30 4 (2009 7 ) J Korean Oriental Med 2009;30(4):13-27 Original Article 선영재1, 최영곤1,2 , 정미영1,2 , 황세희1, 이제현4, 조정희5, 임사비나1,2,3 1경희대학교 대학원 한방응용의학과, 2경희대학교 동서의학연구소, 3경희대학교 대학원 기초한의과학과, 4동국대학교 한의과대학 본초학교실, 5전라남도한방산업진흥원 The Effect of Trans -cinnamaldehyde on the Gene Expression of Lipopolysaccharide-stimulated BV-2 Cells Using Microarray Analysis Young-Jae Sun 1, Yeong-Gon Choi 1,2 , Mi-Young Jeong 1,2 , Se-Hee Hwang 1, Je-Hyun Lee 4, Jung-Hee Cho 5, Sabina Lim 1,2,3 1Dept. of Applied Eastern Medicine, Grad. School, 2WHO Collaborating Ctr. for Traditional Medicine, East-West Med. Res. Institute, 3Dept. of Basic Eastern Medicinal Science, Grad. School, Kyung Hee University, 4Dept. of Herbology, Col. of Eastern Medicine, Dongguk University, Gyeongju, Republic of Korea, 5Jeollanamdo Development Institute for Traditional Korean Medicine Objectives: Trans -cinnamaldehyde (TCA) is the main component of Cinnamomi Ramulus and it has been reported that TCA inhibits inflammatory responses in various cell types. Inflammation-mediated neurological disorders induce the activation of macrophages such as microglia in brain, and these activated macrophages release various inflammation-related molecules, which can be neurotoxic if overproduced. In this study, we evaluated gene expression profiles using gene chip microarrays in lipopolysaccharide (LPS)-stimulated BV-2 cells to investigate the anti- inflammatory effect of TCA on inflammatory responses in brain microglia. Methods: A negative control group was cultured in normal medium and a positive control group was stimulated with 1 / LPS in the absence of TCA. TCA group was pretreated with 10 / TCA before 1 / LPS stimulation. The oligonucleotide microarray analysis was performed to obtain the expression profiles of 28,853 genes using gene chip mouse gene 1.0 ST array in this study. Results: In positive control group, 1522 probe sets were up-regulated in the condition of the cutoff value of 1.5-fold change and 341 genes with Unigene ID were retrieved. In TCA group, 590 probe sets were down-regulated from among 1522 probe sets and 33 genes with Unigene ID were retrieved, which included 6 inflammation-related genes. We found out that Id3 gene is associated with transforming growth factor- β (TGF- β) signaling pathway and Klra8 gene is related to natural killer cell-mediated cytotoxicity pathway. Conclusions: The results mean that TCA inhibits inflammatory responses through down-regulating the expressions of inflammation-related genes in LPS-stimulated BV-2 cells. Key Words : trans -cinnamaldehyde, lipopolysaccharide, microglia, BV-2, microarray Received ：15 January 2009 Revised ：14 May 2009 Accepted ：26 May 2009 Correspondence to ：임사비나(Sabina Lim) 서울시 동대문구 회기동 경희대학교 대학원 한방응용의학과 Tel ：+82-2-961-0324, Fax ：+82-2-961-7831, E-mail ：[email protected] 13 (470) 30 4 (2009 7 ) 서 론 9,10) . , microglia (Cinnamomi Ramulus) trans -cinnamaldehyde (TCA) 1) . 8) , 2) , TCA 3-5) . TCA RAW 264.7 cell Nitric oxide (NO) Inducible nitric oxide synthase (iNOS) TCA 3) . , TCA TCA endothelial cell cyclooxygenase-2 (COX-2) , Lipopolysaccharide (LPS) Prostaglandin E2 (PGE2) murine microglial cell line BV-2 4) , murine macrophage cell TCA human blood monocyte interleukin-1 (IL-1), tumor necrosis factor- α (TNF- α) microarray . 