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1 대한한방내과학회지제 37 권 3 호 (2016 년 6 월 ) J. Int. Korean Med. 2016:37(3) 중만분소환추출물이 Palmitate 로유발된비알코올성지방간 HepG2 cell 모델에미치는영향 이지원 1, 최창원 1, 전상윤 1, 한창우 2, 하예진 3 1 동신대학교한의과대학내과학교실, 2 부산대학교한방병원내과학교실, 3 원광대학교한의과대학내과학교실 Effect of Jungmanbunso-hwan Extract on HepG2 Cell Model of Nonalcoholic Fatty Liver Disease Caused by Palmitate Ji-won Lee 1, Chang-won Choi 1, Sang-yun Jeon 1, Chang-woo Han 2, Ye-jin Ha 3 1 Dept. of Oriental Internal Medicine, College of Korean Medicine, Dong-Shin University 2 Dept. of Oriental Internal Medicine, Korean Medicine Hospital, Pusan National University 3 Dept. of Oriental Internal Medicine, College of Korean Medicine, Won-Kwang University ABSTRACT Objectives: This study was performed to investigate the anti-lipogenic effect and the mechanism of Jungmanbunso-hwan extract (JMBSH) on a cellular model of non-alcoholic fatty liver disease (NAFLD) caused by palmitate in HepG2 cells. Methods: The JMBSH was prepared, andhepg2 cells were treated with various concentrations of JMBSH in order to perform an MTT assay. The HepG2 cells were cultivated in palmitate-containing media with or without extract of JMBSH. The intracellular lipid content in the HepG2 cells was examined. The effects of JMBSH on sterol regulatory element-binding transcription factor-1c (SREBP-1c), acetyl-coa carboxylase (ACC), fatty acid synthase (FAS), stearoyl-coa desaturase-1 (SCD-1), and AMP-activated protein kinase (AMPK) activation in HepG2 cells were measured. Results: JMBSH did not reduce HepG2 cell viability under 1,000 μg/ml. JMBSH considerably decreased intracellular lipid accumulation caused by palmitate in HepG2 cells. JMBSH repressed expression of SREBP-1c, which mediates the induction of lipogenic genes (ACC, FAS, and SCD-1). JMBSH also activated AMPK, which plays animportant role in the regulation of hepatic lipid metabolism. Conclusions: This study suggested that JMBSH relieves hepatic steatosis by repressing SREBP-1c, which mediates the induction of lipogenic genes. The anti-lipogenic effect of JMBSH may also be related to the activation of AMPK. Therefore, JMBSH could potentially be applied to NAFLD treatment after further clinical studies. Key words: Jungmanbunso-hwan, NAFLD, SREBP-1c, AMPK Ⅰ. 서론 지방간이란간조직에중성지방이간무게의 투고일 : , 심사일 : , 게재확정일 : 교신저자 : 최창원전라남도순천시조례동 동신대학교부속순천한방병원 TEL: FAX: hepa0707@hanmail.net 이논문은 2016 년도동신대학교일반대학원한의학석사학위논문임. 5% 이상과다하게축적된경우를말하며, 유발원인중당뇨, 비만, 고지혈증등의대사이상에의해발병된경우를비알코올성지방간질환 (Non-alcoholic fatty liver disease) 이라고한다 1,2. 비알코올성지방간질환은단순지방간증 (hepatic steatosis) 과함께지방간염 (steatohepatitis), 간섬유화 (hepatic fibrosis), 간경변증 (liver cirrhosis) 에이르는보다넓은의미의개념으로사용되고있다

