Korean J. Plant Res. 31(2): (2018) Print ISSN Online ISSN Original Research Artic

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1 Korean J. Plant Res. 31(2): (2018) Print ISSN Online ISSN Original Research Article 박수빈 1, 송훈민 1,3, 김하나 1, 박광훈 2, 손호준 2, 엄유리 2, 박지애 1,3, 정진부 1,4 * 1 안동대학교생약자원학과, 2 국립산림과학원산림약용자원연구소, 3 국립백두대간수목원, 4 안동대학교농업과학기술연구소 Anti-Inflammatory Effect of Biji (Soybean curd residue) on LPS-Stimulated RAW264.7 Cells Su Bin Park 1, Hun Min Song 1,3, Ha Na Kim 1, Gwang Hun Park 2, Ho-Jun Son 2, Yurry Um 2, Ji Ae Park 1,3 and Jin Boo Jeong 1,4 * 1 Department of Medicinal Plant Resources, Andong National University, Andong 36729, Korea 2 Forest Medicinal Resources Research Center, National Institute of Forest Science, Yeongju 36040, Korea 3 Baekdudaegan National Arboretum, Bonghwa 36209, Korea 4 Agricultural Science and Technology Research Institute, Andong National University, Andong 36729, Korea Abstract - In this study, we evaluated anti-inflammatory effect of biji in LPS-stimulated RAW264.7 cells. Biji inhibited the generation of NO and PGE 2 through the suppression of inos and COX-2 expression. In addition, biji attenuated the expression of TNF-α and IL-1β induced by LPS. Biji blocked LPS-mediated IκB-α degradation and subsequently inhibited p65 nucleus accumulation in RAW264.7 cells, which indicates that biji inhibits NF-κB signaling. In addition, biji suppressed p38 phosphorylation induced by LPS. Our results suggests that biji may exert anti-inflammatory activity through blocking the generation of the inflammatory mediators such as NO, PGE 2, inos, COX-2, TNF-α and IL-1β via the inhibiting the activation of NF-κB and p38. From these findings, biji has potential to be a candidate for the development of chemoprevention or therapeutic agents for inflammatory diseases. Key words - Anti-inflammation, Biji, MAPK, NF-κB 서언 전세계적으로고령화사회에진입하고있는가운데경제력이있는노인층이증가하고, 건강에대한소비자들의니즈 (needs) 가확대되면서다양한복합원인에의한만성및난치성질환의증가로질병예방에위한식의약에대한연구개발과관련분야의성장은지속될것으로판단되고있고, 최근 Wellbeing 트렌드와건강에대한관심고조로인해우수한효능이기능성소재개발이요구되고있는실정이다. 비지 (Biji) 는두부또는두유를제조하는과정에서생산되는부산물로제조과정에서대두로부터수용성물질이빠져나간상태이지만인체에유용한성분이남아있는것으로알려져있다 * 교신저자 : jjb0403@anu.ac.kr Tel (Kim et al., 1996). 그러나비지는동물용사료로일부활용되고있으나쉽게부패되어대부분은폐기물로처리되고있는실정이다 (Kim et al., 1996). 최근비지가항비만 (Matsumoto et al., 2007, Yun et al., 2010, Choi et al., 2011), 항고지혈 (Matsumoto et al., 2007), 항산화및면역활성 (Shi et al., 2012) 효과가있는것으로보고되어비지를유용한자원으로이용하기위한기능성연구가진행되고있다. 염증은감염또는조직손상을비롯한다양한외부자극에대한생체내방어적반응이지만 (Zedler and Faist, 2006; Mariathasan and Monack, 2007), 과도한염증반응은활성화된염증세포가 nitric oxide (NO), prostaglandin E 2 (PGE 2 ), interlukin-1beta (IL-1β), tumor necrosis factor-alpha (TNF-α) 등만성염증매개인자의다량분리를유도하여과민성알러지질환, 만성염증, 류마티스관절염, 암등과같은다양한질환을유도한다고알려져있다 (Gracie et al., 1999; Kinne et al., 2000). 만성염증매개인자 c 본학회지의저작권은 ( 사 ) 한국자원식물학회지에있으며, 이의무단전재나복제를금합니다. This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