5) . mouse (macrophage) Oligonucleotide probe set microarray chip whole transcript sense target labeling assay 11) gene ontology (GO) TNF- . LPS α, IL-1 iNOS COX-2 eicosanoids, reactive oxygen , TCA LPS species (ROS), NO, superoxide (O 2) 6) , micr- clustering oglia pathway (microglial activation) TCA TNF- α, IL-1, IL-3, IL-5, IL-6, IL-8 . fibroblast growth factor- β (FGF- β), transforming growth factor- β1 (TGF- β1), nerve 재료 및 방법 growth factor (NGF) , brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), 1. 시료의 제조 NT-4/5 NO TCA TCA 7,8) . HPLC (CNS) microglia . (Cinnamomi Ramulus) , microglia Cinnamomum cassia PRESL , , , , 14 6 : Trans-Cinnamaldehyde Lipopolysaccharide BV-2 cell : Microarray (471) . 200 g 80% 4. TCA 의 세포독성 실험 1500 3 . BV-2 cell 96well well 5000 1% 7 g FBS DMEM (low glucose) 24 ( 3.5%) . Dime- pre- incubation . TCA thylsulfoxide (DMSO) 20 / BV-2 cell TCA (1, 3, 10, 20, 30 . TCA Sigma / ) 48 MTS (3-(4,5-dimet- (MO, USA) . TCA hylthiazol-2-yl)- (3-carboxymethoxyphenyl)-2(4-sulf- Dulbecco’s modified Eagle’s medium (DMEM, Sigma ophenyl)-2H) assay . Cell Titer 96 chemical Co., MO, USA) Aqueous Cell Proliferation Assay kit (Promega, USA) BV-2 cell . MTS solution 20 4 . 2. 桂枝 추출물의 HPLC 분석 ELISA plate reader (Microplate manager, BioRad, Coumarin, TCA, USA) 490nm . cinnamic acid, glacial acetic acid Sigma-Aldrich Chemical Co. (MO, USA) . HPLC 5. 세포 배양과 실험군 분류 methanol (99.8%, Merck, Darmstadt, Germany) BV-2 cell 100 dish (Corning- Costar, USA) , (Mili-Q 10% FBS DMEM system, USA) . 3 HPLC (DIONEX SUMMIT HPLC System, Germany) . P680 HPLC pump, ASI-100 Automated Sample 10% FBS DMEM Injector, UVD 340U UV/VIS-detector, column (250 12-14 . BV-2 cell mm × 4.6 mm i.d.; particle size, 5μm; Phenomenex negative control , 1 Inc., CA, USA) , data CHRO- / LPS positive control , MELEON 6.70 Chromatography Management Software TCA LPS 1 (Dionex) . methanol TCA (10 / ) TCA - 0.01% acetic acid (50:50, v/v) . , 1.0 ml/min , UV 275nm 3 . TCA standard LPS 2 LPS curve HPLC 0.406, 0.081, 0.016 . , mg/mL . TCA 1 TCA 3. Cell culture . Mouse microglial cell line BV-2 cell 100 U/ 24 . penicillin, 100 / streptomycin (Gibco BRL, Grand Island, NY, USA) 10% fetal bovine serum 6. BV-2 cell 에서의 유전자 발현양 분석 (FBS; Gibco BRL, Grand Island, NY, USA) 1) Total RNA DMEM 37°C, 5% CO 2 incubator . BV-2 cell pellet phosphate buffer (PBS) 15 (472) 30 4 (2009 7 ) RNeasy Mini kit (QIAGEN, USA) (6) Second-Cycle, First-Strand cDNA synthesis protocol total RNA . Second-Cycle, First-Strand cDNA GeneChip total RNA spectrophotometer (Ultro- spec WT cDNA Synthesis Kit . 2000, Pharmacia Biotech Inc. USA) NaNoDrop (7) cRNA Single-Stranded DNA ND-1000 (NanoDrop Technologies, USA) Cleanup . cRNA Single-Stranded DNA Cleanup 2) Microarray Analysis GeneChip WT cDNA Amplification Kit Genechip Mouse Gene 1.0 ST array GeneChip Sample Cleanup Module . whole transcript sense targe labeling assay (Affymetrix, NanoDrop ND-1000 USA) manual Single-Stranded DNA 5.5 . (1) total RNA (8) Fragmentation of Single-Stranded DNA BV-2 cell total RNA 300 Single-Stranded DNA Fragmentation Gene- ng/3 . Chip WT Terminal Labeling Kit (Affymetrix Inc. USA) . 45 0.2 strip (2) total RNA T7-(N) 6 Primer, Poly-A RNA tube , 3 2% agarose control Mix gel ethidium- bromide total RNA 300 ng 3 T7-(N) 6 (ET- Br, Sigma-Aldrich, USA) fragme- Primer, Poly-A RNA control ntation . 