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Effect of Ethanol Extracts from Defatted Perilla frutescens on LPS-induced Inflammation in Mouse BV2 Microglial Cells
Biomed Sci Lett 2018;24:398-404
Published online December 31, 2018;  https://doi.org/10.15616/BSL.2018.24.4.398
© 2018 The Korean Society For Biomedical Laboratory Sciences.

Sung-Gyu Lee*, and Hyun Kang†,*

Department of Medical Laboratory Science, College of Health Science, Dankook University, Cheonan-si, Chungnam 31116, Korea
Correspondence to: Hyun Kang. Department of Medical Laboratory Science, College of Health Science, Dankook University, Cheonan-si, Chungnam 31116, Korea. Tel: +82-41-550-3015, Fax: +82-41-559-7934, e-mail: hkang@dankook.ac.kr
Received October 13, 2018; Revised December 10, 2018; Accepted December 13, 2018.
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.
 Abstract

To evaluate the antioxidant and anti-neuroinflammatory effects of defatted Perilla frutescens extract (DPE) in lipopolysaccharide (LPS)-stimulated BV-2 microglial cells. Cell viabilities were estimated by MTT assay. LPS-stimulated BV-2 microglia were used to study the expression and production of inflammatory mediators such as nitric oxide (NO), inducible NO synthase (iNOS), Cyclooxygenase-2 (COX-2), and prostaglandin E2 (PGE2). Pretreatment with DPE prior to LPS treatment significantly inhibited excessive production of NO (10, 25, 50, 75, and 100 μg/mL) in a dose-dependent manner, and was associated with down regulation of expression of iNOS and COX-2. DPE also suppressed the LPSinduced increase in PGE2 level (10, 25, 50, 75, and 100 μg/mL) in BV-2 cells. Therefore, DPE can be considered as a useful therapeutic and preventive approach for the treatment of several neurodegenerative diseases.

Keywords : Defatted Perilla frutescens, Anti-inflammatory activity, Microglial cells, INOS, COX-2
꽌濡

깮솢닔以쓽 利앷 쓽猷뚭린닠쓽 닔以 뼢긽쑝濡 씤媛꾩쓽 닔紐낆씠 利앷맖뿉 뵲씪 끂솕愿젴 吏덈퀝뿉 愿븳 愿떖씠 넂븘吏怨 엳쑝硫, 듅엳 끂솕쓽 二쇰맂 썝씤쑝濡 븣젮吏 궛솕쟻 뒪듃젅뒪뿉 쓽빐 諛쒕퀝릺뒗 눜뻾꽦 뇤吏덊솚뿉 愿븳 뿰援ш 솢諛쒗엳 吏꾪뻾릺怨 엳떎(Li et al., 1999). 씤泥 궡뿉 솢꽦궛냼媛 怨쇰떎븯寃 깮꽦릺硫 빆궛솕 떆뒪뀥쓽 湲곕뒫씠 븯릺硫댁꽌 씤泥 궡쓽 빆궛솕怨꾩쓽 遺덇퇏삎씠 諛쒖깮븯寃 릺怨 씠뒗 빑怨 誘명넗肄섎뱶由ъ븘쓽 DNA 빀꽦 벑쓽 꽭룷湲곕뒫쓣 諛⑺빐븯寃 릺뼱 꽭룷媛 궗硫명븯寃 맂떎(Klein and Ackerman, 2003).

誘몄꽭븘援먯꽭룷(microglial cell)뒗 뇤 뿼利앹뿉 愿뿬븯뒗 二쇱슂 硫댁뿭꽭룷濡, 뇤떊寃 떆뒪뀥뿉꽌 닕二쇰줈遺꽣쓽 諛⑹뼱 議곗쭅옱깮뿉 二쇱슂븳 뿭븷쓣 븳떎(Kreutzberg, 1996). 洹몃윭굹 誘몄꽭븘援먯꽭룷쓽 怨쇳솢꽦솕뒗 뿼利앷낵 媛숈 뇤넀긽 썑 鍮좊Ⅴ寃 씪뼱굹硫, 눜뻾꽦떊寃쎌쭏솚쓽 썝씤씠 릺怨 엳떎(Chao et al., 1995). 誘몄꽭븘援먯꽭룷쓽 留뚯꽦쟻씤 솢꽦솕 씠뿉 닔諛섎릺뒗 뿼利 쑀룄 씤옄뱾쓽 諛쒗쁽怨 깮꽦 븣痢좏븯씠癒몃퀝(Alzheimer’s disease, AD), 뙆궓뒯蹂(Parkinsonism disease, PD), 떎諛쒖꽦 寃쏀솕利(multiple sclerosis, MS) 벑쓽 떎뼇븳 떊寃쎌쭏솚쓽 議곗쭅븰쟻 吏몴濡 利앸챸릺怨 엳떎(Nakajima and Kohsaka, 1993; Gonzalez-Scarano and Baltuch, 1999; Drew et al., 2003). 뵲씪꽌, 誘몄꽭븘援먯꽭룷濡쒕꽣 遺꾨퉬릺뒗 뿼利 留ㅺ컻臾쇱쓣 議곗젅븯뒗 寃껋 떊寃쏀눜뻾꽦 吏덊솚쓽 吏꾪뻾쓣 삁諛⑺븯嫄곕굹 떊寃 넀긽쓽 移섎즺瑜 쐞븳 諛⑸쾿쑝濡 젣떆릺怨 엳떎.

