pissn 2287-3368 / eissn 2287-3376 ORIGINAL ARTICLE Korean Journal of Acupuncture Vol.30, No.4, pp.243-251, 2013 http://dx.doi.org/10.14406/acu.2013.039 동의대학교한의과대학경락경혈학교실 Anti-Inflammatory Effect of Bower Actinidia in LPS-Stimulated RAW264.7 Cells Young-Jun Kim, Choon-Ho Song Department of Meridian & Acupoint, College of Oriental Medicine, Dong-Eui University Objectives : Bower Actinidia has been widely used for treatment of inflammatory diseases, such as jaundice, cystolithiasis. However, the mechanism of its anti-inflammatory activity has not been clarified. In this study, we investigated the inhibitory effect of Bower Actinidia pharmacopuncture extract(ba) on LPS-induced inflammation. Methods : The effect of BA was analyzed by ELISA, RT-PCR and Western blotting in LPS-stimulated RAW264.7 cells. Results : We found that BA suppressed not only the mrna expression of pre-inflammatory cytokines, cyclooxygenase-2(cox-2) and inducible nitric oxide synthase(inos), but also the phosphorylation of ERK, JNK and p38. Conclusions : These results suggest that BA exerts an anti-inflammatory effect through the regulation of the mitogen-activated protein kinase(mapk) pathway, thereby decreasing production of pre-inflammatory cytokines. Key words : Bower Actinidia, pharmacopuncture, inflammation, MAPK, RAW264.7 cells 서론 염증이란생체에해로운자극에대한방어반응으로생체의세포와조직이외부에서자극을받을때그영향을국소화하여손상된부위를정상으로회복하여유지하려는생체의정상적인방어기전으로, 주된증상은통증, 발열, 발적, 종창, 기능상실등이나타난다 1). Lipopolysaccharide(LPS) 는그람음성세균외막의주성분으로단핵구, 대식세포, 수지상세포, 비만세포및장상피세포의표면분자와반응하게되면 2), interleukin-1β(il-1β) 와 tumor necrosis factor-α(tnf-α) 등의전염증성 cytokine(pro-inflammatory cytokine) 의발현이유도되고, nitric oxide(no), PGE 2 등의물질을 생성하게하여염증반응에관여하게된다 3). 서양의학에서는급ㆍ만성염증질환에진통, 해열, 항염증효과가있는비스테로이드소염제 (Non-steroidal anti-inflammatory drug, NSAID) 를전세계적으로흔히사용하고있다 4). NSAID 는 COX (cyclooxygenase) 효소를차단함으로써프로스타글란딘 (prostaglandin) 의합성을감소시켜염증, 통증, 발열과연관된다양한종류의질환에흔히사용되는약제이지만광범위한사용과함께약물에의한부작용도지속적으로증가하는추세에있는데 5), 위장관계부작용, 이명, 부종등다양한부작용을유발하기에 6) 최근에는한약재를포함한다양한식물로부터낮은독성과높은항염증작용이있는새로운약물을찾기위한연구가많이이루어지고있는추세 Received November 19, 2013, Revised December 11, 2013, Accepted December 14, 2013 Corresponding author: Choon-Ho Song Deptartment of Meridian & Acupoint, College of Oriental Medicine, Dong-Eui University, San 45, Yangjeong-dong, Busan 614-052, Korea Tel: +82-51-850-8643, Fax: +82-51-853-4036, E-mail: chsong@deu.ac.kr This work was supported by Dong-Eui University Foundation Grant(No. 2013AA098). CC 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. Copyright 2013, SOCIETY FOR MERIDIAN AND ACUPOINT 243