2 이지원 최창원 전상윤 한창우 하예진 2004년우리나라의비알코올성지방간질환유병률은 11.5% 였으나, 2010년 23.6% 로대략 2배정도로증가하였다 4. 간조직에비가역적인손상을유발할수있다는점과더불어우리나라에서의유병률도급증하고있어비알코올성지방간질환치료의중요성이점차대두되고있다. 이러한상황에서인슐린감작제, 지질저하제, 항산화제등다양한약물이비알코올성지방간질환치료제로활용되고있으나, 표준치료로공인된것은아직까지없다 5. 다만비만, 고지혈증및인슐린저항성이비알코올성지방간질환의발병원인으로작용하므로 2, 치료를위해서는식이조절과운동등을통한생활습관개선이요구된다 6. 비알코올성지방간질환은한의학적으로濕痰의대사장애로기인된다고보고있으며, 그에따라淸熱利濕, 健脾消導或消痰除濕의치법을활용한다 7. 중만분소환은元代李杲의 蘭室祕藏 8 에최초로수록된처방으로, 그뒤 醫學入門 9 을비롯한여러후세의서 에서中滿, 鼓脹, 氣脹, 水脹, 熱脹의치료에활용되었다. 중만분소환은의서마다본초구성에약간의차이는있지만黃芩 ( 酒炒 ), 厚朴, 黃連, 枳實, 半夏 ( 薑製 ) 澤瀉陳皮知母人蔘白朮薑黃砂仁乾薑茯苓豬苓甘草등의본초로구성되어있다 즉, 補氣健脾및祛痰의효능이있는육군자탕, 滲濕利水의효능이있는사령산, 淸熱和胃降逆의효능이있는사심탕, 理氣化濕和胃의효능이있는평위산이합하여진것으로 15 체내의濕熱및痰濁을제거하는작용을한다. 중만분소환에대한국내의연구는중만분소환이생쥐의肝損傷에미치는영향 15 이전부로활용가능성에대한조사가부족한상황이다. 이에저자는체내의濕熱및痰濁제거에효능이있는중만분소환의비알코올성지방간질환에대한효능과작용기전을연구하고자 HepG2 cell에 Palmitate 처리로지방증을유도한후, 중만분소환추출물을투여하여세포내지방량및 SREBP-1c (sterol regulatory element-binding transcription factor-1c), ACC(acetyl-CoA carboxylase), FAS(fatty acid synthase), SCD-1(stearoyl-CoA desaturase-1), AMPK(AMP-activated protein kinase) 에미치는영향을관찰하여유의한결과를얻었기에보고하는바이다. Ⅱ. 재료및방법 1. 재료 1) 세포주 Human hepatocellular carcinoma 세포주인 HepG2 cell은 American Type Culture Collection(Manassas, VA, USA) 에서구입하였다. 2) 약재본실험에사용한중만분소환 (Jungmanbunso-hwan) 은 醫學入門 9 에수록된처방으로, 약재들의비율은동일하게하되그용량은 1/2로줄여구성하였다 (Table 1). 약재는동신대학교부속순천한방병원에서구입하여사용하였다. Table 1. Composition of Jungmanbunso-hwan Herbal medicine name Pharmacognostic name Amount (g) 黃芩 ( 酒炒 ) Scutellariae Radix 12 厚朴 Magnoliae Cortex 10 黃連 Coptidis Rhizoma 10 枳實 Aurantii Immaturus Fructus 10 半夏 ( 薑製 ) Pinelliae Rhizoma 10 澤瀉 Alismatis Rhizoma 6 陳皮 Citri Pericarpium 6 知母 Anemarrhenae Rhizoma 6 人蔘 Ginseng Radix 5 白朮 Atractylodis Macrocephalae Rhizoma 5 薑黃 Curcumae Longae Rhizoma 5 砂仁 Amomi Fructus 4 乾薑 Zingiberis Rhizoma 4 茯苓 Poria 4 豬苓 Polyporus 2 甘草 Glycyrrhizae Radix 2 Total amount

