大韓本草學會誌제 29 권제 5 호 (2014 년 9 월 ) Kor. J. Herbology 2014;29(5):55-63 ISSN 1229-1765(Print), ISSN 2288-7199(Online) http://dx.doi.org/10.6116/kjh.2014.29.5.55. 당귀수산 ( 當歸鬚散 ) 주정추출물이외상성창상 ( 外傷性創傷 ) 병태모델의회복능력에미치는영향 박지원 #, 심부용, 김동희 * 대전대학교한의과대학병리학교실 The effects of Danggwisusan on restoration ability in wound induced animal models Ji Won Bak #, Boo Yong Sim, Dong Hee Kim * Department of Pathology, College of Oriental Medicine, Daejeon University ABSTRACT Objectives : The purpose of this study was verification of the restoration ability effect of Danggwisusan extract(dg) in wound induced Rat. Methods : It needed to make a scar(around 2 2cm2 ) on the top of the fascia in the back of the rats and then the rats were divided into 4groups(n=6). Control was not treated at all, where as DG was orally medicated DG, Terra was percutaneously applied Terramycin, and DG + Terra was both orally medicated DG and percutaneously applied Terramycin per day for three weeks. Results : 93% or higher cell viability was observed in all tested groups from 1, 10, 100 μg / ml in RAW 264.7cells. The DG decreased NO and cytokine production activity dose dependently. The production of IL-1β, IL-6 and TNF-αwere decreased by 46%, 46% and 40% in DG treated 100 μg / ml. The size of wound was significantly decreasing in DG, Terra, DG + Terra. WBC was significantly reduced in DG and DG + Terra. Monocyte was significantly reduced in DG, Terra and DG + Terra. Neutrophil was also reduced in DG, DG + Terra but not meaningless. The mrna expression of MMP-1 was significantly reduced in Terra, MMP-2 was significantly reduced in DG, Terra, DG + Terra, and MMP-9 was significantly reduced in DG + Terra. Conclusions : According to the results, we thought that DG showed anti-inflammatory activities on the RAW 264.7cells in mouse macrophage and in adult rat wound. Moreover, the progress of recovery was found visually, heamatologically, genetically and histopathologically. In conclusion, it could be thought that DG has effect on the treating of wound. Key words : Anti-inflammatory, Cyotokine, Danggwisusan, Matrix metalloprotease(mmp), Wound 서론 1) 산업발달로인한환경적인변화와자연재해나외부적요인에의해창상및화상과같은피부질환이급증하고있다. 인체를구성하고있는피부는외부환경의여러유해한자극으로부터인체의내부장기를보호하고, 체내의수분이나전해질등의외부유출을박는장벽기능과체온조절및외부변화를감지하는감각기능을가진인체조직이다. 외부요인으 로부터우선적인방어기능을함으로써인체조직기관중가장먼저손상이되고, 이러한손상이부위가광범위하면생명유지에도큰영향을미친다 1,2). 한의학적인창상의치료법은初期, 成膿, 潰後의세단계로나누며, 치료법칙도이에따라消, 托, 補의세가지의기본법칙으로분류된다. 그중消法은병변의손상을초기에消散시키고, 托法은화농되어있지않거나부패한조직을조속히화농시켜消退하며, 補法은창상후기에국한적으로파궤된후 *Corresponding author : Dong Hee Kim. Department of Pathology, College of Oriental Medicine, Daejeon University Tel : +82-42-280-2623 FAX : +82-42-280-2624 E-mail : dhkim@dju.kr #First author : Ji Won Bak. Department of Pathology, College of Oriental Medicine, Daejeon University Tel : +82-42-280-2828 FAX : +82-42-280-2624 E-mail : jiwon3240@naver.com Received:18 August 2014 Revised:11 September 2014 Accepted:12 September 2014