이다 3). 獼猴藤은다래과식물미후도 (Actinidia chinensis Planch.) 의덩굴및덩굴즙액으로, 性은寒, 無毒하고, 味는甘하다. 獼猴藤은和胃開胃, 淸熱利濕하는효능이있어消化不良, 反胃, 嘔吐, 黃疸, 石淋등을主治한다 7). 약침요법은침구요법과약물요법을결합한新鍼療法의일종으로, 치료경혈및체표반응점에특정한약에서정제추출한약물을주입기로일정량주입하여질병을치료하는한방의료행위로, 침구및약물요법과병행할수있고상승효과가크기때문에현재는그응용범위가확대되어근골격계질환, 전신및면역질환뿐만아니라종양의치료에까지폭넓게활용되고있다 8). 미후등에관한연구로사상체질의학에서태양인병증에미후등식장탕을활용한증례보고 9-11) 등이있었으나, 아직까지전반적으로미후등에대한연구는부족한실정이다. 이에저자는염증억제작용에대한미후등약침추출물 ( 이하 BA) 의활용가능성을살펴보고더나아가적극적인임상활용을위해 BA의항염증효과를살펴보았다. 실험과정에서 RAW264.7 대식세포에 BA를적용하여세포생존율, 전구염증매개 cytokine 유전자, COX-2 와 inos 발현및 MAPK(mitogen-activated protein kinase) 활성화등의관찰을통해유의한결과를얻었기에보고하는바이다. 재료및방법 1. 약재의추출미후등은옴니허브 ( 대구, 한국 ) 에서구입하였고, 동의대학교한의과대학본초학교실로부터식물학적동정을받았으며, 표본은동의대학교한의과대학생리학교실에보관하고있다. 미후등 100 g 에 2 L의 75% ethanol 을加하여 24 시간동안추출하였다. 추출된용액을원심분리를통하여상층액을분리하고, 0.2 mm 여과지에서여과하여회전진공농축기 (N-1100V, EYELA, JAPAN) 로 200 ml 가되도록감압농축한후냉동건조하여 4.6 g의분말을얻었다. 추출물의수득률은 4.6% 로 20 o C에보관하였다가실험직전생리식염수나배지에희석하여 0.2 μm syringe filtering 후실험에사용하였다. 2. 시약본실험에사용된 LPS(Escherichia coli O55:B5), HEPES, Igepal CA-630, Sodium deoxycholate, NaCl, Tris-HCl, Sodium pyrophosphate, NaVO 4, NaF, Leupeptin, Phenylmethylsulfonyl fluoride 는 Sigma(St Louis, MO, USA) 로부터, fetal bovine serum(fbs), penicillin, streptomycin, Dulbecco's Modified Eagle medium(dmem) 및 trypsin-edta solution 은 Gibco BRL (Grand Island, NY, USA) 로부터, MTT, TRizol 및 DMSO는 Amresco(Cochran Road Solon, OH USA) 로부터, reverse transcription polymerase chain reaction(rt-pcr) kit(accupower RT/PCR PreMix) 는 Bioneer(Daejeon, Korea) 로부터, anti-phospho-erk(p-erk), anti-erk, anti-phospho-jnk(p-jnk), anti- JNK, anti-phospho-p38(p-p38), anti-p38, anti-phospho-pi3k, anti-pi3k, anti-phospho-akt, anti-akt, anti-phospho-iκbα, anti-iκbα, anti-nfκbp65 및 anti-β-actin primary antibody 는 Cell Signaling Technology(Beverly, MA, USA) 으로부터, HRPconjugated secondary antibody 는 KPL(Gaithersburg, MD, USA) 로부터구입하여사용하였다. 3. 세포배양 RAW264.7 세포는한국세포주은행 (Korea Cell Line Bank, KCLB, No. 40071, Seoul, Korea) 에서분양받았으며세포의배양을위하여열비활성화우태혈청과 1% penicillin 및 streptomycin 을포함한 DMEM 배지에서배양하였다. 세포는 37 o C 5% CO 2 조건하에서배양하였고, 2일마다배지를교환하였으며, 세포의증식에따른과밀도현상을해소하기위하여계대배양하였다. 4. MTT assay RAW264.7 세포를 10% FBS를포함한 DMEM 에현탁시킨후 96 well plate(corning, USA) 에 2 10 4 cells/ml 의세포수가되도록분주하여 37 o C 5% CO 2 배양기에서 24시간배양한후 BA를농도별 (0, 1, 2.5, 5, 7.5 및 10 mg/ml) 로처리한후 4시간동안배양하였다. 5 mg/ml의 MTT를추가하여 4시간동안배양한후 tetrazolium bromide salt 를제거하고 DMSO 를 200 μl 씩분주하여 well 에생성된 formazan 을녹인후 spectra max2 microplate reader(molecular Devices, USA) 를사용하여 540 nm에서흡광도를측정하였으며, 3 회의측정으로그에대한평균값과표준편차를구하였다. 5. Reverse transcription polymerase chain reaction (RT-PCR) RAW264.7 세포 (5 10 5 cells/ml) 에 BA(1, 2 및 4 mg/ml) 를처리하여 1시간동안배양한후자극제 LPS(1 μg/ml) 를처리하고 244 www.kjacupuncture.org
