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pissn 2287-3368 / eissn 2287-3376 ORIGINAL ARTICLE Korean Journal of Acupuncture Vol.31, No.4, pp.168-178, 2014

1 pissn / eissn ORIGINAL ARTICLE Korean Journal of Acupuncture Vol.31, No.4, pp , 마황천오약침액이 3T3-L1 지방세포분화및유전자발현에미치는영향 동의대학교한의과대학생리학교실 Effects of Mahuang-Chuanwu(Mahwang-Cheonoh) Pharmacopuncture Solution on Adipocyte Differentiation and Gene Expression in 3T3-L1 Adipocytes Kyung-Hwa Kang Department of Physiology, College of Korean Medicine, Dong-Eui University Objectives : Mahuang-Chuanwu(Mahwang-Cheonoh) Pharmacopuncture(MCP) has been used to treat obesity in Clinical Korean Medicine. MCP solution(mcps) is also expected to have strong anti-obesity activities. However, little is known about the mechanisms of its inhibitory effects on adipocyte differentiation and lipogenesis. Methods : In the present study, we examined the effects of MCPS on differentiation and lipogenesis of 3T3-L1 adipocytes. To elucidate the mechanism of the effects of MCPS on lowering lipid content in 3T3-L1 adipocytes, we examined whether MCPS modulates the expressions of transcription factors to induce lipogenesis and adipogenic genes related to regulate the accumulation of lipids. Results : Our results showed that MCPS significantly inhibited differentiation and lipogenesis of 3T3-L1 adipocytes in a dose-dependent manner. MCPS suppressed the mrna expressions of cytidine-cytidine-adenosine-adenosine-thymidine(ccaat)/enhancer binding proteins α(c/ebpα), C/EBP β, C/EBPδ, and peroxisome proliferator-activated receptor γ(pparγ) genes related to the induction of adipose differentiation. MCPS inhibited the mrna expressions of adipose-specific ap2, adipsin, lipoprotein lipase(lpl), CD36, TGF-β, and leptin genes related to the fat formation. MCPS downregulated the mrna expressions of liver X receptor(lxr) α and fatty acid synthase(fas) genes related to the induction of lipogenesis. In addition, MCPS reduced the production of adipocyte-induced pro-inflammatory cytokines. Conclusions : MCPS could regulate the accumulation of lipids and expression of adipogenic genes via inhibition of transcript factors related to induction of adipose differentiation. Key words : Mahuang-Chuanwu(Mahwang-Cheonoh) Pharmacopuncture (MCP), 3T3-L1 adipocytes, differentiation, adipogenesis, lipogenesis 서 론 식사와생활습관의변화로비만인구가전세계적으로증가하고, 점차비만연령이낮아지는추세에있다. 또한비만은당뇨병, 고지혈증과심혈관계질환등의심각한대사성질환을동반하고, 그발병의직접적인유발원인으로인식되고있어비만의조절이사회적 Received September 25, 2014, Revised November 27, 2014, Accepted November 27, 2014 Corresponding author: Kyung-Hwa Kang Department of Physiology, College of Korean Medicinem, Dong-Eui Universitym, 52-57, Yangjeongro, Busanjin-gu, Busan , Korea Tel: , Fax: , ghkang@deu.ac.kr This work was supported by Dong-eui University Foundation Grant(2014AA132). 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. 168 Copyright 2014, SOCIETY FOR MERIDIAN AND ACUPOINT