3 중만분소환추출물이 Palmitate 로유발된비알코올성지방간 HepG2 cell 모델에미치는영향 3) 시약및기기 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(mtt), Nile Red, Palmitate 는 Sigma-Aldrich Co.(St. Louis, MO, USA) 에서구입하였다. β-actin antibody, SREBP-1 antibody, AMPK antibody, horseradish peroxidase(hrp)-conjugated goat anti-rabbit IgG는 Santa Cruz Biotechnology Inc.(Santa Cruz, CA, USA) 에서구입하였다. Phospho-AMPK antibody 는 Cell Signaling Technology Inc.(Danvers, MA, USA) 에서구입하였다. 그외 Olympus FV-1000 confocal laser scanning microscope(tokyo, Japan), NE-PER nuclear extraction kit(thermo Scientific, IL, USA), SuperSignal chemiluminescence detection kit(thermo Scientific, IL, USA), RNeasy Mini Kit (Qiagen, Hilden, Germany) 등이본연구를위해사용되었다. 2. 방법 1) 중만분소환추출물 (JMBSH) 제작중만분소환총량 101 g에 2.0 L 물을넣고 100 에서 1시간동안추출후여과하였다. 여과액을모아감압농축하고동결건조하여 10 g( 수율 10%) 의건조추출물을얻었다. 2) 세포배양 Human hepatocellular carcinoma 세포주인 HepG2 cell을 100 U/mL penicillin, 100 μg/ml streptomycin 및 10% heat inactivated fetal bovine serum을함유한 Dulbecco's modified Eagle's medium(dmem) 을이용하여, 37, 5% CO 2 환경에서배양하였다. 3) 세포활성도의측정세포활성도를측정하기위해 MTT assay 를시행하였다. HepG2 Cell을 96-well plate 에 10 4 cells/well 의밀도로 seeding 하였다. 하룻밤경과후준비된 JMBSH 1, 10, 100, 500, 1,000 μg/ml를투약하고 16시간배양하였다. 각 well에 MTT 500 μg/ml를처리한뒤, 세포들이 MTT를환원하여생성한 formazan crystal 을 DMSO를사용하여용해하였다. Background subtraction 630 nm로하고 570 nm에서의흡광도를측정하였다 4) 지방산처리지방증을유도하기위해 HepG2 cell에 Palmitate 를처리하였다. Palmitate 를 isopropanol 에 50 mm 농도가되도록용해시킨후최종투약농도 0.5 mm으로투여하였다. 1% bovine serum albumin을함유한 DMEM 을배양액으로사용하여 24시간배양하였다. 5) 세포내지방량측정 Palmitate 0.5 mm을함유한배지에서 24시간동안 HepG2 cell을배양하고, JMBSH 처리군에는 Nile Red staining 16시간전추출물 1,000 μg/ml 을투여하였다. Palmitate 및 JMBSH 처리된 HepG2 cell을 15분간상온에서 4% paraformaldehyde로고정하였다. PBS로세척한뒤, 5분간상온에서 Nile Red(100 ng/ml) 로처리하여세포내중성지방을염색하였다. Olympus FV-1000 confocal laser scanning microscope(tokyo, Japan) 로촬영했으며, Image J 1.48b software로정량화하였다. 6) SREBP-1c, AMPK에대한 Western blot 분석 JMBSH 투여에따른 SREBP-1c 와 AMPK의변화를관찰하고자 Western blot 분석을시행하였다. Palmitate 0.5 mm을함유한배지에서 HepG2 cell 을 24시간배양하였고, JMBSH 투여군에는검사 16시간전각각추출물 700, 1,000 μg/ml 을처리한후 Western blot으로 SREBP-1c 및 pampk protein level을측정하였다. Total cell lysate는 protease inhibitor Cocktail(Roche, Indianapolis, IN, USA) 를포함한 radioimmunoprecipitation assay(ripa) buffer(50 mm Tris-HCl(pH 8.0), 150 mm NaCl, 2 mm EDTA, 1% sodium orthovanadate, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS) 를사용하여추출하였다. Nuclear protein 은 NE-PER nuclear extraction kit(thermo Scientific, IL, USA) 를이용하여추출하였다. Bradford assay로단백량을정량한후 sodium dodecyl sulfate polyacrylamide gel 444

이지원 최창원 전상윤 한창우 하예진 electrophoresis(sds-page) 로전개하였고, Polyvinylidene fluoride(pvdf) membrane에전사하였다. 5% skim milk가들어있는 Tris-buffered saline(tbs) 로 1시간동안상온에서 blocking 하였다.

