陰心包經의間使 (PC5, n=3), 足太陰脾經의商丘 (SP5, n=3), 足少陰腎經의復溜 (KI7, n=3), 足厥陰肝經의中封 (LR4, n=3) 에자침하지않은정상군과, 침자를시행한침자군으로각각분리하였으며, 모든군들은사육단계부터호흡마취과정까지모두동일하게처치되었다. 3. 경혈및침자 12개경맥의五輸穴에서經穴에해당하는 6개혈위에대하여인체에상응하게취혈하였다. LU8 는아래팔앞가쪽면의노뼈붓돌기와노동맥사이로서, 완횡문상 1 寸에, SP5 는안쪽복사아래앞쪽과안쪽복사융기의연결선의가운데함중에, HT4 는아래팔요측, 완횡문에서몸쪽으로 1.5 寸에, KI7는발꿈치힘줄앞쪽에서안쪽복사융기위쪽으로 2 寸에, PC5 는긴손바닥근힘줄과요측손목굽힘근힘줄의사이로완횡문에서몸쪽으로 3 寸에, 中封은앞정강근힘줄안쪽, 안쪽복사과앞쪽의오목한곳에각각취혈하였다 18,19). 침자시술전에 isoflurane (Choongwae, Korea) 과 O 2 로혼합된가스마취를시행하였고, 가스마취는 3% isoflurane 으로호흡마취를유도한후에 2% isoflurane 으로마취상태를계속유지시킨후각경혈좌우모두에직자로자침하고, 5분간유침하였다. 침자에사용된침은호침 ( mm, Dongbang, Korea) 이며, 실험기간동안숙련된전문가 1인이계속하여시술하였다. 38

3 白鼠의經穴 (LU8, SP5, HT4, KI7, PC5, LR4) 針刺가 Nitric Oxide Synthase 및 Norepinephrine 의변화에미치는影響 4. 혈장준비침자후 5분간의유침과정이끝난후에심장천자하여 6 ml의혈액을채혈하였다. 채혈한혈액은 K3 EDTA(Green VAC-tube EDTA K3 liquid 3.0 mm 13 75, Greencross, Korea) 에넣고 3,000 rpm에서 20분동안 4 o C에서원심분리 (Vision Scientific Co., LTD, Korea) 하여혈장을얻어이를 NE content 의분석을위해급속냉동시킨후 -80 o C에보관하였다. 5. 조직준비심장천자를통하여혈액을채혈한후각군들에해당하는경혈부위에서깊이 2 10 mm 의조직 (1 1 cm) 을적출하였다. nnos, inos, enos assay를얻기위한조직은 1X Protease Cocktail inhibitor(bd, USA) 가포함된 NP40 lysis buffer 500 μl를넣어 homogenization 하였다. 이 sample을 20분간 ice상태에놓아둔후 12,000 rpm에서 20분동안 4 o C에서원심분리 (Centrifuge 5415R, Germany) 한후 supernatants 을분리하였다. NE production 을얻기위한조직은해당경혈부위를포함하는조직을분리하여 0.1 M perchloric acid 0.5 ml와함께 homogenize (KIKA Works[Asia] Sdn. Bhd., Malaysia) 하였고, 이를 10,000 rpm에서 20분동안 4 o C에서원심분리 (Centrifuge 5415R, Germany) 하여 supernatants 만분리하였다. 6. Assay of NOS 1) Immunohistochemistry 경혈부위의조직은골격이포함된근육절편을적출하여 Bouin 용액에 24시간고정하였으며, 2.5% 질산 (nitric acid) 용액에서 30 일동안 3일간 1회씩질산액을교환하면서탈회 (decalcification) 를실시하였다. 파라핀블록의제작을위해채취한조직은 Tissue Processor(Tissue-Tex II, Japan) 를사용하였다. 파라핀블록은 microtome 을사용하여 10 μm 두께로절편을제작한후 2% BSA 와 15% 의 Goat Serum 으로 3시간동안반응시킨후 Rabbit anti-nnos(invitrogen. USA), anti-inos (Abcam, England), anti-enos(abcam, England) andtibody 를 0.05% Sodium azide 와 2% BSA 가함유된 PBS 로희석시켜조직을 48시간동안 4oC 에서반응을시킨후 50 mm PBS 에세척하고, biotinylated universal Secondary Antibody(Quick Kit : Vector Laboratories, Burlingame, USA) 에실온에서 10분동안반응시켰다. 그리고 50 mm PBS 에세척하고, Streptavidin peroxidase preformed complex(quick Kit: Vector Laboratories, Burlingame, USA) 에 10분동안반응시킨후에다시 50 mm PBS에세척하고, 조직을 3,3'-diaminobenzidine 발색용액 (Vector Laboratories, Burlingame, USA) 으로반응시킨후, 반응이끝난후조직을 PBS 로세척하여 hematoxyline 으로대조염색후슬라이드를 dehydtration 한후에 200배의광학현미경 (Eclipse 80i, Nikon, Japan) 으로관찰하였다. 