2 Effects of MCPS on Adipocyte Differentiation and Gene Expression 이슈로대두되고있다 1). 비만은체내과잉에너지가지방세포의증식과비대에의해지방조직으로과다축적된상태로비정상적인지방대사를유발하고내분비이상을초래한다. 그러므로최근비만의조절을위해지방세포의분화증식과세포내지질축적의기전을제어하는방법에대한연구가많이이루어지고있다. 한의학에서는비만의조절을위해다양한치료방법이사용되고있는데, 약침요법도이러한치료법의하나로환자의질병을근거로혈위의치료작용과약물의약리작용을결합하여질병을치료하는방법이다. 약침요법은약리효과가소화기관을통과하지않고나타나므로적은양의약물로도경구섭취에비해우수한치료효과를나타내는특징이있다 2). 마황 (Ephedra sinica Stapf) 은대표적인신온해표약 ( 辛溫解表藥 ) 으로발산풍한 ( 發散風寒 ), 선폐평천 ( 宣肺平喘 ), 이수소종 ( 利水消腫 ) 의효능을가지고비만, 호흡기질환, 피부질환, 순환기질환등에많이응용되고있으나, 부작용으로빈맥, 혈관수축, 일시적인고혈압, 기관지확장, 불면증, 자한, 식욕감퇴, 심계항진, 두통등이나타날수있는것으로보고되고있다 3). 동의보감에서마황의최고사용용량은 24 g이며, 이는에페드린함량으로환산할경우미국 FDA 에서의약품으로허용하고있는최고에페드린의함량기준이하이므로비만과체중조절을위해적절히사용할경우효과적인치료가가능할것으로생각된다 4). 천오 (Aconitum carmichaeli Debx) 는대표적인온리약 ( 溫裏藥 ) 으로거풍습 ( 祛風濕 ), 산풍사 ( 散風邪 ), 온경지통 ( 溫經止痛 ) 의효능을가지고풍한습비 ( 風寒濕痺 ) 나역절풍 ( 歷節風 ), 중풍 ( 中風 ) 등에응용되고있으며, 수족마비, 호흡마비, 심계항진, 혈압강하등의부작용을가지고있다 5). 그러나, 마황추출물및약침제제의안전성은면역독성, 발열성시험, 용혈독성시험, 아급성및급성독성시험등의실험연구를통해평가되었으며 6-8), 초오추출물및약침제제의안전성도독성시험실험연구를통해평가되었다 9,10). 또한마황천오약침제제의안전성및유효성에대한설문조사분석연구를통해서도안정성이인정되었다 11). 이와같이마황과천오는풍한 ( 風寒 ) 을발산하고경락을소통하게하여기혈순환을원활하게하고신진대사를활발하게하는작용을통해비만에응용가능할것으로생각된다. 현재한의학에서임상적으로비만치료에마황과천오가많이응용되고있으며, 마황천오약침에대한선행연구로비만치료에대한문헌적고찰과비만환자를대상으로한연구가보고되었다 12,13). 비만치료를위한마황과천오의사용에대한지속적인연구를통해적절하게임상에서응용한다면다양한난치성질환을극복하 는데도움이될것으로생각된다. 이에저자는마황천오약침제제가지방세포의분화와지방생성에미치는억제효과를조사하기위하여지방전구세포의분화, 지방구생성과매개물질분비와이와관련있는유전자의발현에미치는영향을조사하여다음과같은결과를보고하는바이다. 실험방법 1. 재료 3T3-L1 mouse preadipocytes는 American Type Culture Collection(ATCC, USA) 에서분양받아사용하였고, Dulbecco's Modified Eagle's Medium(DMEM), bovine calf serum(bcs), fetal bovine serum(fbs), penicillin-streptomycin mixture 및 dimethylsulfoxide(dmso) 는 GibcoBRL(USA) 로부터구입하였다. Insulin, isobutylmethylxanthine(ibmx), dexamethasone(dex), tetrazolium bromide salt(mtt), dimethylsulfoxide(dmso), bovine serum albumin(bsa) 는 Sigma(USA) 로부터구입하였다. 2. 마황천오약침액 (Mahuang-Chuanwu Pharmacopuncture Solution, MCPS) 조제 MCPS 는대한약침학회에서구입하여사용하였다. 조제방법은간략히설명하면마황과천오를원적외선으로건조하여압출성형기법으로평균직경 10 μm 의초미세분말을추출하였고, 수율은 25% 였다. 건조된분말 100 g을반응조하부에넣고 3차증류수 1.5 L를부어서 impellar, 반응조하부, 반응조상부, 냉각관 ( 직류, 환류 ), 분액여두를설치하고약 60분동안약재를불린후 105 o C로 120분간전탕하여찌꺼기를따로분리하였다. 전탕액을무균실에있는증류추출기에넣고 107 o C로 3시간이상추출하였다. 추출된약침액은무기염류를침강시키기위해서하루동안냉장보관하고, 무균실에서무기염류를제외한상층액을분리하여염도 0.98%, ph 로조절한후 0.45 μm, 0.2 μm, 0.1 μm 필터로 3차여과하여 MCPS 1,000 ml을얻었다. 멸균된 vial병에여과된약침액을각각 20 ml씩소분하여 120 o C로 30분간고압멸균하여 -20 o C에저장하였다가실험에사용하였다. 3. 세포생존률측정 3T3-L1 전구지방세포에대한 MCPS 의독성여부를알아보기위하여 MTT assay를시행하였다. 24 well plate(corning, NY, USA) 에 cells/well 의농도로세포를 DMEM 배양액에분주 169