56 大韓本草學會誌 Vol. 29 No. 5, 2014 장기간에걸쳐유합되지않는경우에사용한다고알려져있다 3). 또한창상치유에관한연구로이 4) 의고압산소, 김 5) 의맥동초음파와냉적용, 한 6) 의스테로이드와비스테로이드소염진통제, 유 7) 의연고제등을이용한다양한치유방법과효과에대한보고는많으나, 한의학적인효과에대한연구결과는미흡한편이다. 當歸鬚散은明代에저술된李 8) 의 醫學入門 에처음수록된처방으로打撲損傷, 氣血凝結, 胸腹脇痛을치료한다고알려져있고, 이처방을구성하는약재들은각기破瘀血, 行血破瘀, 理氣, 淸熱凉血, 活血化瘀, 通血脈하는약효를가진약물들로조성되어있다 8-10). 전반적인藥性은平溫無毒하고, 味는辛苦多甘鹹少하고, 歸經은血과관계가깊은肝, 心, 脾등에편중되어있으며活血祛瘀, 通經活絡, 理氣止痛등의효능이있어瘀血로인한腫痛을치료한다고알려져있다 11-13). 이에當歸鬚散이조직간의出血과관련된병증및瘀血性腫痛의효능이입증되어, 나아가外傷性創傷治癒에대한효과를객관적으로입증하고자실험에임하였다. 이에저자는當歸鬚散이外傷으로인한창상에서의한의학적인실험적연구결과에대한보고가미흡하여, in vitro 실험으로 lipopolysaccharide(lps) 로유도된 Raw 264.7에서當歸鬚散의항염증효능을관찰하고, in vivo 실험으로외상성창상을유발시킨병태모델에자연치유군, 당귀수산투여군, 테라마이신도포군, 당귀수산과테라마이신동시처치군으로실험군을나눠항염증, 창상의면적, 혈액학적검사, 유전자발현정도및조직학적검사를통하여외상성창상에대한당귀수산의효능을검증하고자한다. 1. 약재 재료및방법 본실험에사용한당귀수산 (Danggwisusan 이하 DG로표기 ) 의구성약재들은 옴니허브에서구입하였고, 대전대학교 TBRC-RIC에서정선후사용하였다. 그내용과분량 (1첩) 은다음과같다 (Table 1). Table 1. The Prescription of DG Herbal medicine name Pharmacognostic name Weight(g) 2주간의안정기를가지면서순화를시켰으며, 희생당일까지일반고형사료 ( 퓨리나, Korea) 를공급하고자유식이하면서물을충분히공급하였다. 동물사육실의조건은 conventional system으로 22 ± 2, 1일중 12시간은 200-300 Lux로조명하고, 12시간은모든빛을차단하였다. 본실험은대전대동물실험윤리위원회의승인 ( 동물사용윤리위원회승인번호 DJUARB 2013-027) 을받아동물윤리준칙에의거하여실험하였다. 일반사료의kg당조성내용과분량은다음과같다 (Table 2). Table 2. The Components of Normal Diet Components Percentage(%) Crude protein 20.0 Crude fat 4.5 Crude fiber 6.0 Crude calcium oxide 7.0 Calcium 0.5 Phosphorus 1.0 Total amount 39.0 3. 시약및기기사용된시약은 dulbecco's modified eagle's medium (DMEM : Gibco BRL Co., U.S.A.), 우태아혈청 (fetal bovine serum: FBS, Invitrogen Co., U.S.A.), lipopolysaccharide (LPS : Sigma Co., U.S.A.), cell viability assay kit (Daeillab sevice, Korea), nitric oxide detection kit (Intron Biotechnology, Korea), penicillin (Hyclone, Co., U.S.A.), mouse cytokine milliplex map immunoassay kit (Millipore Co., U.S.A.), total RNA prep kit (Intronbio., Korea), terramycin(pt. Pfizer., Indonesis) 을사용하였으며, 기기는 rotary vacuum evaporator (Büchi B-480 Co., Switzerland), freeze dryer (EYELA FDU-540 Co., Japan), ELISA reader (Molecular Devices Co., U.S.A.), Luminex (Millipore Co., U.S.A.), PCR machine (Thermal Dynamic., U.S.A), Digimatic Caliper (Mitutoyo, Japan), 주정추출에사용된주정은 주정판매월드 (Jeonju, Korea) 에서구입하여사용하였다. 當歸尾 Angelicae Radix 6 蘇木 Caesalpiniae Lignum 4 烏藥 Linderae Radix 4 赤芍藥 Paeoniae Radix Rubra 4 香附子 Cyperi Rhizoma 4 桃仁 Persicae Semen 3 紅花 Carthami Flos 3 甘草 Glycyrrhizae Radix et Rhizoma 2 桂枝 Cinnamomi Ramulus 2 4. 시료추출 DG 2첩에 80% 주정 (C 2H 5OH) 1000 ml을넣고 3시간동안환류추출후여과액을얻어 rotary vacuum evaporator 에서감압농축하였다. 농축된용액을 freeze dryer로동결건조하여분말 20.09 g을얻었으며, 얻어진분말은초저온냉동고 (-80 ) 에서보관하면서실험에따라필요한농도로증류수에희석하여사용하였다. Total amount 32 5. 세포독성측정 2. 동물및사료 실험에사용된랫은수컷 6주령의 Wistar Rat (170-200g) 을 라온바이오 (Korea) 사에서구입하여실험하였다. 동물은 Raw 264.7 세포는 96 well plate에 2 10 4 cells/well로분주하여 24시간동안배양하였다. 새로운배양액으로교체하였고, DG를각각 1, 10, 100 ( μg / ml ) 의농도로처리하여다시