4 이지원 최창원 전상윤 한창우 하예진 electrophoresis(sds-page) 로전개하였고, Polyvinylidene fluoride(pvdf) membrane에전사하였다. 5% skim milk가들어있는 Tris-buffered saline(tbs) 로 1시간동안상온에서 blocking 하였다. 각각의 specific primary antibody와 4 에서하룻밤동안반응시킨후 horseradish peroxidase(hrp) 가결합되어있는 2차항체와 1시간동안상온에서반응시켰다. SuperSignal chemiluminescence detection kit(thermo Scientific, IL, USA) 를사용하여 band를검출했으며, Lamin A/C 와 Actin을 control 로사용했다. 7) ACC, FAS, SCD-1에대한 semi-quantitative RT-PCR JMBSH 투여에따른 ACC, FAS, SCD-1의발현을확인하기위해 semi-quantitative RT-PCR 을시행하였다. Palmitate 0.5 mm을함유한배지에서 HepG2 cell을 24시간배양하였으며, JMBSH 투여군에는검사 16시간전각각추출물 700, 1,000 μg/ml 을처리한후 semi-quantitative RT-PCR로 ACC, FAS, SCD-1 의 mrna level을측정하였다. total RNA를추출하기위해 RNeasy Mini Kit(Qiagen, Hilden, Germany) 를사용했고, M-MLV Reverse Transcriptase(Promega, Madison, WI, USA) 를사용하여추출된 total RNA를역전사하여 cdna를합성하였다. TaqPCRx DNA Polymerase(Invitrogen, Carlsbad, CA, USA) 와각각의 primer(bioneer) 를사용하여합성된 cdna 를증폭하였으며, 사용된각 primer 는다음과같다 (Table 2). 증폭된 DNA 는 1.2% agarose gel에전기영동및 ethidium bromide 로염색한후자외선으로 band 를확인하였으며 GAPDH 를 control로사용했다. 으로판단하였다. Table 2. Primer Sequences Used in Semi-quantitative RT-PCR Primer Primer sequence SREBP-1c 5'-CAGTGGAGGGAACACAGACG-3 5'-AAAGACTGGGCTGTCAGGCT-3 ACC 5'-GGAACAGTGTGCGGTGAAAC-3 5'-TCACTAGTGATCCGAGCAGC-3 FAS 5'-GACATCGTCCATTCGTTTGTG-3 5'-GTTGACATTGTACTCGGCGG-3 SCD-1 5'-GCCCCTCTACTTGGAAGACG-3 5'-CGAGCTTTGTAAGAGCGGTG-3 GAPDH 5'-AAGGGTCATCATCTCTGCCC-3 5'-GTGATGGCATGGACTGTGGT-3 Ⅲ. 결과 1. 세포활성도에미치는영향 JMBSH 이세포활성도에미치는영향을확인하기위해 MTT Assay 를시행한결과, 대조군의 MTT 환원율 100±6.09 에대해 JMBSH 1 μg/ml의농도에서 ±3.93%, 10 μg/ml 에서 ±3.09%, 100 μg/ml에서 98.87±8.75%, 500 μg/ml에서 98.74± 6.92%, 1,000 μg/ml에서 97.19±7.36% 가측정되어 1,000 μg/ml 이하의농도에서는 HepG2 cell에세포독성을유발하지않는것으로나타났다 (Fig. 1). 3. 통계분석통계에는 PASW Statistics Data Editor v18 Korean(SPSS Inc, Chicago, IL, USA) 프로그램을사용하였다. One-way analysis of variance(anova) test 로그룹간의평균을비교하였으며, 모든검정에서 P<0.05인경우유의한통계적차이가있는것 Fig. 1. Cytotoxic effect of JMBSH on HepG2 cells. To evaluate cytotoxic effect of JMBSH on HepG2 cells, an MTT assay was performed. The cells were treated with various concentration of JMBSH for 16h prior to MTT assay. Data are presented as the mean±sem (n=3). 445

중만분소환추출물이 Palmitate 로유발된비알코올성지방간 HepG2 cell 모델에미치는영향 2. 세포내지질축적에미치는영향세포내지방은 Nile Red에의해붉게염색되어관찰되었다. 세포내지질축적을관찰한결과, 대조군의형광강도 1±0.27에대해 JMBSH 1,000 μg/ml 만투여한경우에는 0.82±0.17, Palmitate만처리한 경우에는 6.06±0.