3 하여 24 시간동안안정화시킨후, MCPS 을농도별 (0, 5%, 10%) 로처리하여 48 시간동안반응시켰다. 배양액을제거한후 5 mg/ml 의 MTT 를각 well 에넣고잘섞어준후최대 4시간 37 o C incubator 에서배양한후 tetrazolium bromide salt를제거하고각 well에 DMSO 를 100 μl 씩분주하여생성된 formazan 이잘녹을수있게충분히흔들어모두녹인후 ELISA(Molecular Devices, Sunnyvale, California, USA) 를사용하여 540 nm에서흡광도를측정하였다. 4. 세포배양및분화지방세포의분화를위해, 3T3-L1 전구지방세포는 5% CO 2, 37 o C 배양기에서 DMEM(10% FBS, 100 unit/ml of penicillin G sodium, 100 μg/ml of streptomycin sulfate) 으로배양하였다. 100% confluent 한상태에서 2일후 0.5 mm IBMX, 1 μm DEX 와 1 μg/ml insulin 의분화유도물질 (MDI) 이포함된 DMEM 으로교환하여 2 3 일동안분화를유도하였으며, 그후매 2일마다 1 μg/ml insulin 이포함된 DMEM으로교환하였다. 분화 8일후세포는분석을위해사용되었다. 실험군은 3T3-L1 전구지방세포에분화유도를하지않은미분화군, 3T3-L1 전구지방세포에 0.5 mm IBMX, 1 μm DEX 와 1 μg/ml insulin 을처리하여 8일동안분화를유도한분화유도군 (MDI), 3T3-L1 전구지방세포에 8일동안분화유도와 MCPS 각각 5% 와 10% 의농도로처리한군 (MCPS) 으로나누었다 (Fig. 1). 5. Oil-Red O 염색세포내지방구생성을확인하기위하여 Oil red O 염색을실시하였다. 배양된세포는 phosphate-buffered saline(pbs) 로세척한 후 3.7% formalin 으로 5분간고정하고 deionized water 로세척하여 Oil red O를처리한후실온에서 30분간염색하였다. 그후염색액을제거하고 deionized water 로 3회세척하여염색된세포를 microscopic image(olympus, Japan) 로관찰하였다. 또한정량을위해 DMSO 를가하여지방을추출한후 microplate reader (Molecular Devices, Sunnyvale, California, USA) 를사용하여 490 nm에서흡광도를측정하였다. 6. ELISA assay 24 well plate(corning, NY, USA) 에 cells/well의농도로세포를 DMEM 배양액에분주하고 24시간동안안정화시켰다. MDI에의한분화 8 일후세포상층액을사이토카인분석을위해사용되었다. IL-1β, TNF-α, GM-CSF, IFN-γ, IL-6를 ELISA kit(bd Biosciences Pharmingen, San Diego, CA, USA) 를이용하여측정하였다. 7. RNA 추출및 RT-PCR Cell 에 TRizol 을넣고 2분간방치하였다가세포를떼어낸후 chloroform 을넣고 10초간강하게 vortexing 하고 4 o C 14,000 rpm에서 15분간원심분리하였다. 원심분리후, 상층액을취하여동량의 isopropanol 을혼합하여흔들어준후실온에 10분간방치하였다. 그후 4 o C 14,000 rpm에서 10분간원심분리하여상층액을제거하고 75% ethanol 을넣고섞어준후 4 o C 7,500 rpm에서 5분간원심분리하였다. RNA pellet 은 10분간실온에서건조시키고 DEPC(diethyl pyrocarbonate)-dw 에녹여 260 nm에서흡광도를측정하여 RNA 농도를계산하였다. 1 μg의 RNA 를취하여 RT-PCR kit(bioneer, Daejeon, Korea) 를사용하여 45 o C에서 30 Fig. 1. The Experimental Scheme of Differentiation of 3T3-L1 Preadipocytes. The 2-day post-confluent 3T3-L1 preadipocytes were treated with MDI(0.5 mm IBMX, 1 μm DEX and 1 μg/ml insulin) for 48 hrs. Cells were then replaced with maintenance media containing 1 μg/ml insulin every 2 days for 8 days. To investigate effect of MCPS on adipogenesis, the differentiating 3T3- L1 cells were treated every 2 days with MCPS for 8 days