당귀수산 ( 當歸鬚散 ) 주정추출물이외상성창상 ( 外傷性創傷 ) 병태모델의회복능력에미치는영향 57 24시간동안배양하였다. 배양후 10 μl의 WST solution 을첨가하여세포배양기 (37, 5% CO 2) 에서 30분간반응시켰다. 반응후 450 nm에서 ELISA reader기를이용해흡광도의변화를측정한후대조군에대한세포생존율을백분율로표시하였다. 6. 항염증효능측정 1) Nitric oxide(no) 측정 NO의농도는배양액내의 nitric oxide 농도를 griess reagent system을이용하여측정하였다. Raw 264.7 세포를 96 well plate에 2 10 4 cells/well 로분주하여 24시간동안배양한후새로운배양액으로교체하였고 DG를각각 1, 10, 100 ( μg / ml ) 의농도와 LPS 1 μg / ml의농도를처리하여다시 24시간동안배양하였다. N1 buffer 와 N2 buffer 를각각 10분간반응후 540 nm에서흡광도를측정하였다. Nitrite standard 의농도별표준곡선을이용하여배양액의 NO 농도를결정하였다. 2) Cytokine 생성량측정 Raw 264.7 cells을 12 well plates에 2 10 5 cells/ ml이되도록분주하고, 24시간동안배양한후, DG를 1, 10, 100 ( μg / ml ) 의농도로처리하고, LPS 1 μg / ml을처리하였다. 24 시간동안배양한후세포배양액을수거하여배양액에함유된 IL-1β, IL-6, TNF-α를 custom-made 4-plex cytokine Milliplex panel을이용하여측정하였다. 7. 창상유발및실험군분류마취제 ( 졸레틸 0.5 ml + 럼푼 0.1ml ) 을이용하여 Rat를마취한후, 배부 ( 背部 ) 의피부를깨끗이제모한다. 제모된피부를알콜로깨끗이소독한후, 척추를기준으로고관절정중앙에 2 2 cm2의정사각형크기로작도한다. 수술용가위를이용하여피부를근막위까지제거하여창상을유발하였다. 실험군은자연치유를한 Control 군, DG만투여한 DG군, 테라마이신 (Oxytetracycline HCL 5mg + Polymaxin B Sulfate 10000 I.U) 을처치한 Terra군, DG를투여하고테라마이신을함께처치한 DG + Terra군으로총 4그룹으로나누고 (n=6), 각케이지에한마리씩수용하였다. 9. 혈액내사이토카인및면역세포측정실험종료후 ether 로마취한상태에서심장천자법을이용하여채혈하였다. 전혈중일부는서울의과학연구소에분석의뢰하여총백혈구, 백혈구중호중구, 단핵구, 림프구를측정하였고, 나머지전혈은실온에서 30분간굳힌뒤 3,000 rpm에서 15분간원심분리하여혈청을분리하였다. 분리된혈청을가지고 IL-1β, IL-6, TNF-α 는 custom-made 6-plex cytokine Milliplex panel 을이용하여 Luminex로측정하였다. 10. RNA 분리및 RT-PCR 분석쥐의배부창상피부조직에서발현된창상인자를측정하기위해 RT-PCR 을시행하였다. 분리한피부조직을동결시킨뒤조직을파쇄하여 total RNA prep kit을이용하여 RNA를추출하였다. 역전사반응은추출한 RNA를 RT premix kit의 mixture를사용하여 first-strand cdna를합성하였으며, M-MLV RT를불활성화시킨후합성이완료된 cdna를사용하였다. RT-PCR 은 DNA polymerase 1U/tube 에 250 mm dntps mix, RT buffer (10 mm Tris-HCl, ph 9.0, 30 mm KCl, 1.5 mm MHCl 2) 를포함한 mixture에각샘플과 primer 를넣고 PCR을시행하였다. MMP-1은 55 에서 2분, 95 에서 10분, 60 에서 15초, MMP-2은 61 에서 2분, 95 에서 10분, 60 에서 15초, MMP-9은 57 에서 2분, 95 에서 10분, 60 에서 15초, GAPDH는 57 에서 2분, 95 에서 10분, 60 에서 15초동안각 30 cycles 조건으로 PCR을수행하였으며, 사용된 primer 는아래와같다 (Table 3). 1% agarose gel에전기영동후유전자발현의여부를 UV로촬영하여각그룹별로 band를확인하고, RNA 발현을나타내었다. Table 3. The sequences of primers in used this study. Primer F/R Sequences F GAG ACG TGG ACC GAC AAC AG MMP-1 R AGG CCC ATA TAA AGC CTG GA F TGG CGA GTA CTG CAA GTT CC MMP-2 R GTA AGA GGT GCC CTG GAA GC F CCT GCA GTG CCC TTG AAC TA MMP-9 R TCC AAC AAG AAA GGA CAG CG F GTT ACC AGG GCT GCC TTC TC GAPDH R CAC CCC ATT TGA TGT TAG CG 8. 약물처치및창상면적측정창상을유발한후, 다음날부터모든군은 3주간자유식이를하였고, DG군과 DG + Terra군은 DG를 3주간매일 1회 2ml (200mg/ kg ) 씩오전 10시에경구투여하였다. Terra군과 DG + Terra 군에는테라마이신을창상유발후 1주동안은 2일주기, 유발후 2주, 3주동안은 3일주기로창상부위에충분히도포하였다. DG투여량은 1첩을성인체중 60kg에 1회투여용량으로하고,1첩으로부터얻은시료를마우스체중 30g으로기준하여산출하였다. 창상면적은유발후 1일, 4일, 8일, 11일, 15일, 19일, 22일총 3주간 Digimatic Caliper를이용하여측정평균산출하였다. 11. 조직처리및 H&E 염색실험종료후, 각실험군별로적출한창상피부조직을 10% 중성포르말린에 48시간고정하여고정이완료된조직들을흐르는수돗물에서 12시간수세한뒤조직내고정액을완전제거하였다. 조직의탈수를위해 60% 에서부터 100% 알코올에이르기까지농도상승순으로탈수하고, xylene에투명과정을거친다음파라핀블럭을제작하였다. 제작된블럭은박절기 (microtome) 를이용해 3 4 μm두께로절편을만들어탈파라핀및함수과정을거친다음 hematoxyline 과 eosin (H&E) 염색을실시하여광학현미경상에서관찰및사진촬영하였다.