3시간또는 24시간동안배양하였다. 상층액을제거한후 1 ml의 TRizol 을넣고 2분동안방치한후 chloroform 을넣고 10초동안 vortexing 하고 12,000 rpm 에서 15분동안원심분리한후, 상층액을취하여동량의 isopropanol 을혼합하여흔들어주었다. 12,000 rpm에서 10분동안원심분리하여상층액을제거하고 pellet 은 DEPC(diethyl pyrocarbonate)-dw 20 μl에녹여 RT-PCR 에사용하였다. RT-PCR kit(bioneer, Korea) 를사용하여 45 o C에서 30 분, 94 o C에서 5분동안반응시킨후 94 o C에서 30초동안 denaturation시키고, 55 62 o C에서 30초동안 annealing 시킨다음, 72 o C에서 1분동안 extension 시키는 cycle 을 30 35회반복한뒤, 마지막 extension 은 72 o C에서 5분동안 PCR machine(geneamp, PCR system 9700, USA) 에서수행하였다. 각 PCR products 는 2% agarose gel 에 loading 하여 100V 조건에서 30분동안전기영동을통하여분석하였다. 각각의 primer 의염기서열은다음과같다 (Table 1). 6. Western blot RAW264.7 세포 (5 10 5 cells/ml) 에 BA(1, 2 및 4 mg/ml) 를처리하여 1시간동안배양한후자극제 LPS(1 μg/ml) 를처리하여배양하였다. MAPK 활성을측정하기위하여 LPS(1 μg/ml) 에 30 분동안노출시켰다. 반응이종료된후배지를제거하고 cold PBS 로세척한후 cell lysates 는 lysis buffer(10 mm ph 7.4 Tris- HCl, 5 mm NaF, 1 mm Na 3VO 4, 1 mm EDTA and 1 mm EGTA) 를첨가하여단백질을추출하였다. 단백질량을 Bradford 법으로정량하여 20 50 μg 의단백질을 l0% sodium dodecyl sulfate-polyacrylamide gel electrophoresis(sds-page) 로분리하고, Hypond-PVDF membrane(amersham, Little Chalfont, UK) 으로 transfer하였다. Transfer 된 membrane은 Tris-buffered saline Tween-20(TBST)(20 mm Tris, ph 7.6, 136 mm NaCl, 0.1% Tween 20) 에용해된 5% skim milk에 1시간동안실온에서 blocking한후 anti-phospho-erk와 anti-erk, anti-phospho- JNK 와 anti-jnk, anti-phospho-p38과 anti-p38 MAP kinase와 β- actin primary antibody(1 : 1,000 dilution) 로 4 o C에서 overnight 반응한후 TBST 로 3회 washing 하고, HRP-conjugated secondary antibody(1 : 1,000 dilution) 로 1시간동안실온에서반응시켰다. TBST 로 3회세척한후면역반응성단백질밴드는 X- ray films 에서 enhanced chemiluminescence(ecl) reagents(amersham, Little Chalfont, UK) 을이용하여검출하였다. 7. mrna 및단백질밴드의정량 mrna 발현및면역반응성단백질밴드는 Scion Image Release Beta3b software(maryland, USA) 를사용하여정량하였다. 각밴드영역에서측정된 pixels 의수를정량하여 GAPDH 와 β-actin 에대한비율로나타내었다. 8. 統計분석본실험에서얻어진결과는 SPSS 17.0K for Windows 통계프로그램패키지를사용하여평균치 ± 표준편차로나타내었고유의수준은 p<0.05 로하였다. 각실험군간의통계학적분석은 one way-anova 와 Tukey s multiple comparison test로사후검정을실시하였다. 결과 1. 세포생존률에미치는영향 RAW264.7 세포의생존률에미치는영향을알아보기위하여 MTT assay 를실시하였다. 대조군에서 1.18±0.03의흡광도를나타내었으며, BA(0, 1, 2.5, 5, 7.5 및 10 mg/ml) 의농도를처리한세포에서각각 1.18±0.05, 1.34±0.04, 1.23±0.04, 1.10±0.05 및 1.15±0.11 의흡광도를나타내어모든농도에서정상세포에비해유의한영향을보이지않았다 (Fig. 1). Table 1. The Primers for RT-PCR Analysis Gene Forward primer(from 5' to 3') Reverse primer(from 5' to 3') Size IL-1β AAG CTC TCC ACC TCA ATG GAC A GTC TGC TCA TTC ACG AAA ABB GAG 453 IL-6 TCC AGT TGC CTT CTT GGG AC GTG TAA TTA AGC CTC CGA CTT G 139 TNF-α GCG ACG TGG AAC TGG CAG AAG TCC ATG CCG TTG GTT AGG AGG 354 inos CTG CAG CAC TTG GAT CAG GAA CCT G GGG AGT AGC CTG TGT GCA CCT GGA A 311 COX-2 TTG AAG ACC AGG AGT ACC GC GGT ACA GT CCC ATG ACA TCG 324 GAPDH CCA CAG TCC ATG CCA TCA C TCC ACC ACC CTG TTG CTG TA 568 www.kjacupuncture.org 245