4 Effects of MCPS on Adipocyte Differentiation and Gene Expression Table 1. The Primers for RT-PCR Target gene Oligonuclotide sequences (5' to 3' direction) Target gene Oligonuclotide sequences (5' to 3' direction) C/EBPβ F GTTTCGGGAGTTGATGCAATC CD36 F TTGTACCTATACTGTGGCTAAATGAGA R AACAACCCCGCAGGAACAT R CTTGTGTTTTTGAACATTTCTGCTT C/EBPδ F GATCTGCACGGCCTGTTGTA TGF-β F ATGGAGCTGGTGAAACGGAA R CTCCACTGCCCACCTGTCA R ACTGCTTCCCGAATGTCTGA PPARγ F CGCTGATGCACTGCCTATGA Leptin F CCAAAACCCTCATCAAGACCA R TGCGAGTGGTCTTCCATCAC R CTCAAAGCCACCACCTCTGT C/EBPα F GTGTGCACGTCTATGCTAAACCA LXRα F AGGAGTGTCGACTTCGCAAA R GCCGTTAGTGAAGAGTCTCAGTTTG R CTCTTCTTGCCGCTTCAGTTT ap2 F CCAATGAGCAAGTGGCAAGA FAS F TGGTGGGTTTGGTGAATTGTC R GATGCCAGGCTCCAGGATAG R GCTTGTCCTGCTCTAACTGGAAGT Adipsin F CTGCTGGACGAGCAGTGG GAPDH F CCACAGTCCATGCCATCAC R GATGACACTCGGGTATAGACGC R TTCACCACCCTGTTGCTGTA LPL F GGCCAGATTCATCAACTGGAT R GCTCCAAGGCTGTACCCTAAG 분, 94 o C에서 5분간반응시킨후 94 o C에서 30초간 denaturation 시키고, o C에서 30초간 annealing 시킨다음, 72 o C에서 1 분간 extension 시키는 cycle 을 회반복한뒤, 마지막 extension은 72 o C에서 5분간 PCR machine(geneamp, PCR system 9700)(Applied Biosystems, Foster City, CA, USA) 에서수행하였다. 각 PCR products는 0.5 TBE buffer(45 mm Trisborate, 1 mm EDTA) 를사용하여 2% agarose gel 에 loading 하여 100 V 조건에서 30분간전기영동한후 etidium bromide 염색을한후분석하였다. 각각의 primer 는 Bioneer(Bioneer Co., Daejeon, Korea) 에의뢰하여합성하였다 (Table 1). 8. 통계분석통계분석은 SPSS 12K for Windows 통계프로그램패키지를사용하여평균치 ± 표준편차로나타내었고유의수준은 p<0.05로하였다. 각실험군간의통계학적분석은 One way-anova 와 Dunnett's Multiple Comparison Test 검정을실시하였다. 실험결과 1. 지방세포분화억제효과 1) 지방전구세포증식에미치는영향 : 3T3-L1 전구지방세포에 5 및 10% 의 MCPS 를 48시간동안처리하여지방전구세포증식에미치는영향에대해조사한결과, 아무런처리를하지않은대조군의경우 0.67±0.04 의흡광도를나타낸데비하여 5 및 10% 의 MCPS를처리한군에서각각 0.66±0.05 와 0.64±0.04 의흡광도 Fig. 2. Effects of MCPS on the Cell Viability in 3T3-L1 preadipocytes. 3T3-L1 preadipocytes were treated with the indicated concentrations of MCPS for 48 hrs. Cell viability was evaluated using a colorimetric assay based on MTT assay. Values are means±sd of three independent experiments. 를나타내어대조군에비해유의한차이를나타내지않았다. 따라서 5 및 10% 의 MCPS 농도에의해세포의증식이저해되지않았음을확인할수있었다 (Fig. 2). 2) 지방세포의형태학적변화에미치는영향 : 100% 가득찬 3T3-L1 을 2일동안분화를유도하고분화양상을현미경으로관찰하여세포형태의변화를관찰한결과, 전구지방세포의세포밀도가 100% confluent된세포형태는길게뻗은섬유아세포의형태를보여주었으나, 지방세포로분화유도한 5일, 6일과 8일째세포의형태는시간이지남에따라세포모양이둥그러지고세포질내지방구들이점차많이형성됨을현미경으로관찰할수있었다. 반면 MCPS 군은농도에의존적으로세포내지방구의생성이억제되는것을관찰할수있었다 (Fig. 3). 3) 지방세포내중성지방생성에미치는영향 : Oil red O를이용 171

Fig. 3. Effects of MCPS on the Microscopic Morphological Changes of 3T3-L1. Preadipocytes Differentiation. The 2-day post-confluent 3T3-L1 preadipocytes(day 0) were treated with MDI(0.