58 大韓本草學會誌 Vol. 29 No. 5, 2014 12. 통계처리실험결과는실험결과는 SPSS 11.0 의 unpaired student's T-test 및 ANOVA test를사용하여통계처리하였으며 p < 0.05, p < 0.01 및 p < 0.001 수준에서유의성을검정하였다. 결과 1. 세포독성 RAW 264.7 세포의세포생존율을측정한결과, 대조군을 100.0 ± 1.0% 로나타냈을때, DG 투여군 1, 10, 100 ( μg / ml ) 농도에서 105.5 ± 3.1%, 98.4 ± 3.5%, 93.4 ± 2.1% 의세포생존율을나타내었다 (Fig. 1). Fig. 3. Effects of DG extract on LPS-stimulated IL-1βproduction in Raw 264.7 cells. Cells were treated with LPS (1 μg / ml ) and 1, 10, 100 ( μg / ml ) of DG for 24 hours. The results are expressed as mean ± S.D (Significance of results, * p < 0.05, ** p < 0.01, *** p < 0.001). 3) IL-6 생성에미치는영향 DG의 IL-6 생성량을측정한결과, 대조군이 8779.2 ± 651.1 pg/ ml, 정상군이 2.8±1.6 pg/ ml, DG 투여군은 1, 10, 100 ( μg / ml ) 농도에서 3626.2 ± 382.3 pg/ ml, 4851.0 ± 610.4 pg/ ml, 4727.7 ± 449.7 pg/ ml로나타나, 대조군에비해모든농도에서농도의존적이며유의성 ( ** : p < 0.01, *** : p < 0.001) 있는감소를나타내었다 (Fig. 4). Fig. 1. Cell viability of DG extract in Raw 264.7 cells. The results were expressed as mean ± S.D. 2. 항염증효능에미치는영향 1) Total Nitric oxide(no) 생성에미치는영향 DG 투여군의 NO 생성량은대조군을 100 ± 2.7% 로나타냈을때, 정상군은 36.8 ± 2.6%, DG 투여군은 1, 10, 100 ( μg / ml ) 농도에서 95.7 ± 1.2%, 93.7 ± 1.1%, 86.1 ± 2.5% 로나타나, 농도의존적인감소와 100 μg / ml농도에서유의성있는 ( ** p < 0.01) 감소를나타내었다 (Fig. 2). Fig. 4. Effects of DG extract on LPS-stimulated IL-6 production in Raw 264.7 cells. Cells were treated with LPS (1 μg / ml ) and 1, 10, 100 ( μg / ml ) of DG for 24 hours. The results are expressed as mean ± S.D(Significance of results, ** p < 0.01, *** p < 0.001). 4) TNF-α 생성에미치는영향 DG의 TNF-α생성량을측정한결과, 대조군이 1686.0 ± 198.4 pg/ ml, 정상군이 251.6±33.7 pg/ ml, DG 투여군은 1, 10, 100 ( μg / ml ) 농도에서 1375.1 ± 88.6 pg/ ml, 1341.7 ± 92.2 pg/ ml, 1004.5 ± 127.8 pg/ ml로나타나, 대조군에비해모든농도에서농도의존적이며유의성 ( * : p < 0.05, ** : p < 0.01) 있는감소를나타내었다 (Fig. 5). Fig. 2. Effects of DG extract on NO production in LPS-stimulated Raw 264.7 cells. Cells were treated with LPS (1 μg / ml ) and 1, 10, 100 ( μg / ml ) of DG for 24 hours. The results are expressed as mean ± S.D (Significance of results, ** p < 0.01). 2) IL-1β생성에미치는영향 DG의 IL-1β생성량을측정한결과, 대조군이 36.9 ± 2.1 pg/ ml, 정상군이 13.2 ± 1.2 pg/ ml, DG 투여군은 1, 10, 100 ( μg / ml ) 농도에서 30.2 ± 2.2 pg/ ml, 22.0 ± 2.4 pg/ ml, 19.6 ± 1.6 pg/ ml로나타나, 대조군에비해모든농도에서농도의존적이며유의성 ( * p < 0.05, ** : p < 0.01, *** : p < 0.001) 있는감소를나타내었다 (Fig. 3). Fig. 5. Effects of DG extract on LPS-stimulated TNF-αproduction in Raw 264.7 cells. Cells were treated with LPS (1 μg / ml ) and 1, 10, 100 ( μg / ml ) of DG for 24 hours. The results are expressed as mean ± S.D (Significance of results, * p < 0.05, ** p < 0.01).