誘몄꽭븘援먯꽭룷쓽 怨쇰룄븳 솢꽦솕뒗 lipopolysaccharides (LPS), β-amyloid related proteins, human immunodeficiency virus (HIV)쓽 쇅遺 떒諛깆쭏씤 gp120怨 媛숈 臾쇱쭏뱾뿉 쓽빐 씪뼱굹뒗 寃껋쑝濡 븣젮졇 엳쑝硫, nitric oxide (NO), prostaglandin E2 (PGE2), interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) 벑怨 媛숈 뿼利앹꽦 留ㅺ컻 씤옄 諛 솢꽦궛냼醫(reactive oxygen species, ROS)쓽 遺꾨퉬瑜 珥됱쭊븯뿬 떊寃쎈룆꽦쓣 쑀諛쒗븳떎(Galea et al., 1996; Laflamme and Rivest, 2001). LPS뿉 쓽븳 떊寃쎌뿼利앸컲쓳 룆꽦臾쇱쭏濡 옉슜븯뒗 NO굹 PG쓽 깮꽦뿉 쁺뼢쓣 誘몄튂뒗 inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2)뿉 쓽빐 議곗젅릺湲곕룄 븯뒗뜲 씠윭븳 씪젴쓽 怨쇱젙 nuclear fator (NF)-κB activator protein (AP)-1쓽 떊샇쟾떖 議곗젅 湲곗쟾怨 諛젒븳 뿰愿꽦씠 엳뒗 寃껋쑝濡 븣젮졇 엳떎(Colton, 2009; Park and Chun, 2016).

뱾源(Perilla frutescens var. japonica Hara)뒗 轅怨쇱쓽 븳빐 궡씠 삉뒗 뵪濡쒖꽌 以묎뎅 以묐궓遺, 씪蹂, 룞궓븘떆븘 벑뿉꽌 옱諛곕릺뼱 솕쑝硫 슦由щ굹씪 쟾뿭뿉꽌룄 떎뼇븳 醫낆쓽 뱾源④ 옱諛곕릺怨 엳떎(Ohara et al., 2003). 뱾源⑤뒗 遺덊룷솕 吏諛⑹궛씤 삱젅궛(oleic acid), 由щ젅궛(linoleic acid), 由щ젋궛(linolenic acid)씠 90% 씠긽쓣 씠猷④퀬 엳뼱 泥대궡뿉꽌쓽 냼솕 씉닔媛 鍮좊Ⅴ怨 룞臾쇱꽦 吏諛⑹뿉꽌 떎웾 뼸쓣 닔 뾾뒗 븘닔 吏諛⑹궛룄 異⑸텇엳 怨듦툒諛쏆쓣 닔 엳떎(Lee et al., 2002). 뱾源⑥쓽 슜룄瑜 蹂대㈃ 옂怨 醫낆떎 遺遺 떇슜 諛 빟슜쑝濡 씠슜릺怨 엳吏留, 뿬윭 媛吏 媛怨듬떒怨꾨 嫄곗퀜 泥쒖뿰떇슜깋냼, 솕옣뭹쓽 썝猷뚮굹 怨듭뾽슜 옱猷뚮줈룄 솢슜릺怨 엳떎(Ohara et al., 2003). 吏諛⑹궛 씠쇅쓽 뱾源⑥쓽 꽦遺꾩쑝濡쒕뒗 sesamol, sesamin, sesamolin 벑쓽 lignan 솕빀臾쇱쓣 鍮꾨’븳 뿬윭 럹꽦 솕빀臾쇱씠 븿쑀릺뼱 엳쑝硫, 씠뱾쓽 빆궛솕 옉슜씠 蹂닿퀬릺怨 엳떎(Hwang and Ko, 1980; Wang and Choe, 2012).

뵲씪꽌 蹂 뿰援ъ뿉꽌뒗 뱾源 遺궛臾쇱쓽 씠슜媛移섎 넂씠뒗 諛⑸쾿쑝濡 뱾源⑤컯 異붿텧臾쇱뿉꽌 빆떊寃쎌뿼利 솢꽦씠 엳뒗吏瑜 뿰援ы븯뒗 寃껋씠 紐⑹쟻씠뿀쑝硫, 뼢썑 湲곕뒫꽦 떇뭹 諛 쓽빟뭹쑝濡쒖꽌쓽 媛쒕컻 媛뒫꽦룄 寃넗븯떎.