Cells were then replaced with maintenance media containing 1 μg/ml insulin and MCPS for every 2 days.

5 Fig. 3. Effects of MCPS on the Microscopic Morphological Changes of 3T3-L1. Preadipocytes Differentiation. The 2-day post-confluent 3T3-L1 preadipocytes(day 0) were treated with MDI(0.5 mm IBMX, 1 μm DEX and 1 μg/ml insulin) for 48 hrs in the absence or presence of indicated concentrations of MCPS. Cells were then replaced with maintenance media containing 1 μg/ml insulin and MCPS for every 2 days. Accumulated lipid droplets in adipocytes were observed 5 days, 6 days and 8 days after the initiation of differentiation and morphological changes associated with adipogenesis were photographed using Inverted Microscope(Carl Zeiss, Germany). Fig. 4. Effects of MCPS on the Adipogenesis and Lipid Accumulation of 3T3-L1. Adipocytes. The 2-day post-confluent 3T3-L1 preadipocytes(day 0) were differentiated into adipocytes in the absence or presence of indicated concentrations of MCPS. (A) Adipocytes were stained with Oil red O to determine the degree of adipocyte differentiation. (B) Stained intracellular lipid was solubilized in isopropanol and absorbance was measured to evaluated lipid con# tent. Values are means±sd. p 0.05 vs. preadipocytes, *p 0.05 vs. MDI

Effects of MCPS on Adipocyte Differentiation and Gene Expression 하여세포염색을시행하여지방세포의형태를관찰하고세포내중성지방의양을정량하였다. 관찰한결과, 분화군에비해 5 및 10% 의 MCPS 군에서지방세포내지방구의형성이억제되는것을관찰하였다. 또한지방세포내염색된중성지방의양을정량한결과, 미분화군의흡광도는 0.

6 Effects of MCPS on Adipocyte Differentiation and Gene Expression 하여세포염색을시행하여지방세포의형태를관찰하고세포내중성지방의양을정량하였다. 관찰한결과, 분화군에비해 5 및 10% 의 MCPS 군에서지방세포내지방구의형성이억제되는것을관찰하였다. 또한지방세포내염색된중성지방의양을정량한결과, 미분화군의흡광도는 0.05±0.00 로나타났으나, 분화군에서흡광도는 0.65±0.23 으로미분화군에비해유의하게증가하는것으로나타났다 (p<0.05). 반면, 5 및 10% 의 MCPS 군의흡광도는각각 0.29±0.15 와 0.05±0.00으로지방생성을농도의존적으로유의하게억제하였다 (p<0.05)(fig. 4). 2. 지방세포분화관련전사인자및유전자발현조절효과 1) 지방분화전사인자발현에미치는영향 : Cytidine-cytidineadenosine-adenosine-thymidine(CCAAT)/enhancer binding proteins β(c/ebpβ), C/EBPδ, C/EBPα 와 peroxisome proliferator-activated receptor γ(pparγ) 의발현양상을살펴보면, C/EBPβ, C/EBPδ, C/EBPα 와 PPARγ mrna 는분화군은미분화군에비하여발현이현저하게증가하였고, MCPS 군은분화군에 비해농도의존적으로발현감소를보였다 (Fig. 5). 