당귀수산 ( 當歸鬚散 ) 주정추출물이외상성창상 ( 外傷性創傷 ) 병태모델의회복능력에미치는영향 59 3. 창상치유에미치는영향실험종료후창상면적을측정한결과, Control 군은 80%, DG 투여군은 93%, Terra 투여군은 92%, DG + Terra 투여군은 94% 의감소를나타내었고, DG, Terra, DG + Terra 투여군에서각각 10일, 14일, 17일, 21일째유의성있는 ( * p < 0.05, ** p < 0.01, *** p < 0.001) 감소를나타내었다 (Table 3, Fig. 6-A, B). Table 4. The change of wound size in DG extract and terramycin groups. day Control DG Terra DG + Terra 1 100.0 ± 30.5 100.0 ± 22.5 100.0 ± 20.6 100.0 ± 23.2 4 96.2 ± 21.5 95.3 ± 22.5 93.7 ± 23.0 96.2 ± 25.2 7 82.8 ± 17.9 69.6 ± 22.1 74.5 ± 19.3 63.2 ± 17.0 10 52.3 ± 5.9 37.9 ± 7.2 ** 38.7 ± 8.3 * 32.9 ± 13.5 * 14 36.7 ± 2.1 14.1 ± 3.6 *** 14.9 ± 4.7 *** 13.7 ± 6.8 *** 17 24.3 ± 3.0 9.3 ± 2.3 *** 10.4 ± 3.2 *** 9.9 ± 4.9 *** 21 19.8 ± 7.0 6.7 ± 1.4 ** 8.3 ± 1.8 ** 6.2 ± 2.9 ** Fig. 6. Effects of DG extract and terramycin on levels of IL-1βin the blood of wound-induced Rats. The results are expressed as mean ± S.D.(n=6). (Significance of results, * p < 0.05). 2) IL-6 생성에미치는영향혈액내 IL-6 생성량을측정한결과, Control군이 916.3 ± 137.7 pg/ ml, DG군이 871.7 ± 67.2 pg/ ml, Terra군이 812.2 ± 69.1 pg/ ml, DG + Terra군이 805.5 ± 61.0 pg/ ml로나타났고, Terra군과 DG + Terra군에서유의성 ( * : p < 0.05) 있는감소를나타내었다 (Fig. 8). Fig. 8. Effects of DG extract and terramycin on levels of IL-6 in the blood of wound-induced Rats. The results are expressed as mean ± S.D.(n=6). (Significance of results, * p < 0.05). 3) TNF-α생성에미치는영향혈액내 TNF-α생성량을측정한결과, Control군이 66.4 ± 11.1 pg/ ml, DG군이 50.4 ± 9.6 pg/ ml, Terra군이 47.3 ± 6.7 pg/ ml, DG + Terra군이 45.2 ± 6.1 pg/ ml로나타났고, 모든군에서에서유의성 ( ** : p < 0.01, *** : p < 0.001) 있는감소를나타내었다 (Fig. 9). Fig. 6-A, B. The change of wound size in DG extract and terramycin groups. The results were expressed as mean ± S.D.(n=6). (Significance of results, * p < 0.05, ** p < 0.01, *** p < 0.001). 4. 혈액내사이토카인및면역세포에미치는 영향 1) IL-1β 생성에미치는영향 혈액내 IL-1β생성량을측정한결과, Control군이 106.0 ± 9.7 pg/ ml, DG군이 89.1 ± 9.5 pg/ ml, Terra군이 85.3 ± 9.8 pg/ ml, DG + Terra군이 83.7 ± 9.9 pg/ ml로나타났고, Terra군과 DG + Terra군에서유의성 ( * : p < 0.05) 있는감소를나타내었다 (Fig. 7). Fig. 9. Effects of DG extract and terramycin on levels of TNF-αin the blood of wound-induced Rats. The results are expressed as mean ± S.D.(n=6). (Significance of results, ** : p < 0.01, *** : p < 0.001). 4) 총백혈구생성에미치는영향혈액내의총백혈구생성량은 Control 군이 11.78 ± 1.29 Thous/uL, DG군이 5.98 ± 1.85 Thous/uL, Terra 군이 9.73 ± 2.15 Thous/uL, DG + Terra군이 6.13 ± 1.32 Thous/uL 로나타나, DG군과 DG + Terra군에서유의성있는 ( ** p <