2 韓資植誌 Korean J. Plant Res. 31(2) : 117~123(2018) 의발현은염증신호에의한 nuclear factor-kappb (NF-κB) 와 mitogen-activated protein kinase (MAPK) 신호전달의활성화에기인한다고알려져있다 (Dong et al., 2002). 그리하여만성염증매개인자들의발현과이들유전자들의발현에있어주요신호전달분자인 NF-κB 와 MAPK 의활성조절은항염증제개발을위한주요한핵심분자타겟으로인식되고있고, 이들인자들의활성을억제하는신규후보소재의탐색연구가활발히진행되고있다 (Park et al., 2012). 현존하는항염증제는히드로코르티손, 베타메타손등의스테로이드계와아스피린, 이부르로펜등의비스테로이드계로이들항염증제는위염, 신장염, 심장질환등의부작용을초래하여천연으로부터안전한항염증관련식의약소재개발에대한연구가진행되고있다 (Makins and Ballinger, 2003; Dogne et al., 2006). 본연구는비지의항염증관련식의약소재적용을위해기초자료를확보하고자비지추출물의세포기반항염증활성을측정하였다. 재료및방법실험재료마우스대식세포 RAW264.7 의배양을위해사용된배지인 Dulbecco s Modified Eagle s Medium (DMEM)/F-12 1:1 Modified Medium (DMEM/F-12) 는 Lonza (Walkersville, MD, USA) 에서구매하였다. Lipopolysaccharide (LPS) 는 Sigma Aldrich (St. Louis, MO, USA) 에서구매되었고 western blot 분석을위한항체인 IκB-α, p65, p-erk1/2, total-erk1/2, p-p38, total-p38, TBP 및 β-actin 은 Cell Signaling (Bervely, MA, USA) 에서구매되었다. 비지추출물제조본연구에서사용된비지는경북안동에위치한안동농협더햇식품사업소에서국내산청자콩 (Glycine max, cv. Cheongjakong) 을이용하여두부생산공정중두유와비지를분리하는작업에서배출된생비지를열풍건조 (Forced Convenction Oven, Program-type, ENBIO Lab., Korea) 하여사용하였다. 비지추출물은건조된비지를증류수로비지무게당 20배를첨가하여 4 에서 48시간교반하면서추출 (Shaking Incubator, Hyang Scientific Equipment Co., LTD, Korea) 하였다. 추출후상등액을동결건조 (EYELA Freeze Dryer, SUNIL EYELA CO., LTD, Korea) 하여실험재료로사용하였다. 본연구에사용된비지원료인청자콩은중원대학교박재호교수에의해동정되었으며, 시료의표본은 ( 표본번호 Jeong-1001 (ANH)) 안동대학교생약자원학과에보관하고있다. RAW264.7 세포배양본연구에서사용된마우스대식세포 RAW264.7 은한국세포주은행에서구매되었고, RAW264.7세포는 Namkoong 등의방법 (Namkoong et al., 2015) 에따라배양하였다. RAW264.7세포는 10% fatal bovine serum (FBS), 100 U/ ml penicillin and 100 μg / ml streptomycin 이포함된 DMEM/F-12 배지로 37 에서 5% CO 2 하에서유지하였다. 시료처리를위해비지추출물은 1 phosphate-buffered saline (PBS) 에용해하였고, 대조구는 1 PBS 를사용하였다. Nitric oxide (NO) 측정 NO는 Namkoong 등의방법 (Namkoong et al., 2015) 을변경하여측정하였다. 마우스대식세포 RAW264.7 을 12-well plate 에 /well 이되도록분주하고 24시간동안배양하였다. 24 시간후비지추출물을 0, 0.2, 0.4, 0.8 또는 1.6 mg / ml농도로처리하고 2시간동안배양하였다. 2 시간후 LPS를 1 μg / ml의농도로처리하고 18시간배양하였다. Nitric oxide 의측정은 Griess reagent system (Promoga) 을이용하여측정하였다. Prostaglandin E 2 (PGE 2 ) 측정 PGE 2 는 Namkoong 등의방법 (Namkoong et al., 2015) 을변경하여측정하였다. 마우스대식세포 RAW264.7 을 12-well plate 에 /well이되도록분주하고 24시간동안배양하였다. 24시간후비지추출물을 0, 0.2, 0.4, 0.8 또는 1.6 mg / ml농도로처리하고 2시간동안배양하였다. 2 시간후 LPS를 1 μg / ml의농도로처리하고 18시간배양하였다. PGE 2 의측정은 ELISA Kit 를이용하여측정하였다. SDS-PAGE 및 western blot 분석비지추출물과 LPS 가처리된 RAW264.7 세포에서단백질을추출하기위해서세포를 1 phosphate-buffered saline (PBS) 로 3회세척한후, protease inhibitor cocktail (Sigma- Aldrich) 과 phosphatase inhibitor cocktail (Sigma-Aldrich) 이포함된 radioimmunoprecipitation assay (RIPA) buffer (Boston Bio Products, Ashland, MA, USA) 를 4 에서 30분간 -118-