2) 지방세포특이적유전자발현에미치는영향 : ap2, Adipsin, lipoprotein lipase(lpl), CD36, transformation growth factorβ(tgf-β) 와 Leptin 의발현양상을살펴보면, ap2, Adipsin, LPL, CD36, TGF-β 와 Leptin mrna 의경우분화군은미분화군에비하여발현이증가하였고 MCPS 군은분화군에비해농도의존적으로발현감소를보였다 (Fig. 6). 3) 지방합성유전자발현에미치는영향 : Liver X receptor (LXR)α 와 fatty acid synthase(fas) 의발현양상을살펴보면 LXR α와 FAS mrna 는분화군은미분화군에비하여발현이증가하였고 MCPS 군은분화군에비해농도의존적으로발현감소를보였다 (Fig. 7). 3. 전구염증사이토카인생산에미치는영향 3T3-L1 성숙지방세포에서생산이증가하는전구염증사이토카인 tumor necrosis factor-alpha(tnf-α), interleukin(il)-1β, IL-6, interferon(ifn)-γ 와 granulocyte-macrophage colony- Fig. 5. Effects of MCPS on the Expression C/EBPβ, C/EBPδ, C/ EBPα, and PPARγ in mrna Level. The 2-day post-confluent 3T3-L1 preadipocytes(day 0) were differentiated into adipocytes in the absence or presence of indicated concentrations of MCPS for 8 days. The mrna levels were determined by RT-PCR as described in ʻMaterial and Methods

The mrna levels were determined by RT-PCR as described in ʻMaterial and Methods. Fig. 7. Effects of MCPS on the Expression of LXRα and FAS in mrna Level.

7 Fig. 6. Effects of MCPS on the Expression of ap2, Adipsin, LPL, CD36, TGF-β, and Leptin in mrna Level. The 2-day post-confluent 3T3-L1 preadipocytes(day 0) were differentiated into adipocytes in the absence or presence of indicated concentrations of MCPS for 8 days. The mrna levels were determined by RT-PCR as described in ʻMaterial and Methods. Fig. 7. Effects of MCPS on the Expression of LXRα and FAS in mrna Level. The 2-day post-confluent 3T3-L1 preadipocytes(day 0) were differentiated into adipocytes in the absence or presence of indicated concentrations of MCPS for 8 days. The mrna levels were determined by RT-PCR as described in ʻMaterial and Methods. stimulating factor(gm-csf) 의양을정량하였다. 그결과 TNF-α 의양은전구지방세포에서 52.90±22.29 pg/ml 이었으나, 성숙지방세포에서 ±28.39 pg/ml 로전구지방세포에비해유의한증가를보인반면 (p<0.01), 5와 10% MCPS 군은각각 86.43±71.41 pg/ml(p<0.05) 과 58.04±36.65 pg/ml(p< 0.01) 로성숙지방세포에비해유의한감소를보였다. IL-1β 의양은전구지방세포에서 3.31±2.13 pg/ml 이었으나, 성숙지방세포에서 ±52.61 pg/ml 로전구지방세포에비해유의한증가를보인반면 (p<0.01), 5와 10% MCPS 군은각각 7.61±2.91 pg/ml 과 5.83±3.30 pg/ml로성숙지방세포에비해유의한감소를보였 174