60 大韓本草學會誌 Vol. 29 No. 5, 2014 0.01) 감소를나타내었다 (Fig. 10). Fig. 10. Effects of DG extract and terramycin on levels of WBC in the blood of wound-induced Rats. The results are expressed as mean ± S.D.(n=6). (Significance of results, ** : p < 0.01). 5) 호중구생성에미치는영향혈액내백혈구에대한호중구의비율은 Control 군이 2.80 ± 1.01%, DG군이 2.28 ± 0.55%, Terra군이 2.97 ± 0.19%, DG + Terra 군이 1.87 ± 0.46 % 로나타나, DG군과 DG + Terra군에서는감소를나타내었으나유의성은없었다 (Fig. 11). Fig. 13. Effects of DG extract and terramycin on levels of monocyte in the blood of wound-induced Rats. The results are expressed as mean±s.d.(n=6). (Significance of results, * : p < 0.05). 5. 유전자발현에미치는영향 창상피부조직에서의 MMP-1, 2, 9 발현양을측정한결과, DG, Terra, DG + Terra 모든군이 Control 군에비해모두감소를나타내었고, MMP-1에서는 Terra군, MMP-2 에서는 DG, Terra, DG + Terra군, MMP-9에서는 DG + Terra군에서유의성 ( * p < 0.05, ** p < 0.01) 있는감소를나타내었다 (Fig. 14). Fig. 11. Effects of DG extract and terramycin on levels of neutrophi in the blood of wound-induced Rats. The results are expressed as mean ± S.D.(n=6). 6) 림프구생성에미치는영향혈액내백혈구에대한림프구의비율은 Control 군이 90.20 ± 8.72%, DG군이 95.43 ± 1.24%, Terra군이 93.38 ± 3.20%, DG + Terra군이 97.77 ± 0.77 % 로나타나, 모든군에서큰차이를보이지는않았다 (Fig. 12). Fig. 14. Effects of DG extract and terramycin MMP-1, 2, 9 gene expression in wound tissue. The results are expressed as mean ± S.D.(n=6). (Significance of results, * : p < 0.05, ** : p < 0.01). 6. 조직병리학적관찰 Fig. 12. Effects of DG extract and terramycin on levels of lymphocyte in the blood of wound-induced Rats. The results are expressed as mean ± S.D.(n=6). 7) 단핵구생성에미치는영향혈액내백혈구에대한단핵구의비율은 Control 군이 1.45 ± 0.61%, DG군이 0.30 ± 0.14%, Terra군이 0.30 ± 0.19%, DG + Terra 군이 0.25 ± 0.11 % 로나타나, 모든군에서유의성있는 ( * p < 0.05) 감소를나타내었다 (Fig. 13). 창상피부조직을 Hematoxylin & Eosin 염색하여관찰한결과, Control 군에서는콜라겐의증착과피부의재상피화가두텁게일어났으며, 만성염증이나타났다. DG, Terra, DG + Terra 모든투여군에서콜라겐의증착과만성염증이감소함을나타내었고, 피부의재상피화역시 DG, Terra, DG + Terra 투여군모두에서감소하고, 색이옅어지며, 혈관의재형성이나타났다 (Fig. 15).
당귀수산 ( 當歸鬚散 ) 주정추출물이외상성창상 ( 外傷性創傷 ) 병태모델의회복능력에미치는영향 61 로운교원질과기질단백질을형성하여육아조직이만들어지는데, 이때 collagen, proteoglycan, elastin이포함된기질을형성하게된다 20,21). 또한혈관확장이혈관신생에필수적이며, 창상치유초기에는혈관투과성이증가하는데산화질소 (NO) 는혈관신생및염증반응에관여함으로써긍정적인역할을한다 15). NO는인체에서다양한생리활성을나타내는인자이기도 Fig. 15. Histopathological findings of wound tissue section on DG extract and terramycin treated in wound-induced Rats. 고찰 창상은외부의물리적요인에의해조직이손상을받아세포학적또는해부학적연속성이파괴된상태이며, 炎症期, 上皮化期, 增殖期, 成熟期크게네단계의치유과정으로구분하나명확한시기와순서가정해져있지않고서로중첩되어연속적으로진행된다. 창상치유는외상에대한인체내조직반응을수반하는복구과정이며, 재상피화, 창상수축, 유아조직형성및교원질합성등여러과정을지나조직을회복시키는복잡한생물학적과정이다. 이러한과정은동시에이루어지며, 반흔을형성하거나, 조직의재생을유도하여창상면적이감소된다 14,15). 창상치유과정은콜라겐및비콜라겐성물질들의침착뿐아니라 matrix metalloproteinases(mmps) 와 tissue inhibitors of metalloproteinases(timps) 에의한세포외기질 (extracellular matrix) 의개형이필요하다 1,16). 