옱猷 諛 諛⑸쾿

떎뿕옱猷

蹂 뿰援ъ뿉 궗슜맂 뱾源⑤컯 異⑸턿 쓬꽦 肄붾찓媛 쉶궗뿉꽌 깮 뱾源⑤ 李⑹쑀 썑 궓 遺궛臾쇱쓣 닔嫄고븯뿬 嫄댁“ 썑 궗슜븯떎.

뿉깂삱 異붿텧臾 젣議

뱾源⑤컯 誘뱀꽌湲곕 씠슜븯뿬 議곕텇뇙븯뿬 떆猷 100 g뿉 빐 10諛곗쓽 70% 뿉깂삱쓣 꽔뼱 3씪 룞븞 異붿텧븳 썑, 뿬怨쇱(Whatman No. 2, Maidstone, England)瑜 궗슜븯뿬 뿬怨쇳븯떎. 뿬怨쇳븳 異붿텧븸쓣 媛먯븬냽異뺢린(N-1000S-WD, Eyela Co., Tokyo, Japan)瑜 궗슜븯뿬 냽異 썑, 룞寃곌굔議(FDU-1100, Eyela Co., Tokyo, Japan)븯뿬 뱾源⑤컯 異붿텧臾(defatted Perilla frutescens extract, DPE)쓣 젣議고븯떎.

珥 뤃由ы럹 諛 뵆씪蹂대끂씠뱶 븿웾 痢≪젙

DPE쓽 珥 뤃由ы럹 솕빀臾쇱쓽 븿웾 Folin-Denis踰뺤쓣 쓳슜븯뿬 痢≪젙븯떎(Folin and Denis, 1912). DPE瑜 슜븸 1 mL뿉 끃씠怨 냽룄蹂꾨줈 씗꽍 썑, 씗꽍븳 깦뵆쓣 96 wellplate뿉 60 μL뵫 遺꾩<븯떎. 利앸쪟닔 1:1 씗꽍븳 Folin 떆빟쓣 룞웾 泥④븯怨 3遺꾧컙 諛섏쓳떆궓 썑 10% sodium carbonate(Na2CO3)쓣 룞웾 꽔怨 1떆媛 룞븞 諛섏쓳떆耳 700 nm뿉꽌 씉愿묐룄瑜 痢≪젙븯떎. Gallic acid瑜 씠슜븳 몴以怨≪꽑 떆猷 痢≪젙 諛⑸쾿怨 룞씪븯寃 痢≪젙븯뿬 옉꽦븯떎.

珥 뵆씪蹂대끂씠뱶 븿웾 Nieva Moreno 벑쓽 諛⑸쾿쓣 蹂삎븯뿬 痢≪젙븯떎(Nieva et al., 2000). DPE瑜 냽룄蹂꾨줈 씗꽍븳 떆猷 100 μL 80% 뿉깂삱 860 μL쓣 샎빀븳 샎빀臾쇱뿉 10% aluminium nitrate (Sigma Co.) 20 μL 1 M potassium acetate (Sigma Co.) 20 μL쓣 媛븯뿬 떎삩뿉 40遺 諛⑹튂븳 뮘 415 nm뿉꽌 씉愿묐룄瑜 痢≪젙븯떎. 珥 뵆씪蹂대끂씠뱶 븿웾쓽 몴以臾쇱쭏濡 quercetin (Sigma Co.)쓣 룞씪븳 諛⑸쾿쑝濡 痢≪젙븯뿬 븿웾쓣 援ы븯떎.

꽭룷二 諛곗뼇

꽭룷깮議댁쑉 諛 빆뿼利 슚뒫 寃利앹뿉 궗슜맂 꽭룷二쇰뒗 誘멸뎅 븯踰꾨뱶 쓽怨쇰븰뿉꽌 遺꾩뼇諛쏆 microglia cell line BV-2瑜 궗슜븯떎. BV-2 꽭룷뒗 RPMI1640 諛곗(Gibco, BRL, USA)뿉 10% fetal bovine serum (FBS; Gibco), 100 μg/mL penicillin (Gibco) 洹몃━怨 100 μg/mL streptomycin (Gibco)쓣 泥④븯뿬 5% CO2, 37°C 諛곗뼇湲곗뿉꽌 꽭룷媛 80~90% 옄옄쓣 븣 怨꾨 諛곗뼇븯떎.

꽭룷깮議댁쑉 痢≪젙

LPS 諛 DPE媛 꽭룷깮議댁뿉 誘몄튂뒗 쁺뼢쓣 솗씤븯湲 쐞빐 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide(MTT) 떆빟쓣 씠슜븯뿬 痢≪젙븯떎. 96-well plate뿉 1×105 cell/mL 꽭룷瑜 180 μL뵫 遺꾩<븯뿬 12떆媛 씠긽 CO2 諛곗뼇湲곗뿉꽌 諛곗뼇븳 떎쓬, DPE瑜 냽룄蹂꾨줈 泥섎━븯뿬 24떆媛 諛곗뼇븯떎. 諛곗뼇븳 썑 諛곗뼇븸쓣 젣嫄고븯怨 0.5 mg/mL MTT媛 븿쑀릺뼱 엳뒗 諛곗 200 μL瑜 泥④븳 떎쓬 4떆媛꾨룞븞 諛곗뼇 썑 諛곗뼇븸쓣 젣嫄고븯怨 dimethylsulfoxide (DMSO) 100 μL瑜 泥④븯뿬 깮꽦맂 formazone 寃곗젙쓣 슜빐떆궓 썑, microplate reader瑜 씠슜븯뿬 540 nm뿉꽌 痢≪젙븯떎. 꽭룷깮議댁쑉 LPS瑜 泥섎━븯吏 븡 議곌뎔怨 鍮꾧탳븯뿬 諛깅텇쑉(%)濡 굹궡뿀떎.