Fig. 1. Effects of BA on the cell viability in RAW264.7 cells. The cells were treated with the indicated concentrations(0, 1, 2.5, 5, 7.5 and 10 mg/ml) of BA for 4 h. Cell viability was evaluated using a colorimetric assay based on MTT assay. The results are displayed in the absorbance at 570 nm. Data represent the mean±sd of three independent experiments. 2. 전구염증매개 cytokine 유전자발현에미치는영향 RAW264.7 세포에서염증매개 cytokine 의생산과그유전자의발현에대해조사한결과, IL-1β의생산량은대조군에서 8.25± 0.55 pg/ml 이었으나, LPS 자극군에서 31.42±1.95 pg/ml로유의한상승을보였으며, BA(1, 2 및 4 mg/ml) 전처리후 LPS 자극군에서각각 16.44±0.64, 14.73±1.62 및 15.54±1.27 pg/ml 로모든농도에서 LPS 자극군에비해유의한감소를나타내었다. 또한 IL-1β mrna 발현량은대조군에비해 LPS 자극군에서현저하게증가하였으며, BA(1, 2 및 4 mg/ml) 전처리후 LPS자극군에서유전자의발현이감소하는것으로관찰되었다 (Fig. 2A). IL-6 의생산량은대조군에서 56.64±0.53 pg/ml이었으나, LPS 자극군에서 386.40±34.36 pg/ml 로유의한상승을보였으며, BA(1, 2 및 4 mg/ml) 전처리후 LPS 자극군에서각각 242.50± 11.21, 81.39±35.38 및 58.81±0.86 pg/ml 로모든농도에서 LPS 자극군에비해유의한감소를나타내었다. 또한 IL-6 mrna 발현량은대조군에비해 LPS 자극군에서현저하게증가하였으며, BA(1, 2 및 4 mg/ml) 전처리후 LPS 자극군에서유전자의발현이감소하는것으로관찰되었다 (Fig. 2B). TNF-α의생산량은대조군에서 0.79±0.11 ng/ml이었으나, Fig. 2. Effects of BA on the production and gene expression of inflammatory cytokines in the LPS-stimulated RAW 264.7 cells. The cells were incubated for 1 h in the presence or absence of BA(1, 2 and 4 mg/ml) prior to LPS stimulation(3 h). The production of inflammatory cytokine, IL-1β(A), IL-6(B) and TNFα(C) was detected at 450 nm by ELISA assay. Data represent the mean±sd of three independent experiments. # p<0.05 vs unstimulated control group. *p<0.05 vs LPS- stimulated group. Expression of these genes was assayed by RT-PCR. GAPDH was used as internal control genes. Fig. 3. Effects of BA on PGE2 generation and COX-2 gene and protein expression in the LPS-stimulated RAW 264.7 cells. The cells were incubated for 1 h in the presence or absence of BA(1, 2 and 4 mg/ml) prior to LPS stimulation(4 h for PGE 2 and 24 h for COX-2). PGE 2 generation(a) was determined at 405 nm by ELISA assay. Data represent the mean±sd of three independent experiments. # p<0.05 vs unstimulated control group. *p< 0.05 vs LPS-stimulated group. The level of COX-2 gene(b) and protein(c) was assayed by RT-PCR and Western blot and GAPDH and β-actin was used as internal control genes and proteins respectively. 246 www.kjacupuncture.org