8 Effects of MCPS on Adipocyte Differentiation and Gene Expression Fig. 8. Effects of MCPS on the Levels of TNF-α, IL-1β, IL-6, IFN-γ, and GM- CSF in 3T3-L1 Adipocytes. Cytokines were detected by ELISA. Values are expressed as mean±sd. # p< 0.05 vs. preadipocytes. *p<0.05 vs. MDI. Dunnett's multiple comparison tests after one-way analysis of variance. 다 (p<0.01). IL-6의양은전구지방세포에서 11.89±1.00 pg/ml 이었으나, 성숙지방세포에서 48.53±6.48 pg/ml 로전구지방세포에비해유의한증가를보인반면 (p<0.01), 5와 10% MCPS 군은각각 30.97±14.45 pg/ml 과 14.46±6.96 pg/ml 로성숙지방세포에비해유의한감소를보였다 (p<0.01). INF-γ의양은전구지방세포에서 0.38±0.23 pg/ml 이었으나, 성숙지방세포에서 77.57± 0.23 pg/ml 로전구지방세포에비해유의한증가를보인반면 (p< 0.01), 5와 10% MCPS 군은각각 33.55±24.40 pg/ml과 6.29± 6.44 pg/ml로성숙지방세포에비해유의한감소를보였다 (p< 0.01). GM-CSF 의양은전구지방세포에서 11.82±2.20 pg/ml 이었으나, 성숙지방세포에서 35.31±11.17 pg/ml로전구지방세포에비해유의한증가를보인반면 (p<0.01), 5와 10% MCPS 군은각각 26.56±7.51 pg/ml(p<0.05) 과 19.02±2.32 pg/ml(p<0.01) 로성숙지방세포에비해유의한감소를보였다 (Fig. 8). 고찰 본연구에서비만환자의치료에사용되고있는마황천오약침의효과를실험적으로증명하기위하여 MCPS 가지방세포의분화를통한지방조직생성, 지방생성및분화관련유전자발현에미치는영향을조사하였다. MCPS 의농도에따른지방전구세포에대한세포생존율에대한 효과를살펴보기위하여 MTT assay 를통해 48시간동안살펴본결과 MCPS 는 5% 와 10% 의농도에서지방전구세포의증식을저해하지않는것으로나타났다 (Fig. 2). 그러나, MDI 분화유도에의해지방세포로분화되는과정에서현미경관찰에의해 MCPS 가 5% 와 10% 의농도에서농도의존적으로세포의형태학적변화와세포내지방구의생성을효과적으로억제하는결과를관찰하였다 (Fig. 3). 또한세포내축적된중성지방의양을정량한결과 MCPS 가 5% 와 10% 의농도에서농도의존적으로유의한억제효과를나타내었다 (Fig. 4). 지방세포는백색지방조직의주요구성세포로주로중성지방을저장하고동원함으로써에너지균형을조절하는기능을하며, 내분비작용과근거리분비작용을한다. 성숙지방세포는형태학적으로세포내에큰지방방울이특징인데, 핵이세포의외부가장자리에전치하는동안세포질은지방방울을둘러싼얇은층으로압축되어세포가확장되는것으로알려져있다 14). 또한지방조직의생성은 PPARγ 와 C/EBP isoforms 와같은지방분화전사인자의유도에의해지방전구세포가증식과분화과정을거쳐성숙한지방세포가된다. C/EBP isoforms 는여섯종류의이성질체가알려져있는데조직분포에있어지방세포에만국한되어존재하는것이아니라과립구나간세포에도분포하여말기분화에관여하는것으로알려져있으며, 감염이나상처에대한면역반응에도관여한다고알려져있다 15). 지방전구세포를이용한분화실험에서 C/EBPβ 와 C/EBPδ 가초기에증가를보인후분화후기에 175

9 C/EBPα 의증가가두드러지것으로보고되었다 16). PPAR 는지방산과아이코사노이드에의해활성화되는전사인자이다 17). PPAR subtypes(pparα, PPARγ 및 PPARδ) 는뚜렷한조직발현과생리학적활성을가진다. 그중 PPARγ 는지방조직에서주로발현되어지질리모델링과지방세포분화에필수적인전사인자로지질저장에중요한역할을하며, 탄수화물대사에간접적으로영향을미치며 18), 리간드가 PPARγ 에결합하여활성화되는핵수용체의한구성원으로또다른핵수용체인 retinoid-x-receptor(rxr) 와함께이형이합체를형성하여표적유전자의프로모터에위치한 PPAR response elements(ppres) 에결합하여작용하는것으로알려져있다 19,20). 본연구에서지방세포분화후기에 C/EBPβ, C/EBPδ, C/EBP α와 PPARγ 의 mrna 발현에미치는영향을조사한결과, MCPS 는 5% 와 10% 농도에서농도의존적으로이들전사인자의발현을억제하는것으로나타났다 (Fig. 5). 