NO 깮꽦빐 슚怨

DPE쓽 빆뿼利 슚뒫쓣 遺꾩꽍븯湲 쐞븯뿬 뿼利 쑀諛 씤옄씤 LPS DPE瑜 媛 냽룄蹂꾨줈 泥섎━븳 떊寃쎄탳꽭룷뿉꽌 깮궛릺뒗 NO瑜 뼲젣븯뒗 슚뒫씠 엳뒗吏 솗씤븯떎. NO 痢≪젙 24 well plate뿉 꽭룷瑜 1×105 cell/mL쓣 seeding븳 썑, LPS DPE瑜 냽룄蹂꾨줈 泥④븳 썑, 24 h incubator뿉꽌 諛곗뼇븯떎. 洹 썑, 諛곗뼇븸怨 Griess reagent (Sigma Co.)瑜 媛곴컖 100 μL뵫 룞웾 샎빀븯뿬 諛섏쓳떆궓 썑, 540 nm뿉꽌 씉愿묐룄 媛믪쓣 痢≪젙븯떎.

PGE2 깮꽦웾 痢≪젙

꽭룷 諛곗뼇븸 궡쓽 PGE2 뼇쓣 痢≪젙븯湲 쐞빐 R&D systems(Minneapolis, USA)뿉꽌 commercial competitive enzyme immunoassay kit瑜 援ъ엯븯뿬 떎뿕븯떎. 꽭룷뿉 DPE瑜 泥섎━븯怨 100 ng/mL쓽 LPS瑜 泥섎━븯떎. 18떆媛 썑 꽭룷諛곗뼇븸쓣 뼸뼱 PGE2 痢≪젙뿉 궗슜븯떎. 諛곗뼇맂 諛곗瑜 goat anti-mouse濡 coating맂 96 well plate뿉 媛곴컖 100 μL뵫 loading븯怨, 뿬湲곗뿉 1李⑦빆泥 슜븸 50 μL PGE2 conjugate 50 μL뵫 泥④븯뿬 4°C뿉꽌 overnight 떆耳곕떎. Washing buffer濡 4쉶 꽭泥숉븯怨 substrate solution쓣 200 μL뵫 泥섎━븯뿬 30遺꾧컙 諛섏쓳떆궓 썑, stop solution쓣 50 μL 泥섎━븳 썑 450 nm뿉꽌 痢≪젙븯떎.

Western blot analysis

쟾湲곗쁺룞쓣 쐞븳 떒諛깆쭏 떆猷뚯쓽 異붿텧 꽭룷瑜 ice 긽뿉꽌 tris buffered saline (TBS, 20 mM Tris-HCl, pH 8.0, 137 mM NaCl)쑝濡 3쉶 꽭泥숉븳 썑, lysis buffer (TBS, 1% NP-40, 1 mM sodium orthovanadata, 10 μg/mL aprotinin, 10 μg/mL leupeptin 諛 1 mM PMSF)瑜 泥④븯뿬 4°C뿉꽌 30遺꾧컙 異붿텧븯뿬 12,000×g뿉꽌 10遺꾧컙 썝떖 遺꾨━ 썑 긽벑븸쓣 紐⑥븯떎. 異붿텧맂 떒諛깆쭏쓽 젙웾 Bradford protein assay kit瑜 궗슜븯뿬 젙웾 썑, 룞씪븳 뼇쓽 떒諛깆쭏쓣 궗슜븯뿬 sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)濡 遺꾨━떆耳곕떎. 遺꾨━맂 떒諛깆쭏쓣 nitrocellulose membrane뿉 transfer 븯怨, 씠 membrane쓣 빆泥댁쓽 鍮 듅씠쟻 寃고빀쓣 李⑤떒븯湲 쐞븯뿬 blocking buffer (5% non-fat milk)뿉꽌 30遺꾧컙 諛섏쓳떆궓 썑, 媛 寃利 떒諛깆쭏뿉 븳 빆泥 (anti-iNOS, anti-COX2)瑜 媛븯뿬 4떆媛 룞븞 諛섏쓳떆耳곕떎. 씠뼱꽌 0.1% Tween 20쓣 븿쑀븳 TBST 슜븸쑝濡 20 遺꾧컙 꽭泥숉븳 떎쓬, secondary antibody濡 諛섏쓳떆耳곕떎. 씠뼱꽌 ECL system쑝濡 諛섏쓳떆궓 썑 X-ray 븘由 긽뿉꽌 떒諛깆쭏쓣 솗씤븯떎.