LPS 자극군에서 5.35±0.32 ng/ml 로유의한상승을보였으며, BA(1, 2 및 4 mg/ml) 전처리후 LPS 자극군에서각각 5.10±0.54, 3.25±0.87 및 0.75±0.03 ng/ml 로 BA 2, 4 mg/ml 농도에서 LPS 자극군에비해유의한감소를나타내었다. 또한 TNF-α mrna 발현량은대조군에비해 LPS자극군에서현저하게증가하였으며, BA(1, 2 및 4 mg/ml) 전처리후 LPS자극군에서유전자의발현이감소하는것으로관찰되었다 (Fig. 2C). 3. PGE2 생산과 COX-2 유전자및단백질발현에미치는영향 RAW264.7 세포에서 PGE2 의생산과 COX-2 유전자및단백질발현에대해조사한결과, PGE2 의생산량은대조군에서 626.10± 78.94 pg/ml 이었으나, LPS 자극군에서 782.40±72.00 pg/ml로유의한상승을보였으며, BA(1, 2 및 4 mg/ml) 전처리후 LPS 자극군에서각각 574.60±58.19, 565.50±58.18 및 552.00±73.26 pg/ml로모든농도에서 LPS 자극군에비해유의한감소를나타내었다 (Fig. 3A). COX-2 mrna 의발현량은대조군에서거의발현되지않았으나 LPS 자극군에서현저하게증가하였으며, BA(1, 2 및 4 mg/ml) 전처리한세포에서유전자의발현이감소하는것으로나타났다 (Fig. 3B). COX-2 단백질의발현량은대조군에서약하게발현되었으나 LPS 자극군에서현저하게증가하였으며, BA(1, 2 및 4 mg/ml) 전처리한세포에서단백질의발현이점차감소하는것으로나타났다 (Fig. 3C). 4. NO 생산과 inos 유전자및단백질발현에미치는영향 RAW264.7 세포에서 NO의생산과 inos 유전자및단백질발현에대해조사한결과, NO의생산량은대조군에서 0.15±0.04 μm이었으나, LPS 자극군에서 1.28±0.03 μm로유의한상승을보였으며, BA(1, 2 및 4 mg/ml) 전처리후 LPS 자극군에서각각 0.51±0.11, 0.25±0.01 및 0.13±0.02 μm 로모든농도에서 LPS 자극군에비해유의한감소를나타내었다 (Fig. 4A). inos mrna 의발현량은대조군에서거의발현되지않았으나 LPS 자극군에서현저하게증가하였으며, BA(1, 2 및 4 mg/ml) 전처리한세포에서유전자의발현이감소하는것으로나타났다 (Fig. 4B). inos 단백질의발현량은대조군에서약하게발현되었으나 LPS Fig. 4. Effects of BA on NO generation and inos gene and protein expression in the LPS-stimulated RAW 264.7 cells. The cells were incubated for 1 h in the presence or absence of BA(1, 2 and 4 mg/ml) prior to LPS stimulation(24 h). NO generation(a) was measured at 540 nm by Griess reaction assay. Data represent the mean±sd of three independent experiments. # p<0.05 vs unstimulated control group. *p<0.05 vs LPS-stimulated group. The level of inos gene(b) and protein(c) was assayed by RT-PCR and Western blot and GAPDH and β-actin was used as internal control genes and proteins respectively. Fig. 5. Effects of BA on the activation of MAPK in LPS-stimulated RAW264.7 cells. The cells were incubated for 1 h in the presence or absence of BA(1, 2 and 4 mg/ml) prior to LPS stimulation(30 min). Cytosolic lysates were separated by SDS-PAGE. The phosphorylation and total level of ERK, JNK and p38 was detected by Western blot. β-actin was used as internal control proteins. www.kjacupuncture.org 247
자극군에서현저하게증가하였으며, 1 mg/ml 의 BA를전처리한세포에서는단백질이발현이감소되지않고다소증가하였으나, BA(2 및 4 mg/ml) 전처리한세포에서단백질의발현이감소하는것으로나타났다 (Fig. 4C). 이러한 BA에의한 PGE2 와 NO의생산량억제효과는 PGE2 와 NO의합성에관여하는 COX-2 와 inos 유전자의발현및단백질의발현조절을통해나타나는하는것으로생각된다. 4. MAPK 활성화에미치는영향 LPS로자극한 RAW264.7 세포에서 BA 전처리가 ERK, JNK와 p38의활성화경로에미치는영향을살펴본결과, 대조군에비해 LPS 자극군에서 p-erk, p-jnk 와 p-p38 의발현이증가하였고, BA(1, 2 및 4 mg/ml) 전처리한군은 LPS 자극군에비해 p-erk, p-jnk 와 p-p38 의발현이억제되는것으로나타났다 (Fig. 5). 고찰 염증은손상혹은괴사조직, 미생물과같은이물질을제거하는기본적인보호반응으로, 염증이없으면감염은지속되며창상이회복되지않고상해조직은곪은상처로남게되는데, 실제임상에서염증은부적절하게시작되거나조절이잘되지않기때문에많은질환에서조직손상의원인이되기도한다 12). Macrophages 는인체전반에걸쳐분포하여외부의병원체로부터인체를방어하는역할을담당하며, LPS 는그람음성박테리아의외부세포막을구성하는성분으로 macrophages 의활성화인자로작용하는데, 활성화된 macrophages는 interleukins(ils), TNF-α, COX-2, inos 와같은염증매개인자의생성을촉진하는작용을한다 13,14). COX-2 는염증에관여하는대표적인효소로서, 여러종류의자극에의해나타나는데염증과통증에중요한역할을하는 prostaglandin 의생성을조절하는역할을할뿐만아니라염증의활성화과정에도핵심적인역할을한다 15). 