3 처리하여 lysis 시켜단백질을얻었다. Bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL, USA) 로단백질정량후, 동일량의단백질을 12% SDS-acrylamide 에 loading 하고 PVDF membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA) 에이동시킨후 5% non-fat dry milk 로상온에서 1시간동안 blocking 하였다. 1시간후, 1차항체를 5% non-fat dry milk 에용해시켜 4 에서 14시간동안반응시킨후 membrane 을 0.05% tween-20 이포함된 tris-buffered saline (TBS-T) 로 5분간 3 회세척하였다. 그후 2차항체는 5% non-fat dry milk 에용해시켜 membrane 에상온에서 1시간처리하였고, TBS-T 로 5분간 3회세척후 membrane 은 ECL western blotting substrate (Amersham Biosciences, Piscataway, NJ, USA) 를이용하여단백질을확인하였다. Reverse transcriptase-polymerase chain reaction (RT-PCR) Total RNA 추출은비지추출물과 LPS가처리된 RAW264.7 세포로부터 RNeasy Mini kit (Qiagen, Valencia, CA, USA) 를이용하여수행되었고, cdna 는 1 μg의 total RNA 를 Verso cdna kit (Thermo Scientific, Pittsburgh, PA, USA) 를이용하여제조되었다. PCR 은 PCR master mix kit (Promega, Madison, WI, USA) 를이용하여수행되었고, 사용된 primer 는 Table 1과같다. 통계분석모든결과는 3회반복측정후평균 ± 표준편차로나타내었고, 처리간유의성은 Student s t-test 로검증하여 p-value 값이 0.05 미만일때통계적으로유의하다고판정하였다 (Microsoft Exel 2010). 결과및고찰비지추출물의 inos 와 COX-2 발현억제를통한 NO 및 PGE 2 생성억제효과 NO는감염또는조직손상을비롯한다양한외부자극으로대식세포가활성화되어생성되는것으로, inducible nitric oxide synthase (inos) 에의해 L-arginine으로부터생성된다. inos 에의한과도한 NO는패혈성쇼크, 류마티스관절염등과같은염증성질환을유발한다 (Laskin and Pendino, 1995, Wu and Morris, 1998; Bogdan, 2001). 또한염증반응시 cyclooxygenase-2 (COX-2) 에의해생성되는 PGE 2 도염증관련질병을유발하는것으로보고되고있다 (DuBois et al., 1994; Smith et al., 1994; Harris et al., 2002). 비지추출물의 NO와 PGE 2 생성억제효과를측정한결과, 비지추출물은 LPS 가처리된 RAW264.7 세포에서농도의존적으로 NO와 PGE 2 의생성을억제하였다 (Fig. 1A and 1B). NO 와 PGE 2 의생성은각각 inos 와 COX-2 에의해생성되기때문에, 비지추출물의 NO와 PGE 2 생성억제가 inos 와 COX-2 발현억제에기인하는지확인하기위해 RT-PCR 방법으로유전자발현을조사하였다. 그결과, 비지추출물은 NO와 PGE 2 생성억제와유사하게 inos 와 COX-2의발현을억제하였다 (Fig. 1C). 염증반응을유도하는인자인 NO와 PGE 2, 그리고이염증인자의생성을조절하는 inos 와 COX-2 의조절은항염증관련타겟으로중요하다고여겨지고있기때문에비지추출물의 inos 와 COX-2 발현조절을통한 NO와 PGE 2 생성억제는비지추출물의항염증소재적용에대한가능성을보여주는것으로판단된다. Table 1. Sequence of oligonucleotide primers used for RT-PCR Gene name inos COX-2 TNF-α IL-1β GAPDH Sequence Forward : 5 -aatggcaacatcaggtcggccatcact-3 Reverse : 5 -gctgtgtgtcacagaagtctcgaactc-3 Forward : 5 -ggagagactatcaagatagt-3 Reverse : 5 -atggtcagtagacttttaca-3 Forward : 5 -tccaggcggtgcctatgt-3 Reverse : 5 -cgatcaccccgaagttcagt-3 Forward : 5 -acctgtcctgtgtaaatgaaagacg-3 Reverse : 5 -ttggtattgcttgggatcc-3 Forward : 5 -ggcatggccttccgtgt-3 Reverse : 5 -ggtttctccaggcggca