5 중만분소환추출물이 Palmitate 로유발된비알코올성지방간 HepG2 cell 모델에미치는영향 2. 세포내지질축적에미치는영향세포내지방은 Nile Red에의해붉게염색되어관찰되었다. 세포내지질축적을관찰한결과, 대조군의형광강도 1±0.27에대해 JMBSH 1,000 μg/ml 만투여한경우에는 0.82±0.17, Palmitate만처리한 경우에는 6.06±0.34, JMBSH 1,000 μg/ml 와 Palmitate 를함께처리한경우에는 2.65±0.24 가측정되어, Palmitate 와 JMBSH 을함께투여한군이 Palmitate 처리군에비하여세포내지질축적을유의하게감소시켰다 (Fig. 2). (A) (B) Fig. 2. Effect of JMBSH on intracellular lipid accumulation in HepG2 cells. HepG2 cells were treated with palmitate (0.5 mm, for 24h) and/or JMBSH (1,000 μg/ml for 16h). After incubation, the cells were stained with Nile red. (A) Intracellular lipid component were visualized by fluorescence microscopy (original magnification 400). (B) Cellular steatosis was quantified by measuring three random fields of view with image J 1.48b software and expressed as the mean±sem (n=3). *mark indicates significant difference compared to PA cells (P<0.05). 3. SREBP-1c 에미치는영향 SREBP-1c 는지질합성관련유전자들을조절하는주요전사인자이다 16. Lamin A/C에대한 SREBP-1c 의 protein 비율을분석한결과, 대조군이 1±0.09, JMBSH 700 μg/ml 을투여한경우는 1.09±0.07, JMBSH 1,000 μg/ml을투여한경우는 1.24±0.08, Palmitate 만처리한경우는 4.12±0.46, Palmitate와 JMBSH 700 μg/ml 을함께투여한경우는 2.97±0.12, Palmitate와 JMBSH 1,000 μg/ml을함께투여한경우는 1.53±0.24 로측정되어, Palmitate 와 JMBSH 을함께투여한군이 Palmitate 처리군에비하여 SREBP-1c protein 을유의하게 (P<0.05) 감소시켰다 (Fig. 3). Fig. 3. Effect of JMBSH on SREBP-1c expression in HepG2 cells. The expression level of SREBP-1c protein was measured by western blot analysis. The intensity of each band was measured with image J 1.48b software, and the relative quantity was calculated over lamin A/C. Data are presented as the mean± SEM (n=3). *mark indicates significant difference compared to only palmitate treated cells (P<0.05). 446

6 이지원 최창원 전상윤 한창우 하예진 4. ACC, FAS, SCD-1 에미치는영향 Lipogenesis에관여하는효소들인 ACC, FAS, SCD-1 은 SREBP-1c에의해발현이증가하여지방합성에영향을미친다 16. GAPDH에대한 ACC의 mrna 비율은대조군이 1±0.08, JMBSH 700 μg/ml 을투여한경우는 0.83±0.12, JMBSH 1,000 μg/ml 을투여한경우는 0.38±0.02, Palmitate 만투여한경우는 3.78±0.13, Palmitate와 JMBSH 700 μg/ml 을투여한경우는 0.59±0.24, Palmitate와 JMBSH 1,000 μg/ml을투여한경우는 0.36±0.08이었다. GAPDH에대한 FAS의 mrna의비율은대조군이 1±0.44, JMBSH 700 μg/ml을투여한경우는 0.94±0.06, JMBSH 1,000 μg/ml을투여한경우는 0.69±0.06, Palmitate 만투여한경우는 5.27±0.38, Palmitate와 JMBSH 700 μg/ml 을투여한경우는 0.63±0.06, Palmitate와 JMBSH 1,000 μg/ml을투여한경우는 0.41±0.07이었다. GAPDH 에대한 SCD-1의 mrna 의비율은대조군이 1±0.05, JMBSH 700 μg/ml을투여한경우는 0.63±0.12, JMBSH 1,000 μg/ml을투여한경우는 0.35±0.02, Palmitate만투여한경우는 3.49±0.16, Palmitate와 JMBSH 700 μg/ml 을투여한경우는 0.38±0.06, Palmitate와 JMBSH 1,000 μg/ml을투여한경우는 0.36±0.08이었다. 