또한, tumor necrosis factorα(tnf-α 도염증자극이발생하여활성대식세포에서비정상적으로과도하게분비되면 IL-1β, IL-6, IL-10 등의염증성 cytokine 의분비를촉진시켜염증을활성화시키는작용을하게된다 16). Cytokine 은외부의자극에대하여한개체의세포와유기적, 종합적반응을일으키는데필요한세포상호간의작용을매개하며, 조혈작용과면역반응및일반적염증과정등에관련된모든세포들의작용을조절한다 17). 활성화된 macrophages 는내재면역반응에관련된다른세포들을유인하고활성화시키는 cytokine 을분비하 는데, IL-1 과 IL-6 와같은 cytokine 은체온상승을초래하는단백질의생산을증가시키고, TNF-α 와같은 cytokine 은말초혈관상피의투과성을증대시키며세포나수용성분자들의혈관으로부터조직으로의이동을증진시키는효과가있다 2). 흉선의대식세포나수지상세포에서생성되는 IL-1 은 IL-1α와 IL-1β 의 2종류가있는데흉선림프구의증식을촉진하며 T림프구의분화를보조할뿐만아니라, 세포독성 T 세포의발현을보조하고 NK세포의활성을상승시키며대식세포의세포상해작용을증강함으로써종양세포나바이러스감염세포의배제에도작용하고있다 18). IL-1 의생물학적인작용은제1형 IL-1 수용체라는막수용체에의하여매개되며 NF-κB 와 AP-1 전사인자를활성화시키게된다 19). IL-6 은일차면역반응 B 세포성장및분화인자로작용하는데, 항원자극을받아증식된 B 림프구의항체생산과분비에중요한역할을담당하며형질세포항체분비항진, T 세포활성보조자극등의기능을가진것으로알려졌다 20). TNF-α 는감염을국소화하는데중요한역할을하는데일단감염이혈액을통해퍼진경우에는대식세포에의한전신적인 TNF-α 분비로혈관확장, 혈압강하, 혈관의투과성증가로인한혈장의손실을일으키고 shock 을유발할수도있다 21). LPS 에의해자극을받은 macrophages 는또한 MAPK 활성에영향을미쳐 serine/threonine kinase 가세포밖신호를핵내로전달하게하는데, extracellular signal-regulated kinase(erk), JNK, p38 등이 LPS 에의하여활성화되는대표적인 MAPK 이다 22,23). ERK 신호전달경로에서활성화된 ERK 는다양한전사인자를인산화할수있는데, ERK 가자극인자에의해광범위하게활성화되는반면, p38과 JNK는 stress 반응경로를구성하는부분으로염증성 cytokine 같은인자에의해유도된세포스트레스에의해활성화된다 24,25). ERK, JNK, p38의 3가지각 MAPK는인산화를통해활성화됨으로써염증매개물질생성을유도하게된다 26). 獼猴藤은다래과식물미후도 (Actinidia chinensis Planch.) 의덩굴및덩굴즙액으로, < 本草綱目 >에서氣味는甘, 滑, 寒, 無毒하다고하며, 그주치에대해 < 本草拾遺 >에서 下石淋, 主胃閉, 取汁和薑汁服之佳 라하였고, < 廣西本草選編 >에서 治消化不良, 嘔吐, 黃疸. 한다고하였으며, 和胃開胃, 淸熱利濕하는효능이있어消化不良, 反胃, 嘔吐, 黃疸, 石淋등의치료에활용된다 7). 기존선행된연구로는과실인미후도에서유래한 triterpenes 의신생혈관억제작용 27) 과 in vitro 에서미후도추출물의항산화, 항증식성의평가 28) 등이보고되었으며, 뿌리인미후도근에대하여 triterpenoids의 positive cytotoxic activity 29,30) 와항암효과 31,32) 등이보고되었다. 그리고덩굴인미후등에대한연구는메탄올추출물의 248 www.kjacupuncture.org
항염증효과 33) 와사상체질의학에서태양인병증에미후등식장탕을활용한증례보고 9-11) 외에아직까지연구가부족한실정이다. 약침요법은경혈과체표촉진에의해얻어진양성반응점및혈관에약침추출액을주입하여자침과약물의효능을이용해생체의기능을조정하고병리상태를개선시키는치료법으로현재웅담, 우황, 사향, 녹용, 자하거, 호도, 홍화자, 봉독, 산삼, 오공등다양한약침추출액이연구, 제조되어임상에활용되고있다 8,34). 염증의대표적증상중발열과혈관확장은한의학에서熱證의속성을가졌다라고볼수있는데, 熱證의발병원인과증상에따라달리변증할수있겠지만크게熱證은외부사기의감염으로인한表熱證과內傷雜病으로인한裏熱證이있으며, 주로表熱證에는解表藥을쓰고裏熱證에는淸熱藥을배합하여치료하게된다 35). 미후등은氣味가甘, 滑, 寒, 無毒하고淸熱利濕하는효능이있다고알려졌는데, 이러한성질을통해항염증작용을확인할수있을것으로생각되어본연구에서는미후등약침추출물을 LPS로염증모델을유도한 RAW264.7 세포에처치하여관찰하는방법을선택하였다. 