넻怨꾩쿂由

떎뿕쓽 寃곌낵媛믪 SPSS Window program (ver. 10.0)쓣 씠슜븯뿬 痢≪젙븯쑝硫, 紐⑤뱺 痢≪젙媛믪 룊洹좉컪 ± 몴以렪李⑤줈 굹깉떎. 넻怨꾪븰쟻 遺꾩꽍 student’s t-test瑜 떎떆븯怨, 쑀쓽꽦 P<0.05濡 븯떎.

寃곌낵

뤃由ы럹 諛 뵆씪蹂대끂씠뱶 븿웾鍮꾧탳

DPE뿉 議댁옱븯뒗 珥 뤃由ы럹 諛 뵆씪蹂대끂씠뱶 븿웾 媛곴컖 gallic acid, quercetin쓣 湲곗臾쇱쭏濡 븯뿬 痢≪젙븯떎(Fig. 1). 洹 寃곌낵, DPE쓽 珥 뤃由ы럹 븿웾 48.32 mg/g쓽 븿웾쓣 굹궡뿀쑝硫, 珥 뵆씪蹂대끂씠뱶 븿웾 17.32 mg/g쑝濡 븿쑀븯怨 엳쓬쓣 솗씤븯떎.

Fig. 1.

Total polyphenols and flavonoids contents in DPE. Milligrams of total polyphenol content/g of samples based on gallic acid as standard. Milligrams of total flavonoid content/g of samples based on quercetin as standard. Each value is mean ± S.D. (n=3).



꽭룷깮議댁쑉 痢≪젙

DPE媛 꽭룷깮議댁뿉 겮移섎뒗 쁺뼢쓣 솗씤븯湲 쐞빐 BV-2 꽭룷二쇱뿉 LPS DPE瑜 냽룄蹂꾨줈 泥섎━븯떎. 洹 寃곌낵, LPS 100 ng/mL瑜 떒룆쑝濡 泥섎━븯쓣 븣, 泥섎━븯吏 븡 援곗뿉 鍮꾧탳븯뿬 겙 李⑥씠媛 뾾뿀떎. 삉븳, LPS DPE瑜 媛숈씠 泥섎━븯쓣 븣뿉룄 10, 25, 50, 75, 100 μg/mL 紐⑤뱺 냽룄뿉꽌 cell viability媛 95% 씠긽쑝濡 꽭룷뿉 븳 룆꽦씠 뾾뒗 寃껋쓣 솗씤븯떎(Fig. 2).

Fig. 2.

Effect of DPE on cytotoxicity in BV-2 cells. DPE was treated with various concentrations in BV-2 cells for 24 h. Values are expressed as the mean ± SD (n=3) of determinations made in triplicate experiments.



DPE쓽 NO 깮꽦빐 슚怨

DPE쓽 NO 깮꽦빐 슚怨쇰 솗씤븯湲 쐞븯뿬 LPS濡 옄洹밸맂 BV-2 꽭룷二쇱뿉 DPE瑜 10~100 μg/mL 냽룄蹂꾨줈 泥섎━븳 썑, NO쓽 깮꽦냽룄瑜 솗씤븯떎. LPS瑜 떒룆 泥섎━븯쓣 븣, NO 븿웾씠 23.2 μM濡 利앷릺뒗 寃껋쓣 솗씤븯떎. 씠뿉 諛섑빐, DPE瑜 泥섎━븯쓣 븣 10, 25, 50, 75, 100 μg/mL쓽 냽룄뿉꽌 媛곴컖 17.5, 16.1, 13.2, 8.5, 4.3 μM쓽 냽룄濡 NO媛 媛먯냼릺뒗 寃껋쓣 솗씤븷 닔 엳뿀떎. 듅엳, 50 μg/mL 냽룄뿉꽌遺꽣 NO쓽 깮꽦웾씠 50% 씠긽 媛먯냼릺뒗 寃껋쓣 솗씤븯떎(Fig. 3).

Fig. 3.

Effect of DPE on NO production in BV-2 cells. DPE was treated with various concentrations in BV-2 cells for 24 h. Values are expressed as the mean ± SD (n=3) of determinations made in triplicate experiments. *Statistically significant difference from the LPS control group, as determined by the student’s t-test as P<0.05.



Prostaglandin E2 (PGE2) 깮꽦웾

蹂 뿰援ъ뿉꽌 LPS濡 쑀룄맂 BV-2 꽭룷뿉꽌 DPE쓽 PGE2 깮꽦웾쓣 솗씤븳 寃곌낵, DPE뒗 LPS留뚯쓣 泥섎━븳 援곗뿉 鍮꾪빐 75, 100 μg/mL 냽룄뿉꽌 쑀쓽꽦 엳뒗 PGE2 깮꽦웾 媛먯냼媛 솗씤릺뿀떎(Fig. 4).