이러한 4단계중중요한시기를염증기와증식기로볼수있으며, 염증단계에는호중구및대식구, 증식단계에는섬유아세포가중요한역할을하는데, 섬유아세포는 collagen tenascin, fibronectin 및많은 proteoglycans 를분비하며육아조직을형성하게된다. 이때창상기질은섬유아세포및염증세포에서분비되는 MMPs에의해파괴된다. 그리고개형단계에이르러 MMPs와 TIMPs에의해세포양과혈류양이감소하면서치유가완성된다 17,18). 본실험에앞서 DG 시료의안전성을확인하고자마우스유래대식세포주인 Raw 264.7 세포를대상으로하여시료의독성을확인한바안전성이확인되었다. 염증반응은조직손상에대해반응하는능동적인과정으로생체의조직이나세포가어떤원인에의해손상을받게되면이에대한반응을함으로써손상된부위를극소화시키고복구시키려는국소적반응이다 1). 염증기의초기와후기에는호중구 (netrophil) 와단핵구 (monocyte) 가많이나타나는데, 호중구는세균과상처내손상된기질을탐식하여죽은조직을제거하는역할 (debridement) 을담당하고. 단핵구는조직에서대식세포로바뀌고이런대식세포는여러성장인자들을분해하여혈관신생과육아조직생성을이끌어염증단계에서재생으로넘어가는아주중요한가교역할을한다 19). 증식기에는피부의상피가다시만들어지면서신생혈관이형성되고섬유형성으로손상된피부조직이재생된다. 섬유모세포는상처부위로이동하며증식하면서새 하지만과다생산되면활성산소와결합하여, 과산화질소등의다른독성유리기물질을만들어내어염증질환을유발시키거나악화시키는인자로작용한다 22). 그람-음성균의세포외막에존재하는내독소중의하나인 lipopolysaccharide(lps) 는 Raw 264.7과같은 macrophage 와 monocyte에서 interleukin-1β(il-1β), interleukin-6 (IL-6), tumor necrosis factor-α(tnf-α) 와같은염증성 cytokine 의분비를촉진하며 inos의발현을유도하여 NO를생성한다고알려져있다 22,23). IL-1β는외부항원을감지한대식세포에서분비하는비특이염증반응을개시하는초기면역반응물질로써 LPS나 TNF-α와같은다른사이토카인에의해유도가되며, 대식세포, 호중구등에의해생산이되는염증유발사이토카인이다. IL-6는염증반응시분비되는친염증성사이토카인으로단핵구나대식세포에서분비되며림프구를활성화시켜항체생산을증가시키는것으로, 염증성병변에서항상증가하는것으로보고되고있으며, TNF-α는 T cell과대식세포를활성화하고다른 pro-inflammatory cytokine 들을증가시켜서염증반응을일으키는데중요한역할을한다. 주로활성화된대식세포에서대량생산되지만, 림포이드계의세포 (lymphoid cells), 비만세포 (mast cells), 내피세포 (endothelial cells) 을비롯하여생체에존재하는다양한세포에서도생산된다. 또한, 면역세포들의집합과면역매개물질들의생산을자극하는데중요한역할을담당하며, 인체내의미생물들을포식하는대식세포의능력을증진시키고육아종 (granuloma) 을형성하는등, 숙주의방어기전에있어서도핵심적인역할을수행한다 23-25). 이에 DG가 in vitro 실험에서 LPS로유도한 Raw 264.7 세포에서의 NO, IL-1β, IL-6, TNF-α 생성에미치는영향과 in vivo 실험에서혈청에서의 IL-1β, IL-6, TNF-α 생성에미치는영향을측정한결과, 모두농도의존적인감소와더불어유의성 ( * p < 0.05, ** p < 0.01, *** p < 0.001) 있는감소를나타내어항염증에효능이있음을나타내었다. 앞선연구에따르면전층창상이치유되는과정은기전상으로창상수축과창상변연의기저세포가창상부로이동하는창상상피화를통해치유되므로, 전체적인창상치유의정도는창상수출율과창상상피화율의합으로수식화하여나타냄으로효능을입을할수있다고알려져있다 26). 본연구에서도 DG, Terra, DG + Terra 투여군에서 10일이후부터유의성 ( ** p < 0.01, *** p < 0.001) 있는면적감소효과를나타내었다. 기질금속단백분해효소 (matrix metalloproteinases, MMP) 는각특이성에따라 20여종으로분류되고, 그특성은세포외기질의여러가지단백질을분해하며, 기질금속단백분해효소조직억제제 (tissue-darived inhibitor of MMPs, TIMP) 에의해억제된다. MMP는조직회복, 혈관생성, 조직의발생과정에서형태를형성하는데중요하며, 섬유모세포, 각질세포,
62 大韓本草學會誌 Vol. 29 No. 5, 2014 호중구성백혈구, 대식세포에서분비가된다. 창상치유의과정중그활성이조절받게되면조절기전이파괴되어만성창상등의병적인상태가지속되며, 특히켈로이드와비후성반흔에서증가된콜라겐축적은 MMP-1, 9 mrna와밀접한연관이있다고보고되었다 27,28). 선행연구에따르면급성창상기에는 MMP-1, 2, 9 mrna가급격히증가하고, 창상회복기나육아형성및재형성단계에는점차줄어든다고하였고, 만성창상에서는 MMP-2, 9 mrna가지속적으로높은농도로나타나며, 이는육아조직형성부족을야기한다고보고하였다 29,30). 본실험에서 MMP-1, 2, 9 mrna의발현양은자연치유군에비해 DG, Terra, DG + Terra 투여군모두감소을나타내어, 선행연구와비교, 부합되는결과가나타나, 창상치유와육아조직의재형성이잘이루어지고있다고추측할수있다. 