염색된 NOS의 number 는연구자 2인이 count 하였다. 7. Assay of NE production NE production 은 Lakshmana 와 Raju 의방법 20) 을변형하여측정하였다. HPLC(LC-10Avp[Shimadzu, Japan]) 의 column으로 TSK-gel ODS-80 TM(5 μm, cm, Tosho, Japan) 을사용하였고, 이동상용매는모두 HPLC 급으로서 50 mm sodium acetate : acetonitril : methanol을 92 : 5 : 3 (v/v %) 의비율로혼합하여진공여과후사용하였으며, 유속은 0.8 ml/min 으로하였다. 검체의주입은 autoinjector 로하였고, 검출은 fluorimetric detector(rf-10axl, Shimadzu, Japan) 를사용하였다. 표준물질로서 NE(Sigma, USA) 를사용하였으며, 각물질의함량은 10 ppm(1 ng/100 μl), 50 ppm(5 ng/100 μl), 100 ppm(10 ng/100 μl) 과 0 ppm을 standard 로하여이들과비교한검체의 relative peak areas 를계산하여 weight gram 당으로환산하여계산하였다. 8. 통계처리모든측정값은 Excel statistic program(microsoft, USA) 을이용하여평균치와표준오차 (mean±standard error) 로표시하였고, 각실험군간의통계학적분석은 Window 용 SPSS(SPSS, USA) 를사용하여비모수적방법으로 Mann-Whitney U test를시행하였다. 각실험군은대조군에비하여 α=0.05 수준 (p<0.05) 에서유의성을검정하였다. 결과 1. nnos 발현에미치는영향經渠 (LU8), 靈道 (HT4), 間使 (PC5), 商丘 (SP5), 復溜 (KI7), 中封 (LR4) 에대한鍼刺가경혈조직내 nnos 발현변화를관찰한결과, 침자를시행하지않은 LU8군은 232.0±29.37(number), HT4군은 164.7±18.49(number), PC5군은 21.3±5.70(number), SP5군은 28.3±6.36(number), KI7군은 19.7±2.60(number), LR4군은 91.7±12.45(number) 를나타내었으며, 鍼刺를시행한 LU8 군은 20.7±7.75(number), HT4군은 66.3±22.36(number), PC5군은 39

4 김영선ㆍ최동희ㆍ장호선ㆍ나창수ㆍ신헌태ㆍ조주현ㆍ이경인ㆍ김선민ㆍ표병식ㆍ윤대환 25.3±3.76(number), SP5군은 17.3±1.20(number), KI7군은 65.7±24.91(number), LR4군은 97.7±20.99(number) 를나타내었다. 鍼刺를시행하지않는각각군들과의비교에서 LU8 군과 HT4 군가유의한감소를보였다 (p<0.05)(fig. 1, 2). 2. inos 발현에미치는영향經渠 (LU8), 靈道 (HT4), 間使 (PC5), 商丘 (SP5), 復溜 (KI7), 中封 (LR4) 에대한鍼刺가경혈조직내 inos 발현변화를관찰한결과, 침자를시행하지않은 LU8 군은 87.7±8.11(number), HT4 군은 24.0±6.11(number), PC5 군은 17.3±4.84(number), SP5 군은 17.3±0.88(number), KI7 군은 18.0±1.15(number), LR4군은 48.3±9.40(number) 를나타내었으며, 鍼刺를시행한 LU8 군은 24.7±4.63(number), HT4 군은 36.0±2.31(number), PC5 군은 35.7±14.17(number), SP5 군은 38.0±7.55(number), KI7 군은 47.7±4.37(number), LR4군은 74.3±6.94(number) 를나타내었다. 鍼刺를시행하지않는각각군들과의비교에서 LU8 군은유의한감소를, KI7군은유의한증가를보였다 (p<0.05)(fig. 3, 4). 3. enos 발현에미치는영향經渠 (LU8), 靈道 (HT4), 間使 (PC5), 商丘 (SP5), 復溜 (KI7), 中封 (LR4) 에대한鍼刺가경혈조직내 enos 발현변화를관찰한결과, 침자를시행하지않은 LU8 군은 178.7±5.46(number), HT4 군은 162.7±55.98(number), PC5군은 27.7±3.71(number), SP5군은 Fig. 1. Effect of acupuncture on immunoreactivity for neuronal nitric oxide synthase(nnos) on the acupoints in rats. Each Normal group didn t get any other