이는지방조직의생성과지질저장에필수적인지방세포분화후기에주로발현되는전사인자뿐만아니라분화초기에주로발현되어분화후기까지발현되는초기전사인자를모두제어하는효과를나타낸결과로해석된다. 지방조직생성 (adipogenesis) 은 C/EBPα 와 PPARγ의활성화로인해지방전구세포가증식과분화과정을거쳐성숙한지방세포로되는것으로써이때지질이축적되고실질적인지방세포의표현형을결정하는 ap2, LPL, 그리고 adipsin 과같은지방세포특이적인유전자 (adipose specific gene) 가발현되는것으로알려져있다 14). 본연구에서지방세포특이적유전자인 ap2, Adipsin, LPL, CD36, TGF-β 와 Leptin 의발현양상에대해조사한결과 MCPS 는 5% 와 10% 농도에서농도의존적으로지방세포특이적유전자의발현을감소시켰다 (Fig. 6). 또한지방합성에핵심적인역할을하는핵수용체로지방생성과축적에관여하는유전자인 LXRα 와 FAS 의발현에대한영향을조사한결과 MCPS 는 5% 와 10% 농도에서농도의존적으로지방합성에관여하는유전자의발현을감소시켰다 (Fig. 7). 이와같은결과는성숙지방세포로의분화와지방합성을억제하는효과를나타낸결과로해석된다. 지방조직은각종사이토카인을포함하는아디포카인이라는생리활성물질을분비하여지방대사및당대사에관여하는중요한내분비기관이다. 비만의경우지방세포에서생산하는염증성사이토카인의분비가증가하여인슐린저항성을유발하고, 심혈관계질환및제2형당뇨병등의비만성대사질환의발생에중요한역할을하는것으로알려져있다 21). TNF-α는지방세포에서염증과인슐린저항성을유발하는중 추적매개자로지방세포내염증성신호경로의활성화를통해지방세포의기능을손상시키고인슐린민감성을감소시킨다 22-24). IL-6 은지방분해와간의중성지방분비자극에의해고중성지방혈증을유도하며 25), IFN-γ 는지방세포에서인슐린유도혈당흡수를억제하고당대사이상과인슐린저항성을유발하는것으로알려져있다 26,27). 또한비만인의지방조직에서는대식세포의화학주성인자인 GM-CSF 가과발현되어지방조직으로대식세포의침투와인슐린저항성을증가시키고, TNF-α, IL-1β 와 IL-6 와같은전구염증사이토카인유전자발현을증가시키며염증부위면역세포를침투시켜전구염증사이토카인을유도한다. 특히, 고지방식이를투여한마우스의지방조직에서전구염증사이토카인의전반적인발현에중요한작용을하는것으로알려져있다 28,29). 본연구에서성숙한지방세포에서분비되는대표적인염증성사이토카인인 TNF-α, IL-1β, IL-6, IFN-γ 및 GM-CSF 의양을조사한결과 MCPS 는 5% 와 10% 농도에서농도의존적으로전구염증성사이토카인의분비를억제하는효과를나타내었다 (Fig. 8). 이와같은결과는비만으로인한전구염증성사이토카인의증가를억제함으로서인슐린저항성과같은지방대사및당대사의장애를조절하여비만성대사질환의예방에효과가있을것으로생각된다. 본연구결과를종합해볼때 MCPS 가지방세포분화전사인자와지방합성유전자의조절을통해성숙지방세포로의분화와지방생성을억제하고, 성숙지방세포에서생성되어이차적인비만성대사질환을유발하는염증성사이토카인의분비를억제하는것으로나타났는데, 이는한의학적원리에의한체온조절과수액의조절효능을통해비만조절및관련질환에대한조절가능성을보여주는것으로의미있는결과라고생각된다. 감사의글 This work was supported by Dong-eui University Foundation Grant(2014AA132). References 1. Haslam D, Sattar N, Lean M. Obesity--time to wake up. BMJ 2006 ; 333 : Korean Pharmacopuncture Institute. Pharmacopuncturology: Principles and Clinical Applications. Elsevier Korea LLC: Seoul, 176

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013~020. Áö¹æ¼¼Æ÷ ºÐÈ�ÁßÀÎ 3T3-L1 13 DOI : 10.3831/KPI.2009.12.2.013 3T3-L1 Received : 09. 04. 29 Accepted : 09. 05. 19 Adipocyte-Related Genes and Transcription Factors were affected by Ganoderma lucidum during 3T3-L1 Differentiation