Fig. 4.

Effect of DPE on PGE2 production in BV-2 cells. DPE was treated with various concentrations in BV-2 cells for 24 h. Values are expressed as the mean ± SD (n=3) of determinations made in triplicate experiments. *Statistically significant difference from the LPS control group, as determined by the student’s t-test as P<0.05.



iNOS COX-2 깮꽦뼲젣

DPE쓽 NO PGE2 깮꽦뿉 愿뿬븯뒗 iNOS COX-2쓽 슚냼瑜 솗씤븯湲 쐞븯뿬 씠뱾 슚냼쓽 꽭룷 궡 諛쒗쁽쓣 痢≪젙븯떎. BV-2 cell뿉 DPE瑜 50, 100 μg/mL 2媛쒖쓽 냽룄濡 15떆媛 泥섎━븳 뮘, LPS濡 20遺 룞븞 옄洹뱁븯怨 꽭룷 궡 떒諛깆쭏쓣 遺꾨━瑜 븯뿬 썾뒪꽩 釉붾’ 遺꾩꽍 諛⑸쾿쓣 넻븯뿬 씠뱾 떒諛깆쭏쓽 諛쒗쁽쓣 솗씤븯떎(Fig. 5). 洹 寃곌낵 DPE쓽 泥섎━냽룄뿉 鍮꾨븯뿬 媛먯냼븯뒗 寃껋쑝濡 솗씤릺뿀떎.

Fig. 5.

Effect of DPE on iNOS and COX-2 protein expressional levels in LPS-stimulated BV-2 microglial cells. The expression levels of iNOS and COX-2 production in the LPS-stimulated BV-2 cells by indicated concentrations (50, 100 μg/mL) of the DPE was monitored by western blot analyses with the specific antibodies against iNOS and COX-2. The internal control used was β-actin.


怨좎같

뇤뿉꽌쓽 뿼利 諛섏쓳씠 留뚯꽦쟻쑝濡 吏냽씠 릺硫 NO, ROS, 뿼利앹꽦 궗씠넗移댁씤씠 遺꾨퉬릺怨 씠윭븳 뿼利 쑀諛쒕Ъ吏덈뱾 뇤꽭룷瑜 눜솕떆耳 뿬윭 媛吏 吏덈퀝쓣 쑀諛쒗븯뒗 寃껋쑝濡 븣젮졇 엳떎. 떊寃쎄탳꽭룷뒗 LPS 벑怨 媛숈 뿼利 쑀諛쒕Ъ吏덉뿉 끂異쒖씠 릺硫 뿼利앹쓣 留ㅺ컻븯뒗 臾쇱쭏쓣 깮꽦븯뒗 寃껋쑝濡 蹂닿퀬릺뿀떎(Boje and Arora, 1992; Chao et al., 1995; Hendriks et al., 2005; Glass et al., 2010).

湲곕뒫꽦 臾쇱쭏쓽 몴쟻씤 꽦遺 以 븯굹씤 뤃由ы럹怨 솕빀臾쇰뱾 븳 遺꾩옄 궡뿉 2媛 씠긽쓽 phenolic hydroxyl (-OH)湲곕 媛吏 諛⑺뼢議 솕빀臾쇰줈꽌 떇臾쇱껜뿉 듅닔븳 깋源붿쓣 遺뿬븯怨 빆뿼, 빆궛솕, 빆븫 벑쓽 떎뼇븳 깮由 솢꽦쓣 媛吏꾨떎(Bors and Saran, 1987; Sato et al., 1996). 泥쒖뿰빆궛솕젣 諛 빆뿼利앹젣쓽 뿭븷쓣 븯뒗 뤃由ы럹怨 뵆씪蹂대끂씠뱶 뼚옉쓣 DPE뿉꽌 痢≪젙븳 寃곌낵, 뤃由ы럹怨 뵆씪蹂대끂씠뱶媛 媛곴컖 48.32, 17.32 mg/g쓽 븿웾쑝濡 룷븿릺뼱 엳뒗 寃껋쓣 솗씤븯떎(Fig. 1). 蹂 뿰援ъ뿉꽌 痢≪젙맂 珥 뤃由ы럹 븿웾 Kim(2015) 벑씠 蹂닿퀬븳 븿웾(222.6 mg gallic acid equivalent/100 g) 蹂대떎 빟 21諛 넂 닔以쑝濡 굹궗떎. 삉븳 Lee(1994) 벑쓽 蹂닿퀬뿉꽌뒗 뱾源⑤ 룷븿븳 8媛吏쓽 醫낆떎 뭹醫낆뿉 뵲씪 珥 뤃由ы럹 븿웾씠 빟 120~2,060 mg gallic acid equivalent/100 g쑝濡 굹굹뒗 寃껋쓣 蹂닿퀬븯떎. 씠 媛숈씠 珥 뤃由ы럹 븿웾쓽 李⑥씠뒗 媛 뿰援щ쭏떎 씠슜맂 뱾源⑥쓽 뭹醫, 옱諛 議곌굔, 닔솗떆湲 벑쓽 李⑥씠濡 씤빐 븿웾 李⑥씠媛 떎뼇븳 寃껋쑝濡 깮媛곷맂떎.