treatment. LU8, HT4, PC5, SP5, KI7, LR4 groups were treated acupuncture at each acupoint. Results are shown as mean±s.e. *p<0.05 as compared with the corresponding data of each Non-Acupuncture group. Fig. 3. Effect of acupuncture on immunoreactivity for inducible nitric oxide synthase(inos) on the acupoints in rats. Each Normal group didn t get any other treatment. LU8, HT4, PC5, SP5, KI7, LR4 groups were treated acupuncture at each acupoint. Results are shown as mean±s.e. **p<0.01 as compared with the corresponding data of each Non-Acupuncture group. Fig. 2. Representive microphotographs of immunoreactivity pattern for neuronal nitric oxid esynthase(nnos) in rats. Each Normal group didn t get any other treatment. LU8, SP5, HT4, KI7, PC5, LR4 groups were treated acupuncture at each acupoint

5 白鼠의經穴 (LU8, SP5, HT4, KI7, PC5, LR4) 針刺가 Nitric Oxide Synthase 및 Norepinephrine 의변화에미치는影響 148.0±11.53(number), KI7군은 54.3±6.44(number), LR4군은 103.7±11.46(number) 를나타내었으며, 鍼刺를시행한 LU8 군은 19.3±8.35(number), HT4군은 147.7±19.62(number), PC5군은 32.0±1.73(number), SP5군은 38.0±3.06(number), KI7군은 106.3±24.90(number), LR4군은 152.0±18.25(number) 를나타내었다. 鍼刺를시행하지않는각각군들과의비교에서 LU8 군은유의한감소를, KI7군은유의한증가를보였다 (p<0.05)(fig. 5, 6). 4. 조직내 Norepinephrine 생성에미치는영향經渠 (LU8), 靈道 (HT4), 間使 (PC5), 商丘 (SP5), 復溜 (KI7), 中封 (LR4) 에대한鍼刺가조직내 Norepinephrine 생성변화를관찰한결과, 침자를시행하지않은 LU8 군은 3.5±0.21(ppm), HT4 군은 Fig. 5. Effect of acupuncture on immunoreactivity for endothelial nitric oxide synthase(enos) on the acupoints in rats. Each Normal group didn t get any other treatment. LU8, HT4, PC5, SP5, KI7, LR4 groups were treated acupuncture at each acupoint. Results are shown as mean±s.e. *p<0.05, **p<0.01 as compared with the corresponding data of each Non-Acupuncture group. Fig. 4. Representive microphotographs of immunoreactivity pattern for inducible nitric oxid esynthase(inos) in rats. Each Normal group didn t get any other treatment. LU8, SP5, HT4, KI7, PC5, LR4 groups were treated acupuncture at each acupoint Fig. 6. Representive microphotographs of immunoreactivity pattern for endothelial nitric oxide synthase(enos) in rats. Each Normal group didn t get any other treatment. LU8, SP5, HT4, KI7, PC5, LR4 groups were treated acupuncture at each acupoint