창상피부조직을 Hematoxylin & Eosin 염색하여관찰한결과, control 군에서는콜라겐의증착과피부의재상피화가두텁게일어나고만성염증이생겼으나, DG, Terra, DG + Terra 투여군모두콜라겐의증착과만성염증의감소를나타냈었고, 또한피부의재상피화가감소하며, 색이옅어졌다. 이는 DG가콜라겐의증착과만성염증및피부재상피화를감소시켜병리조직학적으로창상치유에도움이된다고판단된다. 결론 본연구는 DG의외상성창상의회복능력에미치는효능을객관적으로검증하고자실험을통하여다음과같은결론을얻었다. 1. 세포독성평가에서는 93% 이상의세포생존율을나타내어안정성이확인되었다. 2. DG의항염증효능은 in vitro 실험에서 NO 생성저해능과 IL-1β, IL-6, TNF-α 소거능, 그리고 in vivo 실험에서혈청내의 IL-1β, IL-6, TNF-α 소거능모두농도의존적인감소와유의적인결과가도출되었다. 3. 창상면적은자연치유군에비해 DG, Terra, DG + Terra 투여군에서 10일, 14일, 17일, 21일째유의적인감소결과가도출되었다. 4. 혈액검사에서총백혈구는 DG, DG + Terra 투여군에서, 단핵구는 DG, Terra, DG + Terra 투여군에서유의적인감소결과를나타내었고, 호중구에서는 DG, DG + Terra 투여군에서감소하였지만유의성은없는결과를도출하였다. 5. MMP-1, 2, 9 mrna 발현에서는 DG, Terra, DG + Terra 투여군모두감소를나타내었고, MMP-1에서는 DG 투여군, MMP-2에서는 DG, Terra, DG + Terra 투여군, MMP-9에서는 DG + Terra 투여군에서유의적인결과가도출되었다. 6. 병리조직학적검사상 DG, Terra, DG + Terra 투여군모두콜라겐의증착과만성염증의감소를나타냈었고, 또한피부의재상피화가감소하며, 색이옅어지는결과를도출하였다. 이상의외상성창상에대한실험으로, DG, Terra, DG + Terra 투여군이 in vitro, in vivo에서단독치료보다는병행치료시유의한결과가도출되었음을실험적으로입증되었으며, 향후많은피부병증에대한한방과양방의병행치료에대하여심도깊은연구가필요하다고사료된다. 감사의글 본연구는산업통상자원부지정대전대학교난치성면역질환의동서생명의학연구지역혁신센터의지원에의한것입니다. References 1. Lee YG. Effect of Astragali Radix on Dermoepidermal Recovery to Wound Tissues in Rats' Skin. Dongguk University. 2004. 2. Chang JH, Hwang SH, Lee EJ, Nobuaki O, Lee JH. Effect of Panax ginseng C.A. Meyer Extract (Ginseol K-b1) on UVB-induced Skin Damage in Hairless Mice. East Asian Soc Dietary Life. 2012 ; 22(2) : 224-30. 3. Liu ZY. wai ek pi fu ke de bian zheng lun zhi. Seo won dang. 1987 : 35 4. Lee YH, Kim WS. Morphological Observation on the Effects of Hyperbaric Oxygenation to the Cutaneous Wound Healing. Chungnam Med J. 1991 ; 18(1) : 89-97. 5. Kim TY, Jo NJ, Jeong HS, Kim MS. The Effects of Pulsed Ultrasound Treatment and Cold Therapy in the Process of Acute Wound Healing. J Korean Acad Clin Electrophysiol. 2007 ; 5(2) : 23-33. 6. Han CD, Lee WM, Lee HB, Hahn MH, Yu WW. Effects of Steroid and Nonsteroidal Anti-inflammatory Drugs on Acute Wound Healing and Collagen Synthesis in Rat Skin. J Korean Orthop Assoc. 2003 ; 38(4) : 393-9 7. Yoo YC, Yoo SG. The effects of compound madecassol on the wound healing. Arch Plast Surg. 1998 ; 25(8) : 1451-58. 8. Li C. Ui hak ib mun. Bub in moon hwa sa. 2009. 9. Heo Jun. Dong ui bo gam. Bub in moon hwa sa. 2012 : 1046-47, 1052. 10. Herbal medicine. Seoul : Younglimsa. 2007. 154-155, 236-38, 396-99, 462-64, 476-77, 583-85, 629-631. 11. Lee GH. Study on the Effects of Dangguixusan( 當歸鬚散 ) on Experimental Blood Stasis Model Induced
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