4 韓資植誌 Korean J. Plant Res. 31(2) : 117~123(2018) Fig. 1. Inhibitory effect of biji on the generation of NO and PGE 2 through the suppression of inos and COX-2 expression in LPS-stimulated RAW264.7 cells. (A and B) RAW264.7 cells were pretreated with biji at the indicated concentrations for 2 h, and then co-treated with LPS (1 μg / ml ) for 18 h. NO and PGE 2 were measured according to Materials and Methods. (C) RAW264.7 cells were pretreated with biji at the indicated concentrations for 2 h, and then co-treated with LPS (1 μg / ml ) for 18 h. For RT-PCR analysis of the gene expression of inos and COX-2, total RNA was prepared. GAPDH was used as internal control for RT-PCR. *P < 0.05 compared to the cells without Biji. 비지추출물의 TNF-α 와 IL-1β 발현억제효과 TNF-α 와 IL-1β 는주요한만성염증유발사이토카인으로염증관련종양형성에관여하며, 이사이토카인의대량생산은내피세포의염증, 염증질환, 패혈성쇼크등의질환을유발한다고알려져있다 (Delgado et al., 2003). 그리하여이러한염증성사이토카인의발현을조절하는물질은염증성질환을예방할수있는가능성이있다고알려져있다 (Lee et al., 2011). 비지추출물이 TNF-α 와 IL-1β 의생성에미치는영향을평가하기위해, LPS가처리된 RAW264.7 세포에서의비지추출물의 TNF-α 와 IL-1β 의 mrna 발현조절을조사하였다. 비지추출물이처리되지 않은세포에서는 LPS에의해 TNF-α 와 IL-1β 의 mrna 발현이증가되었지만, 비지추출물은농도의존적으로 TNF-α 와 IL-1 β의 mrna 발현을감소시켰다 (Fig. 2). 본결과는비지추출물이 TNF-α 와 IL-1β 의 mrna 발현을억제하여항염증활성을나타내는것으로판단된다. 비지추출물의 RAW264.7 세포에서 NF-κB 의활성억제효과 NF-κB 신호전달은만성염증유발인자의생성에관여하는것으로알려져있다. LPS 와같은외부자극은 IκB-α 의분해를유도하여 p65 의핵내전이를유도하고, 핵내 p65 는만성염증 -120-

5 Fig. 2. Inhibitory effect of biji on the mrna expression of TNF-αand IL-1βin LPS-stimulated RAW264.7 cells. RAW264.7 cells were pretreated with biji at the indicated concentrations for 2 h, and then co-treated with LPS (1 μg / ml ) for 18 h. For RT-PCR analysis of the gene expression of TNF-αand IL-1β, total RNA was prepared. GAPDH was used as internal control for RT-PCR. *P < 0.05 compared to the cells without Biji. Fig. 3. Inhibitory effect of biji on NF-κB signaling in LPS-stimulated RAW264.7 cells. (A) RAW264.7 cells were pretreated with biji at the indicated concentrations for 2 h, and then co-treated with LPS (1 μg / ml ) for 15 min. Cell lysates were subjected to SDS-PAGE and the western blot was performed using antibody against IκB-α. Actin was used as internal control for western blot analysis. (B) RAW264.7 cells were pretreated with biji at the indicated concentrations for 2 h, and then co-treated with LPS (1 μg / ml ) for 30 min. After the treatment, cytoplasm and nucleus were isolated, respectively. Cell lysates were subjected to SDS-PAGE and the western blot was performed using antibody against p65. Actin and TBP were used as internal control for western blot analysis. *P < 0.05 compared to the cells without the treatment, and #P < 0.05 compared to the cells treated with LPS alone

韓資植誌 Korean J. Plant Res. 31(2) : 117~123(2018) 유발인자관련 DNA 에 binding 하여만성염증유발인자의발현을유도한다 (Moynagh, 2005). 그리하여염증질환개선을위해염증을유발하는인자의조절과더불어염증반응에관련된신호전달을조절하는것이중요하다.