6 김영선ㆍ최동희ㆍ장호선ㆍ나창수ㆍ신헌태ㆍ조주현ㆍ이경인ㆍ김선민ㆍ표병식ㆍ윤대환 Fig. 7. Effect of acupuncture on tissue norepinephrine release on the acupoints in rats. Each Normal group didn t get any other treatment. LU8, HT4, PC5, SP5, KI7, LR4 groups were treated acupuncture at each acupoint. Fig. 9. Effect of acupuncture on plasma norepinephrine release in rats. Each Normal group didn t getany other treatment. LU8, HT4, PC5, SP5, KI7, LR4 groups were treated acupuncture at each acupoint. Results are shown as mean±s.e. *p<0.05 as compared with the corresponding data of each Non-Acupuncture group. Fig. 8. Chromatograms of tissue norepinephrine release using fluorimetric detector. Normal group didn t get any other treatment. LU8, SP5, HT4, KI7, PC5, LR4 groups were treated acupuncture at each acupoint. Fig. 10. Chromatograms of plasma norepinephrine release using fluorimetric detector. Normal group didn t get any other treatment. LU8, SP5, HT4, KI7, PC5, LR4 groups were treated acupuncture at each acupoint. 42

7 白鼠의經穴 (LU8, SP5, HT4, KI7, PC5, LR4) 針刺가 Nitric Oxide Synthase 및 Norepinephrine 의변화에미치는影響 4.7±0.47(ppm), PC5군은 4.2±0.17(ppm), SP5군은 4.2±0.50 (ppm), KI7군은 4.2±0.17(ppm), LR4군은 4.0±0.13(ppm) 을나타내었으며, 鍼刺를시행한 LU8 군은 4.1±0.40(ppm), HT4 군은 4.6±0.54(ppm), PC5군은 4.1±0.05(ppm), SP5군은 3.6±0.21 (ppm), KI7군은 3.7±0.28(ppm), LR4군은 4.6±0.51(ppm) 을나타내었다. 鍼刺를시행하지않는각각군들과의비교에서모든실험군들에서유의한변화를보이지않았다 (Fig. 7, 8). 5. 혈장내 Norepinephrine 생성에미치는영향經渠 (LU8), 靈道 (HT4), 間使 (PC5), 商丘 (SP5), 復溜 (KI7), 中封 (LR4) 에대한鍼刺가혈장내 Norepinephrine 생성변화를관찰한결과, 침자를시행하지않은 LU8 군은 52.4±3.46(ppm), HT4 군은 52.7±1.77(ppm), PC5군은 56.5±2.66(ppm), SP5군은 54.4± 0.44(ppm), KI7군은 55.0±1.86(ppm), LR4군은 53.6±0.51 (ppm) 를나타내었으며, 鍼刺를시행한 LU8 군은 64.1±14.98 (ppm), HT4군은 48.2±0.47(ppm), PC5군은 50.1±1.75(ppm), SP5군은 50.1±1.02(ppm), KI7군은 53.6±4.80(ppm), LR4군은 55.5±2.59(ppm) 를나타내었다. 鍼刺를시행하지않는각각군들과의비교에서 SP5 군에서유의한감소를보였다 (p<0.05)(fig. 9, 10). 고찰 十二經脈과任脈과督脈上에존재하는穴을 十四經穴 이라하여, 經穴 이라칭하며, 이經穴들이상호연계되어분류되고이론화된것이經絡이며, 인체내의기혈운행의통로로서유기적정체의기능활동을하는구조적, 기능적인체계의성격을가지고있다. 또한, 經穴은經脈과관계가있어本經의病證을主治하는作用및經脈과소속된臟腑의病證에도반영된다 21). 十二經脈의四肢에서肘膝關節以下에분포한五輸穴은 5개의특정혈자리인井ㆍ滎ㆍ兪ㆍ經ㆍ合을말하며, 四肢末端에서肘膝방향으로차례대로배열되어있다. 즉, 작은곳에서큰곳으로, 얕은곳에서깊은곳으로의經氣의흐름이있다 3,21). 井穴은經氣가出하는곳으로지하의수원에서물이흘러나오는듯하며, 맥기가가늘고얕은것을의미하고四肢末端에분포하며, 滎穴은經氣가流行하는곳으로經氣의흐름이샘물이흐르는듯하며, 掌指와跖趾關節앞에분포하고, 兪穴은經氣가점점盛해지는곳으로물이얕은곳에서깊은곳으로힘있게흘러들어가는것과같으며, 掌指와 跖趾關節뒤에분포하고, 經穴은經氣가盛한곳으로강물이흐르는것같은경기의흐름으로腕과關節以下에분포하고, 合穴은經氣가臟腑부위와會合하는곳으로모든강물이모여바다에흘러들어가는것과같으며, 주로肘膝關節附近에분포한다 3,21). 