위의결과 Palmitate 와 JMBSH을함께투여한군은 Palmitate 처리군에비하여 ACC, FAS, SCD-1 mrna level 을유의하게 (P<0.05) 감소시켰다 (Fig. 4). (A) (B) (C) Fig. 4. Effect of JMBSH on expression of lipogenic genes in HepG2 cells. Expression of lipogenic genes, ACC, FAS, SCD-1 was determined by semi-quantitative RT-PCR. The intensity of each band was measured with image J 1.48b software, and the relative quantity was calculated over GAPDH, a housekeeping gene. Data are presented as the mean±sem (n=3). *mark indicates significant difference compared to only palmitate treated cells (P<0.05). 5. AMPK 활성화에미치는영향 AMP-activated protein kinase(ampk) 는활성화되면인산화형태로존재하게되어 phospho-ampk 가증가하게되며, pampk는 SREBP-1c 의발현을감소시킨다고알려져있다 17,18. Actin 에대한 pampk 의 protein 비율을분석한결과, 대조군이 1±0.03, JMBSH 700 μg/ml을투여한경우는 2.86±0.48, JMBSH 1,000 μg/ml을투여한경우는 18.82±0.97, Palmitate 만처리한경우는 2.12±0.22, Palmitate와 JMBSH 700 μg/ml 을함께투여한경우는 4.73±0.35, Palmitate와 JMBSH 1,000 μg/ml을함께투여한경우는 24.26±0.87로측정되어, Palmitate와 JMBSH 을함께투여한군이 Palmitate 처리군에비해 pampk protein level 을유의하게 (P<0.05) 증가시켰다 (Fig. 5). 447

7 중만분소환추출물이 Palmitate 로유발된비알코올성지방간 HepG2 cell 모델에미치는영향 Fig. 5. Effect of JMBSH on AMPK activation in HepG2 cells. The expression level of pampk protein was measured by western blot analysis. The intensity of each band was measured with image J 1.48b software, and the relative quantity was calculated over Actin. Data are presented as the mean± SEM (n=3). *mark indicates significant difference compared to only palmitate treated cells (P<0.05). Ⅳ. 고찰 비알코올성지방간질환은임상적으로무증상이거나, 비특이적소화기증상및피로감, 우상복부불쾌감이동반되기도한다 19. 비알코올성지방간질환은염증이동반되지않는경우는비교적양호한예후를갖지만 20 지방간염이발생하면환자의절반에서간섬유화, 15% 에서간경변이발생하였고, 3% 에서간부전으로진행되거나간이식을필요로하였다 21. 현재비알코올성지방간질환의공인된치료법은없으며운동, 식이, 체중감량등생활습관의개선과함께당뇨, 고지혈증등의연관질환치료를중심으로하면서간보호제등을사용하고있다 22. 이에비알코올성지방간질환의새로운치료제개발이절실히요구되는상황이다. 최근한의학계에서도비알코올성지방간질환에단미제 혹은복합처방 을이용한동물실험및세포연구가진행되고있다. 비알코올성지방간질환의발생과연관된원인 중하나인당뇨는한방의消渴에해당되며 30 素問奇病論, 素問通評虛實篇, 靈樞逆順肥瘦論 31 등에서비만과식이를원인으로꼽았다. 한의학적으로비만은膏梁厚味로인한脾胃기능실조등의원인으로濕痰이생성되어수액대사장애를야기하여발생한다 32. 