Microglia cell씤 BV-2 꽭룷뒗 뇤쓽 뿼利앷낵 눜뻾꽦 蹂솕뿉꽌 以묒슂븳 뿭븷쓣 븯뒗 以묒텛떊寃쎄퀎 떇꽭룷씠硫, 뿼利 諛쒖깮 떆 솢꽦솕맂 microglia cell 뿼利 留ㅺ컻臾쇱쭏씤 prostaglandins (PG)怨 nitric oxide (NO) 벑쓣 諛쒖깮떆궎寃 맂떎(Hur et al., 2007). 듅엳, LPS 媛숈 궡룆냼媛 以묒텛떊寃쎄퀎뿉 移⑦닾븯寃 릺硫 씤吏뒫젰肉먮쭔 븘땲씪 諛붿씠윭뒪꽦 뇤뿼쑝濡 씤븳 湲곗뼲젰 媛먰눜 뼵뼱뒫젰쓽 븯媛 諛쒖깮릺寃 릺뒗뜲 BV-2 꽭룷뒗 씠 媛숈 吏덊솚 諛⑹뼱뿉 엳뼱 以묒슂븳 꽭룷씪 븷 닔 엳떎(Seo et al., 2013). NO뒗 nitric oxide synthase(NOS)뿉 쓽빐 L-arginine쑝濡쒕꽣 빀꽦릺뒗 2李⑥떊샇 쟾떖 臾쇱쭏濡 뇤 湲곕뒫怨 떊寃쎈낫샇, 떊寃쎈꽦 벑뿉 뿰愿릺뼱 엳떎(Hong et al., 2002). 蹂 뿰援ъ뿉꽌 LPS濡 쑀룄맂 BV-2 꽭룷뿉꽌 DPE쓽 NO 깮꽦웾쓣 솗씤븳 寃곌낵, DPE뒗 LPS留뚯쓣 泥섎━븳 援곗뿉 鍮꾪빐 10, 25, 50, 75, 100 μg/mL 紐⑤뱺 냽룄뿉꽌 냽룄쓽議댁쟻씤 NO 깮꽦웾 媛먯냼媛 솗씤릺뿀떎(Fig. 3). 씠 媛숈 寃곌낵뒗 DPE媛 LPS뿉 쓽빐 怨 깮꽦맂 NO 깮꽦웾쓣 슚怨쇱쟻쑝濡 媛먯냼떆궡뿉 뵲씪 以묒텛떊寃쎄퀎뿉 諛붿씠윭뒪 移⑦닾濡 씤븳 뿼利앹꽦 뇤 떊寃 넀긽씠 諛쒖깮븷 寃쎌슦, 떇꽭룷씤 BV-2 꽭룷瑜 넻빐 슚怨쇱쟻쑝濡 媛먯냼떆궗 닔 엳쓬씠 寃利앸릺뿀떎.

PGE2쓽 利앷뒗 湲됱꽦 뿼利앸떒怨꾩뿉꽌 슚怨쇱쟻씠怨 떊냽븯寃 媛먯냼媛 릺吏 븡쓣 寃쎌슦, 뿼利 珥덇린떒怨꾨 留뚯꽦떒怨꾨줈 쑀룄븯寃 릺誘濡 PGE2뒗 뿼利앸떒怨꾩뿉꽌 媛옣 以묒슂븯떎怨 븷 닔 엳떎(Seo et al., 2013). 듅엳, 以묒텛떊寃쎄퀎뿉꽌 뿼利앹씠 留뚯꽦쑝濡 吏꾪뻾맆 寃쎌슦뿉뒗 뇤 꽭룷 넀긽쓣 諛뷀깢쑝濡 떊寃쎈퀝, 留먯큹떊寃, 泥숈닔 諛 뇤뵾吏덉쓽 蹂꽦쓣 넻빐 떊泥댁뿉 떖媛곹븳 넀긽쓣 珥덈옒븯寃 맂떎. 蹂 뿰援ъ뿉꽌 LPS濡 쑀룄맂 BV-2 꽭룷뿉꽌 DPE媛 PGE2 깮꽦웾뿉 誘몄튂뒗 슚怨쇰 솗씤븳 寃곌낵, DPE뒗 LPS留뚯쓣 泥섎━븳 援곗뿉 鍮꾪빐 75, 100 μg/mL 냽룄뿉꽌 PGE2 깮꽦웾씠 쑀쓽꽦 엳寃 媛먯냼릺뒗 寃껋쓣 솗씤릺뿀떎(Fig. 4). 씠 媛숈 寃곌낵뒗 븵꽑 NO 깮꽦웾 媛먯냼 寃곌낵 留덉갔媛吏濡 DPE媛 PGE2 깮꽦웾 뿭떆 쑀쓽쟻쑝濡 媛먯냼떆궡뿉 뵲씪 以묒텛떊寃쎄퀎쓽 뿼利 諛쒖깮쓣 슚怨쇱쟻쑝濡 뼲젣떆耳 留뚯꽦 뿼利앸떒怨꾨줈 吏꾪뻾릺뒗 寃껋쓣 슚怨쇱쟻쑝濡 媛쒖꽑떆궗 닔 엳쓬씠 엯利앸릺뿀떎.