본연구에서는임상에서다용되고있는치료혈중의하나인오수혈중經渠 (LU8), 靈道 (HT4), 間使 (PC5), 商丘 (SP5), 復溜 (KI7), 中封 (LR4) 은모두陰經에해당하는혈위들로陽經에비하여취혈조건과신경및혈관분포등해부학적조건등이비교적우수하여이들을대상으로해당경혈국소부위와혈장에서침자가신경활성과관련있는 NOS 와 NE의변화에미치는영향을관찰하고자하였다. 산화질소 (nitric oxide, NO) 는신경전달물질로알려져있으며, 혈관내피세포와뇌조직에서도생산된다. NO는우리몸에서많은생리적기능을하는데, 그중에서도혈관의이완과혈류량을조절하고, 혈소판응집억제와성기능장애개선과항미생물작용및항암작용등여러가지의생물학적기능을하고, 다른세포에서살균작용을나타내며, 중추신경계와말초신경계에서뉴런의신경전달물질로도쓰이기도한다 22). 이에대하여 NO가인체내에서도발생하여생리적기능과병리적기능을가지고있는것이알려진바있다고 Yim 23) 은보고하였고, 이는 nitric oxide synthase(nos) 에의하여만들어진다. Nitric oxide synthase(nos) 는 neuronal NOS (nnos), inducible NOS(iNOS), endothelial NOS(eNOS) 3가지의 isotype 이알려져있는데, nnos 와 enos 는항상발현되어있어 constitutivenos(cnos) 라고도한다. 특히신경조직에서 NO를생성하는 nnos 는세포간의정보전달과신경전달을담당하여, 중추신경계와말초신경계및혈관계에작용하는것으로도알려져있다 5-7,23). 중추신경계에서의 nnos 는외부자극에대해민감하게반응하며, 이는신경세포가자극되고, 세포내의칼슘이온이증가하면서효소가활성화되어 NO를합성하고, 주변의조직에확산되어신경전달물질역할을담당하지만, 뇌의저산소증, 허혈증, 뇌졸중등의질병이있는상태에서는세포내로칼슘이온이증가하여많은 NO가생성되고신경세포에손상이나타나기도한다 24,25). enos 는동맥, 소동맥의벽, 또는벽주위의혈관내피와평활근에서발생되어, NO를증가시킴으로혈관의활동성을조절한다고알려져있다 26). inos 의증가는염증에의한조직을손상가속화시킨다고알려져있다 27). 이와관련된연구보고로足三里, 足三里配曲池, 足三里配陰陵泉에전침자극을시행하여 nnos 활성을측정한결과, 뇌줄기영역에서足三里와足三里配曲池전침자극군은정상군에비하여유의한감소를, 足三里配陰陵泉전침자극군은足三里와足三里配曲池전침자극군에비하여유의한증가를보였으며, 소뇌영역에서는足三 43

8 김영선ㆍ최동희ㆍ장호선ㆍ나창수ㆍ신헌태ㆍ조주현ㆍ이경인ㆍ김선민ㆍ표병식ㆍ윤대환 里와足三里配曲池전침자극군이정상군에비하여유의한감소를, 足三里配陰陵泉전침자극군은足三里와足三里配曲池전침자극군에비하여유의한증가를나타내어足三里의배합이중추신경계의 NO system 에각기다른영향을미치는것을 Lee 등 13) 이보고하였고, 전침효과가 nnos 신경세포의 activity 에대한효과를관찰한결과, 대뇌겉질영역의 nnos 에서 100 Hz군이침자군과 2 Hz군데비하여일차운동겉질, 후각뇌주위겉질, 시각겉질, 뇌섬겉질의부위에서유의한감소를, 뇌줄기영역의 nnos 에서 100 Hz군이침자군및 2 Hz군에비하여위둔덕층, 뒤가쪽수도관주위회색질, paralemniscal nucleus 에서유의한감소를, 소뇌영역의 nnos 에서 100 Hz 군이가장떨어지는결과를각각나타내었다고 Kim 등 28) 이보고하였다. 足三里와曲池에침자극을시행한후흰쥐의뇌줄기와소뇌의신경세포에서 nnos 활성을측정하여, 뇌줄기영역중 SuG 와 DLPAG 에서曲池침자군은정상군과足三里침자군보다유의한감소를, 소뇌영역에서足三里침자군및曲池침자군에서 nnos 의활성에대하여유의한차이가없다는것을확인하고, 침자극이경혈선택과 NOS 활성에관계가있다는것을, 경혈의침자극이 NO system 에영향을준다는것을 Kim 등 29) 이보고하였으며, 농도별봉독약침이생쥐의 Type II Collagen 유발관절염에미치는영향에서봉독약침액처리후농도의존적으로발현이감소되었다고 Lee 등 30) 은보고하였다. 본연구에서 LU8 의침자가해당경혈부위내 nnos, inos, enos 활성감소에영향을주었으며, HT8 은 nnos 활성감소에, KI7은 inos, enos 활성증가에영향을주는것으로관찰되었다. 이는오수혈중모든경혈이 NOS 활성과관련되지않는다는것을알수있으며각혈위마다의특징적혈성이작용함을관찰할수있으며, 또한 LU8 침자시 NOS 과관련한 nnos, inos, enos 의모든 isotype 활성에서감소를보이는것은 LU8 와 NO의자극에대한활성과관련된다고추론할수있다. 이는 Lee 등 13) 이보고한曲池, 足三里침자연구와일치된결과로일정혈위에대한침자는뇌뿐만아니라혈위에서도 NOS 와관련된인자의변화를관찰할수있었다. Norepinephrine(NE) 은교감신경의자극으로인해부신수질에서발생되는스트레스성호르몬이며, 이는집중력, 섭식행동, 불면증, 수면등에영향을주며, 스트레스와운동자극에민감한반응을보이며, 청반핵 (locus coeruleus) 과뇌간핵 (brainstemnucleus) 에서의신경전달물질로이용되고있다 31,32). 