고지혈증은한의학적으로嗜食肥甘厚味의外因및肝脾腎機能失調의內因으로인해痰濁, 濕熱, 瘀血이생성되어발병한다 33. 이렇듯비알코올성지방간질환의발생과관계된것으로알려져있는원인질환들모두체내의濕痰과유의한관계가있다. 이에저자는체내의濕熱및痰濁을제거하는효능을갖고있는중만분소환이비알코올성지방간질환치료에활용될수있을것이라생각하고본연구를시행하였다. 본실험에서는 HepG2 cell에 palmitate를처리하여비알코올성지방간질환세포를배양하였다. JMBSH 이간세포내지방축적을줄이는지확인하기위해 palmitate 와함께 JMBSH 을처리하였다. Nile red staining을통해 JMBSH 을함께처리한경우지방축적이유의하게감소하는것을확인하였다. JMBSH 이간내지질축적을감소시키는기전을확인하기위해 Western blot 을통해 JMBSH 이 SREBP-1c 에미치는영향을관찰하였다. SREBP-1c 는간에서지질의합성을유도하는주요전사활성인자로 ACC, FAS, 및 SCD-1과같은유전자들의발현을유도시켜간내지방합성을촉진한다 16. JMBSH 을함께처리한경우 SREBP-1c 가유의하게감소하였음을확인하였다. 또한 semi-quantitative RT-PCR 을통해 JMBSH 을함께처리한경우 SREBP-1c에의해발현이유도되는 ACC, FAS, SCD-1 역시유의하게감소하였음을확인하였다. 따라서 JMBSH이간세포내지방축적을완화시키는작용은 SREBP-1c 억제를통한 ACC, FAS, SCD-1의발현감소로인해나타난다는것을알수있었다. AMPK 는세포내에너지가부족해지면활성화되어해당작용을일으켜에너지균형을회복시키 448

8 이지원 최창원 전상윤 한창우 하예진 게하는효소로서지방및탄수화물대사를조절하는데있어중요한역할을한다 34,35. AMPK 는활성화되면인산화형태로존재하므로 phospho-ampk 가증가하고, 이는 SREBP-1c 의발현을감소시키며 ACC의활성을저해시켜지방산합성을감소시킨다 17,18,36. 또한 carbohydrate responsive element-binding protein(chrebp), sn-glycerol-3-phosphate acyltransferase (GPAT), 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase) 의활성을저해하여 anti-lipogenic effect 를갖는다 본연구에서는 JMBSH 의 anti-lipogenic effect 가 AMPK 와관계가있는지확인하고자 HepG2 cell에 JMBSH을투여하고 pampk의변화를관찰 하였다. 그결과 JMBSH 은 pampk 단백을증가시킨다는점을확인할수있었다. 따라서 JMBSH 은 AMPK를활성화시켜 anti-lipogenic effect를나타내는것으로생각된다 (Fig. 6). 이를종합하면 JMBSH 은 AMPK 를활성화시켜 SREBP-1c 의억제를유발하고, 그에따라 ACC, FAS, SCD-1 의발현을감소시켜 anti-lipogenic effect를나타내는것으로사료된다. 본연구는세포단위실험이므로향후비알코올성지방간질환치료제로서의중만분소환활용가능성에대한지속적인동물실험및임상연구가필요할것으로생각된다. Fig. 6. Possible pharmacological mechanism of JMBSH. It seems that antilipogenic effect of JMBSH is mediated through activation of AMPK at least in part. Ⅴ. 결론중만분소환추출물이비알코올성지방간에미치는영향을연구하기위해 palmitate 처리로 HepG2 cell에지방증을유도한후, 세포내지방량및 SREBP-1c, ACC, FAS, SCD-1, AMPK 에미치는 영향을관찰한결과다음과같은결론을얻었다. 1) 중만분소환추출물은모든농도에서세포독성을유발하지않았다. 2) 중만분소환추출물은세포내지방축적을유의하게감소시켰다. 3) 중만분소환추출물은 lipogenesis 에관여하는주요전사인자인 SREBP-1c 를유의하게억제시켰다. 449

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농림수산식품부장관귀하 이보고서를 팥의대사성질환개선및기능성규명 에관한연구의최종보고서로제출 합니다 년 2 월 11 일 - 1 -

농림수산식품부장관귀하 이보고서를 팥의대사성질환개선및기능성규명 에관한연구의최종보고서로제출 합니다. 2013 년 2 월 11 일 - 1 - 연구기관명 : 부산대학교 연구책임자 : 정명호 연구원 : 손용해 연구원 : 장민경 - 2 - Ⅰ. 제목 팥의대사성질환개선및기능성규명 Ⅱ. 연구개발의목적및필요성 팥의대사성질환개선소재발굴과그기능성을과학적으로증명하기위해, 팥의당뇨,