6 韓資植誌 Korean J. Plant Res. 31(2) : 117~123(2018) 유발인자관련 DNA 에 binding 하여만성염증유발인자의발현을유도한다 (Moynagh, 2005). 그리하여염증질환개선을위해염증을유발하는인자의조절과더불어염증반응에관련된신호전달을조절하는것이중요하다. 비지추출물의 NF-κB 신호전달억제활성을평가하기위해비지추출물의 LPS 유도 IκB-α 분해와 p65의핵내전이억제를조사하였다. 비지추출물이처리되지않은세포에서는 LPS 에의해 IκB-α 의분해가유도되었지만비지추출물이처리된세포에서는유의적으로 IκB-α 의분해가억제되었다 (Fig. 3A). 또한 LPS는 p65의핵내전이를유도하였으나, 비지추출물은 LPS 에의해유도되는 p65 의핵내전이를농도의존적으로감소시켰다 (Fig. 3B). 본결과는비지추출물은 NF-κB 신호전달억제를통해항염증활성을나타내는것을보여준다. 비지추출물의 RAW264.7 세포에서 MAPK 의활성억제효과 Mitogen-activated protein kinases (MAPK) 는세포의증식, 분화및세포사멸등의생물학적반응을조절하는 serine/ threonine kinase 로서 NF-κB 신호전달과더불어염증반응을조절한다고알려져있다 (Gi et al., 2010). 비지추출물의 MAPK 의활성에미치는영향을평가하기위해, MAPK 구성인자인 ERK1/2 와 p38 의인산화를조사하였다. 비지추출물은 LPS에의해서유도되는 ERK1/2 의인산화에대한억제활성이없었으나 p38 의인산화는비지추출물에의해서농도의존적으로급격히억제되는것을확인하였다 (Fig. 4). 본결과는비지추출물은 MAPK 에서 p38 의활성화억제를통해항염증활성을나타내는것을보여준다. 적요 비지는대두가공시생산되는부산물로대부분폐기되고있는실정이지만, 최근비지를유용한자원으로이용하기위한기능성연구가진행되고있다. 그러나비지의항염증에대한연구가미비하여본연구진은비지추출물이마우스대식세포인 RAW264.7 에 LPS에의한염증반응에미치는영향을평가하였다. 본연구에서비지추출물은 NF-κB 와 p38 의활성억제를통해만성염증유발인자인 NO, inos, PGE 2, COX-2, TNF-α 및 IL-1β 의발현을억제하는것으로확인되었다. 따라서비지는독성과부작용이적은항염증관련식의약소재로활용될수있을것으로사료된다. 사사 본연구는농림축산식품부고부가가치식품기술개발사업 ( 과제번호 : ) 에의해이루어진것의일부이며, 이에감사드립니다. Fig. 4. Inhibitory effect of biji on MAPK signaling in LPSstimulated RAW264.7 cells. RAW264.7 cells were pretreated with biji at the indicated concentrations for 2 h, and then co-treated with LPS (1 μg / ml ) for 15 min. Cell lysates were subjected to SDS-PAGE and the western blot was performed using antibody against p-erk1/2, total-erk1/2, p-p38 and total-p38. Total-ERK1/2 and total-p38 were used as internal control for western blot analysis. *P < 0.05 compared to the cells without the treatment, and #P < 0.05 compared to the cells treated with LPS alone. References Bogdan, C Nitric oxide and the immune response. Nat. Immunol. 2: Choi, M.S., J.I. Kim, J.B. Jeong, S.B. Lee, J.N. Jeong, H.J. Jeong, E.W. Seo, T.Y. Kim, O.J. Kwon and J.H. Lim Suppressive effect of by-product extracts from soybean on adipocyte differentiation and expression of obesity-related genes in 3T3-L1 adipocytes. J. Life Sci. 21:

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α α α α α α α α 太陰調胃湯加減方 dbdb 마우스 肝에 대한 아디포사이토카인 및 발현에 미치는 영향 SREBPs 와 섞어서 하고 마커 또한 하여 전기 영동을 하였다 전기영동을 한 후에 에 를 쪼여서 각 를 확인하였다 이 를 프로그램을 이용해 수치화하 여 분석하였다 肝 조직 동결 절편 분리한 조직은 로 시간 동안 고정 시킨 후 에 세척한 후 물기를