이와관련된연구보고로然谷, 陰谷, 少府, 少海鍼刺가 norepinephrine 에미치는영향에대하여관찰한결과, 然谷, 陰谷, 少府, 少海鍼刺중에서腎經의水穴 KI10 군및心經의水穴 HT3 군에서유의한감소를, 腎經의 火穴 KI2 군및心經의火穴 HT8 군에서유의한차이가보이지않았다고 Youn 등 33) 은보고하였으며, 12 經脈의合穴刺鍼후조직및혈장내 Norepinephrine 생성변화를관찰한결과, 조직에서는 normal 군에비하여 HT3군, SP9군, KI10 군은유의하게감소하였으며, PC3군과 LR8군은유의하게증가하였고, 혈장에서는 TE10 군, SP9군, LR8군, GB34 군, BL40 군은이유의하게증가하였다고 Kim 등 34) 은보고하였다. Nitric oxide(no) 는가스형태로 nitric oxide synthase 에의하여생성되는데, NO는 plasma membrane 에침투할수있으며, 주위세포에게까지도영향을미친다 27). 자율신경계의교감신경활성이증가되면피부나장에분포된혈관수축으로인하여혈류가증가하고, 에너지를효과적으로쓸수있도록하며, 동공이산대되고, 기관지가확장됨으로서산소유입량이증가하고, 심수축력과심박동률이증가하여골격근과뇌의관류량이커지게된다. 또한, 부신수질을자극하여혈관내로 epinephrine 과 norepinephrine 을유리시키며, 에너지동원기능을활발하게한다. 즉, 교감신경섬유는많은臟腑에연계되어있으며, 말초신경계로서작용한다 11). 교감신경과반대로부교감신경은장의연동운동을증가시키고, 동공수축, 피부와장의혈관이완을유도하고, 심박동을느리게하기도한다. 교감신경에서나타나는 norepinephrine 은평활근에작용하고, 피부및피하근육등에분포하는말초혈관의긴장을촉진시킨다 11). 본연구에서는오수혈중 6가지의경혈침자가해당경혈부위의 NE 농도변화에는영향을미치지못하였으며다만혈장내 NE 농도는 SP5 군에서유의한감소를보였다. 이로보아 SP5 는체내혈류에서교감신경이관련된 NE 활성감소에일정한영향을주는것으로추론되며, 실험에응용된오수혈은각각의해당경혈부위의 NE농도에영향을주지않는것으로관찰되었다. 이런결과로보아, 오수혈중 LU8, HT8, KI7 에대한침자는신경전달물질의생성과관련된 NOS 에일정한영향을주지만 NE의농도변화에영향을주지않는다는것을관찰할수있었으며, 추후다양한신경전달물질에대한관련성과자침효과에대한기본적인생리활성기전에대한심도있는연구필요하리라고사료된다. 결론 經渠 (LU8), 靈道 (HT4), 間使 (PC5), 商丘 (SP5), 復溜 (KI7), 中封 (LR4) 에대한鍼刺가 nnos, inos, enos 및 Norepinephrine 에미치는영향을관찰한결과다음과같은결과를얻었다. 44

9 白鼠의經穴 (LU8, SP5, HT4, KI7, PC5, LR4) 針刺가 Nitric Oxide Synthase 및 Norepinephrine 의변화에미치는影響 1. nnos 발현변화에서鍼刺를시행하지않는각각의군에비해 LU8 군과 HT4 군가유의한감소를보였다. 2. inos 발현변화에서鍼刺를시행하지않는각각의군에비해 LU8 군은유의한감소를, KI7군은유의한증가를보였다. 3. enos 발현변화에서鍼刺를시행하지않는각각군에비해 LU8 군은유의한감소를, KI7군은유의한증가를보였다. 4. 조직내 Norepinephrine 생성에미치는영향에서鍼刺를시행하지않는각각의군에비해모든실험군들에서유의한변화를보이지않았다. 5. 혈장내 Norepinephrine 생성에미치는영향에서鍼刺를시행하지않는군에비해 SP5군에서유의한감소를보였다. 감사의글 This research was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education, Science and Technology(No ). 참고문헌 1. Introduction of Korean Traditional Medicine. Seoul : Esdang. Na Chang-Su : Lee SR. Study of Acupuncture Points. Seoul : Jisangsa : 36, Jie YW. Yang Wei Jie complete works 3. Gyeonggi : Daesung Publishing Company : 303-6, 310, 311, Cho HS, Shin MS, Kim CJ, Baek SS. Effect of treadmill exercise on inflammatory markers in the substantia nigra in 6-OHDAinduced Parkinson's rats. Exercise Science ; 21(1) : Zhuang W, Eby JC, Cheong M, Mohapatra PK, Bredt DS, Disatnik