이에저자는 BA의항염증효과를확인하고, 활용가능성을살펴보기위해 LPS로유도된 RAW264.7 세포에서 BA가세포생존에미치는영향, COX-2, inos 등의염증관련물질발현에미치는영향, IL-1β, IL-6 와 TNF-α 등의 cytokine 의유전자발현에미치는영향, MAPK 활성화에미치는영향에대해살펴보고그기전을밝혀보고자하였다. BA의세포독성을조사하기위해 BA를각각 0, 1, 2.5, 5, 7.5 및 10 mg/ml 로처치하고 MTT assay 로흡광도를관찰하였을때, 대조군에서 1.18±0.03 의흡광도를나타내었으며, BA를각각 0, 1, 2.5, 5, 7.5 및 10 mg/ml 처치한군에서흡광도는각각 1.18±0.05, 1.34±0.04, 1.23±0.04, 1.10±0.05 및 1.15±0.11 로나타나 BA의세포에대한독성은나타나지않았다 (Fig. 1). BA가전구염증매개 cytokine 유전자발현에미치는영향을알아보기위해자극제 LPS(1 μg/ml) 를처치한 LPS 자극군에서는아무런처리를하지않은대조군에비해 IL-1β, IL-6 와 TNF-β 생산량의유의한상승과 mrna 발현량의상승을보였으며, BA(1, 2 및 4 mg/ml) 전처리후 IL-1β, IL-6와 TNF-α 의생산량과 mrna 발현량은감소를나타내는것이관찰되었다 (Fig. 2). BA가 RAW264.7 세포에서 PGE2 의생산과 COX-2 유전자및단백질발현에대해조사한결과, PGE2 의생산량은대조군에비해 LPS 자극군에서유의한상승을보였으며, BA(1, 2 및 4 mg/ml) 전처리후 LPS 자극군에서모든농도에서 LPS 자극군에비해유의한감소를나타내었고 (Fig. 3A), COX-2 mrna 의발현량은대조군에서거의발현되지않았으나 LPS 자극군에서현저하게증가하였 으며, BA(1, 2 및 4 mg/ml) 을전처리한세포에서유전자의발현이감소하는것으로나타났다 (Fig. 3B). 또한 COX-2 단백질의발현량은대조군에서약하게발현되었으나 LPS 자극군에서현저하게증가하였으며, BA(1, 2 및 4 mg/ml) 을전처리한세포에서단백질의발현이점차감소하는것으로나타났다 (Fig. 3C). BA가 RAW264.7 세포에서 NO의생산과 inos 유전자및단백질발현에대해조사한결과, NO의생산량은대조군에비해 LPS 자극군에서유의한상승을보였으며, BA(1, 2 및 4 mg/ml) 전처리후 LPS 자극군에서모든농도에서 LPS 자극군에비해유의한감소를나타내었고 (Fig. 4A), inos mrna 의발현량은대조군에서거의발현되지않았으나 LPS 자극군에서현저하게증가하였으며, BA(1, 2 및 4 mg/ml) 을전처리한세포에서유전자의발현이감소하는것으로나타났다 (Fig. 4B). 또한 inos 단백질의발현량은대조군에서약하게발현되었으나 LPS 자극군에서현저하게증가하였으며, 1 mg/ml 의 BA를전처리한세포에서는단백질이발현이감소되지않고다소증가하였으나, BA(2 및 4 mg/ml) 을전처리한세포에서단백질의발현이감소하는것으로나타났다 (Fig. 4C). 이러한 BA에의한 PGE2 와 NO의생산량억제효과는 PGE2 와 NO의합성에관여하는 COX-2 와 inos 유전자의발현및단백질의발현조절을통해나타나는하는것으로생각된다. BA가 LPS로자극한 RAW264.7 세포에서 ERK, JNK와 p38의인산화에미치는영향을살펴본결과, LPS 자극군에서 p-erk, p-jnk 와 p-p38 의발현이증가한반면, BA(1, 2 및 4 mg/ml) 전처리후 LPS 자극군에서 p-erk, p-jnk 와 p-p38 의발현이억제되는것으로나타났다 (Fig. 5). 이결과로보아 BA가 MAPK 의활성화를감소시킴으로써염증반응을억제하는것으로생각된다. 이상의연구결과를종합적으로살펴볼때 BA가전구염증매개 cytokine 유전자의발현조절을통한전구염증매개 cytokine 억제, COX-2, inos 의유전자발현조절을통한 PGE2, NO의생산억제, MAPK 활성을억제하는등염증성반응들을차단함으로써항염증효과를가짐을알수있었으며, 향후미후등을염증질환치료에응용하는것이가능할것이라사료된다. 결론 미후등의항염증효과를확인하기위해 LPS로유도된 RAW 264.7 세포의염증모델에서항염작용실험을통하여다음과같은결론을얻었다. 1. 미후등약침추출물의세포독성을확인하기위해 RAW 264.7 www.kjacupuncture.org 249
대식세포에서 MTT assay 를수행한결과미후등약침추출물을농도별로처리했을때세포독성이나타나지않았다. 2. 미후등약침추출물은염증성 cytokine 인 IL-1β, IL-6와 TNF-α유전자의발현과분비를억제하였다. 3. 미후등약침추출물은 COX-2, inos 유전자발현과 PGE2, NO의생산을억제하였다. 4. 미후등약침추출물은 ERK, JNK 와 p38 의인산화를억제하였다. 이상의결과에서미후등은항염증효과를가지고있음을확인할수있었다. 감사의글 This work was supported by Dong-Eui University Foundation Grant(No. 2013AA098). References 1. Kim YH. Pathology. 3/e. Seoul : Hyeonmunsa. 2010 : 51-69. 2. Lee MH. Lippincott's Illustrated Reviews : Immunology. Seoul : Shinilbooks. 2008 : 41-53. 3. Park HJ, Lee JS, Lee JD, Kim NJ, Pyo JH, Kang JM, et al. The Anti-inflammatory Effect of Cinnamomi Ramulus. J Korean Oriental Med. 2005 ; 26 : 140-51. 4. Kim JW. NSAID-induced Gastroenteropathy. Korean J Gastroenterol. 2008 ; 52 : 134-41. 5. Cho JW, Lee ES, Shin WG. Evaluation of NSAID usage and appropriateness for prevention of NSAID-related ulcer complications. Kor J Clin Pharm. 2012 ; 22(3) : 211-9. 