NO뒗 깮由ы븰쟻 빀꽦議곗젅뿉꽌 媛옣 以묒슂븳 뿭븷쓣 븯뒗뜲 씠뱾 neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS) 媛숈 3媛吏쓽 삎깭濡 議댁옱븳떎(Yui et al., 1991). 洹 以, iNOS뒗 뿼利앹“젅쓣 씪쑝궎뒗 以묒슂븳 슚냼 以 븯굹씠떎(Korhonen et al., 2002). iNOS뒗 Ca2+ 鍮 쓽議댁꽦 寃쎈줈瑜 넻빐꽌 옣떆媛꾩뿉 嫄몄퀜 利앷븯뿬 떎웾쓽 NO瑜 삎꽦븳떎(Vegeto et al., 2001). iNOS쓽 諛쒗쁽 murine macrophage, 궡뵾꽭룷, 룊솢洹쇱꽭룷 諛 떖洹쇱꽭룷 벑 留롮 꽭룷뿉꽌 愿李곕릺怨 엳떎. 씤媛꾩쓽 iNOS뒗 뿼利앹꽦吏덊솚씠 엳뒗 솚옄쓽 macrophage뿉꽌 떎웾쓽 NO瑜 깮꽦븯뿬 꽭洹좎묠엯쓣 뼲젣븯嫄곕굹 샊 T-꽭룷利앹떇쓣 뼲젣븯뿬 援냼 뿼利 諛섏쓳쓣 븯뼢떆궎뒗 諛⑹뼱뿉 以묒슂븳 뿭븷쓣 븯怨 엳뒗 寃껋쑝濡 蹂닿퀬릺怨 엳떎(Wang et al., 2002; Hanisch, 2002). COX뒗 COX-1怨 COX-2濡 議댁옱븯뒗뜲, 洹 以 COX-2뒗 삤吏 TNF-α, IL-1β 媛숈 궗씠넗移댁씤怨 諛뺥뀒由ъ븘 룆냼 諛 LPS 媛숈 뿼利 떊샇뿉 諛섏쓳븯뿬 諛쒗쁽맂떎. 삉븳 COX-2뒗 PGE2쓽 留롮 뼇쓣 깮꽦븯硫, 뿼利앹쓣 쑀룄븳떎(Mitchell et al., 1995; Chen et al., 2001). 蹂 뿰援ъ뿉꽌 iNOS COX-2 떒諛깆쭏 LPS 泥섎━ 떆 媛뺥븯寃 諛쒗쁽릺뿀쑝굹 LPS뿉 DPE 50, 100 μg/mL瑜 븿猿 泥섎━ 떆 iNOS COX-2쓽 諛쒗쁽뼇씠 媛먯냼븯뒗 寃껋쓣 솗씤븷 닔 엳뿀떎(Fig. 5).

蹂 뿰援 寃곌낵뿉꽌 蹂댁뿬以 DPE쓽 빆떊寃쎌뿼利 슚怨쇰뒗 뱾源⑤컯씠 誘몄꽭븘援먯꽭룷쓽 솢꽦쓣 뼲젣븯뿬 떊寃 뿼利앸컲쓳쓣 슚쑉쟻쑝濡 젣뼱븷 닔 엳뒗 湲곕뒫꽦 냼옱濡쒖꽌쓽 媛뒫꽦쓣 젣떆븯떎. 洹몃윭굹 뼢썑 빆떊寃쎌뿼利 媛쒖꽑 냼옱濡쒖쓽 媛쒕컻쓣 쐞빐꽌뒗 뇤떊寃쎌넀긽 룞臾쇰え뜽쓣 씠슜븳 뱾源⑤컯異붿텧臾쇱쓽 쑀슚꽦 寃利앷낵 슚怨쇱뿉 븳 遺꾩옄쟻 硫붿빱땲利 뿰援ш 異붽쟻쑝濡 吏꾪뻾릺뼱빞 븷 寃껋쑝濡 궗猷뚮맂떎.

ACKNOWLEDGEMENT

蹂 뿰援щ뒗 以묒냼踰ㅼ쿂湲곗뾽遺쓽 李쎌뾽꽦옣湲곗닠媛쒕컻궗뾽쓽 씪솚쑝濡 닔뻾븯쓬(S2539864, 援궡 떎쑀뱾源 遺궛臾쇰줈遺꽣 愿묐끂솕 냼옱 諛쒓뎬 諛 뒪궓뵒렂뒪 솕옣뭹媛쒕컻).

CONFLICT OF INTEREST

No potential conflict of interest relevant to this article was reported.

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