MH, et al. The susceptibility of muscle cells to oxidative stress is independent of nitric oxide synthase expression. Muscle Nerve ; 24(4) : Bredt DS. Endogenous nitric oxide synthesis : biological functions and pathophysiology. Free Radic Res ; 31(6) : Feng C. Mechanism of nitric oxide synthase regulation: electron transfer and interdomain interactions. Coord Chem Rev ; 256(3-4) : Kim WJ, Yun YS, Kim MS, Kim SH, Choi HR. Immunohistochmical Study of Nitric Oxide Synthase in Salivary Glands. The Korean Journal of Anatomy ; 33 : Hung YC, Guh JH, Cheng ZJ, Chang YL, Hwang TL, Liao CH, et al. Inhibition of the expression of inducible nitric oxide synthase and cyclooxygenase-2 in macrophage by 7HQ derivatives: involvement of ikappab-alpha stabilization. Eur J Pharmacol ; 418 : Liang YC, Huang YT, Tsai SH, Lin-Shiau SY, Chen CF, Lin JK. Supression of inducible cyclooxigenase and inducible nitric oxide synthase by apigenin and related flavonoids in mouse macrophages. Carcinogenesis ; 20 : Dale P, Augustine GJ, Fitzpatrick D, Hall WC, Lamantina AS, Mcnamara JO, et al. Neuroscience. 3rd ed. Seoul : World Science : Jeong IG, Lee JD, Kim CH. Effect of joksamni combination on NADPH-diaphorase, neuronal nitric oxide synthase, neuropeptide y and vasoactive intestinal peptide in the crebral cortex of spontaneously hypertensive rat. The Journal of Korean Acupuncture & Moxibustion Society ; 20(5) : Lee HM, Kim CH, Kim YS. Acupoint combination-related changes of NADPH-d diaphorase and neuronal nitric oxide synthase in the brainstem and cerebellum of spontaneously hypertensive rats. The Journal of Korean Acupuncture & Moxibustion Society ; 20(6) : Kang HC, Lee SG. The effects of fire point(lr2), water point(lr8) through reinforcement-reduction acupuncture stimulation on ANS & EEG. Journal of Oriental Neuropsychiatry ; 21(2) : Chang CH, Huang JL, Ting CT, Chang CS, Chen GH. Atropineinduced HRV alteration is not amended by electroacupuncture on zusanli. Am J Chin Med ; 33(2) : Kim HK, Lee SH, Kim YS. Effects of sa-am acupuncture (Damjeonggyeok) on autonomic nervous system in night nurses. The Journal of Korean Acupuncture & Moxibustion Society ; 24(4) : Knardahl S, Elam M, Olausson B, Wallin BG. Sympathetic nerve activity after acupuncture in humans. Pain ; 75(1) :

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