6. Cho YJ, Moon SL, Park KH, Cho NC, Song YW. Comparison of side effects of non-steroidal antiinflammatory drugs(nsaids) in rheumatoid arthritis patients. J Kor Soc Hosp Pharm. 1998 ; 15(2) : 186-92. 7. State Administration of Traditional Chinesemedicine. Chinese Herbal Medicine. Shanghai : Shanghai Scientific and Technical Publisher. 1999 ; 3 : 538-43. 8. Korean pharmacopuncture institute scholarship committee, Pharmacopuncture medicine laboratory. Pharmacopuncturology. 2/e. Seoul : Elsevier Korea. 2011 : 1-32, 93-115. 9. Park EK, Park SS. 3 Case study that classified and performed a treatment as taeyangin. J Korean Oriental Med. 2001 ; 22(4) : 164-70. 10. Jeon SH, Lee HM, Jo HS, Kim JW. A case report of taeyangin yulkyuk. J Sasang Constitut Med. 2004 ; 16(3) : 118-23. 11. Ahn JH, Kwon EM, Song AN, Kim EH, Park SJ, Jung YH, et al. A case of soeumin rectal cancer misdiagnosed as a taeyangin and mistreated with mihuedungsikjang-tang. J Sasang Constitut Med. 2011 ; 23(4) : 533-40. 12. Kumar V, Abbas A, Fausto N, Aster J. Robbins & Cotran Pathologic Basis of Disease, 8/e. Seoul : Beommun-education. 2011 : 43-4. 13. Kim DY, Jeong WS, Moon HC, Park SJ. Water extract of flowers of magnolia denudata inhibits lps-induced nitric oxide and pro-inflammatory cytokines production in murine peritoneal macrophage by inhibiting NF-κB activation. Korean J Oriental Physiology&Pathology. 2007 ; 21(4) : 916-20. 14. Shin JS, Kim JM, An WG. Anti-inflammatory effect of red ginseng through regulation of MAPK in lipopolysaccharide- stimulated RAW264.7. Korean J Oriental Physiology & Pathology. 2012 ; 26(3) : 293-300. 15. Durrenberger PF, Facer P, Gray RA, Chessell IP, Naylor A, Bountra C, et al. Cyclooxygenase- 2(Cox-2) in injured human nerve and a rat model of nerve injury. J Peripher Nerv Syst. 2004 ; 9(1) : 15-25. 16. Leung L, Cahill CM. TNF-alpha and neuropathic pain-a review. J Neuroinflammation. 2010 ; 7(27) : 1-11. 17. Jeon JC, Lee EY. The effects of euonymi ligunum suberalatum phamacopuncture on nitric oxide and interleukin-6 production in macrophage. The Journal of Korean Acupuncture & Moxibustion Society. 2010 ; 27(4) : 223-31. 18. Kwon HH. Clinical immunology. 2/e. Seoul : Korea Medical Publishing. 2010 : 157-70. 19. Abul KA, Andrew HL, Shiv P. Cellular and Molecular Immunology. 6/e. Seoul : E-Public. 2008 : 271-307. 20. Kim SJ. Immunology. Seoul : Korea Medical Publishing. 1994 : 119-31. 21. Murphy K, Travers P, Walport M. Janeway's immunobiology. 7/e. Seoul : E-Public. 2008 : 78-91. 22. Garrington TP, Johnson GL. Organization and regulation of mi- 250 www.kjacupuncture.org
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