2 5 70°C 5 4°C 2 . (9) Fragmented Single-Stranded DNA Labeling Fragmented Single-Stranded DNA Labeling (3) First-Cycle, First-strand cDNA GeneChip WT Terminal Labeling Kit First-Cycle, First-strand cDNA GeneChip . WT cDNA Synthesis Kit (Affymetrix Inc., USA) . (10) Hybridization Hybridization GeneChip Hybridization, Wash (4) First-Cycle, Second-Strand cDNA and Stain Kit (Affymetrix Inc., USA) . First-Cycle, Second-Strand cDNA GeneChip GeneChip ST Array septa hybridization WT cDNA Synthesis Kit . cocktail 80 septa 45°C, 60 rpm hybridization (5) First-Cycle, cRNA Synthesis and Cleanup oven 17±1 . First-Cycle, cRNA Synthesis and Cleanup GeneChip WT cDNA Amplification Kit Gene- (11) Fluidics Scanning Chip Sample Cleanup Module (Affymetrix Inc., USA) GeneChip ST Arrays Fluidics Protocols . NanoDrop ND-1000 (NanoDrop Fluidics Station 450 (Affymetrix Inc., USA) Technologies, USA) cRNA Fluidics Scripts FS450_0007 (Affymetrix Inc., USA) . 16 6 : Trans-Cinnamaldehyde Lipopolysaccharide BV-2 cell : Microarray (473) GeneChip Operating Software (GCOS, Affymetrix (Normalization) Quantile Normalization Inc., USA) GeneChip Scanner 3000 , log2 . 7G (Affymetrix Inc., USA) scan . Fold Change cutoff value 1.5 detection p-value 7. Data 분석 . (Clustering analysis) (Hierarchical clustering) Gene chip scan data GeneChip Operating . Pathway ( Software (Affymetrix Inc., USA), Expression Console ) Kyoto Encyclopedia Software (Affymetrix Inc., USA) GenPlex v3.0 of Genes and Genomes (KEGG) pathway (ISTECH Inc., Korea) 28,853 pathway P-value . Hybridization control oligonucleotide B2, bioB, bioC, bioDn, CreX, poly A controls, positive 결 과 vs negative area under the curve (pos_vs_neg_auc) . 1. 桂枝 추출물의 HPLC 분석 결과 signal intensity average Coumarin, TCA, cinnamic acid differential expression (AD) 277 nm, 290 nm, and 277 nm . UV 275 nm GenPlex v3.0 software (ISTECH Inc., Korea) , 0.01% acetic acid - BeadStudio Signal methanol (50:50, v/v) . (Preprocessing module normalization), HPLC coumarin, TCA, cinnamic (differentially expressed genes (DEG) finding acid retention time 7.2 , 12.3 , 16.0 module DEG ), (Clust- . chrom- ering module ) pathway atogram Fig. 1. (Pathway module chromatogram ) . AAA BBB Fig. 1. 17 (474) 30 4 (2009 7 ) (mean ± S.E.M.; n = 3), TCA 2) Chip internal controls 161.35 ± 1.55 mg/g . TCA control . control 2. TCA 의 세포독성 평가 hybridization Bio B, C, Dn, CreX 3` 5`, poly-A (lys, phe, BV-2 cell TCA thr, dap) , BV-2 cell TCA (Sigma-Aldrich, pos_vs_neg_auc . USA) 1, 3, 10, 20, 30 / Chip internal control MTS assay . (Fig. 2). TCA 10 / microarray 4. Microarray 실험 결과 . 1) Scan image 3. Gene chip 에 적용된 sample 의 상태 평가 (1) Hybridization gene chip biotin-labeled cDNA gene chip hybri- 1) cDNA Fragmentation dization scan pseudocolor Gene chip target cDNA . 2% agarose gel chip ET-Br cDNA (GeneChip Mouse Gene 1.0 ST array) fragmentation . , negative control cDNA size 4,000 bp . , fragmented cDNA 200 bp fragme- (2) Negative control positive control , TCA ntation . gene chip scan image 120 100 Cell viability (%) Cell viability 80 60 40 20 0 0 10 20 30 40 50 TCA(㎍/ml) Fig. 2. 18 6 : Trans-Cinnamaldehyde Lipopolysaccharide BV-2 cell : Microarray (475) AAABA BBBCCCC Fig.