Effects of Samjunghwan on the IL-1 Gene Expression in the Macrophage

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1 대한한방내과학회지제 27 권 1 호 (2006 년 3 월 ) Korean J.Orient.Int. Med. 2006:27(1): Effects of Samjunghwan on the IL-1Gene Expression in the Macrophage Se-yoon Kim, Ji-cheon Jeong Objectives : Macrophage has an important innate defense role in the immune system. When we are infected with pathogens, macrophage ingests them through phagocytosis or endocytosis, and then secretes many cytokines, such as IL-1, IL-6 and TNF, which are regulators of immune responses. The aim of this study is to determine how Samjunghwan effects the expression of cytokine and other immune-related genes in macrophages. Methods : Cells were treated directly with Samjunghwan and/or LPS at regular intervals. Total RNA of cells was isolated using TRIzol reagent, and the changes in cytokine gene expressions were investigated using RT-PCR, western blot and ELISA. Results : IL-1, IL-1 and genes were inducibly expressed specifically by Samjunghwan in macrophage. Especially, IL-1gene was induced most strongly by treatment with Samjunghwan. Over time, treatment with Samjunghwan showed that the expression levels of IL-1and IL-1genes increased from 1 to 4h, and then decreased from 4 to 15h. However, the expression level of gene increased continuously up to 11h. IL-1, IL-1and genes were expressed synergistically by a simultaneous treatment of both Samjunghwan and LPS in macrophages. Secretion levels of translated IL-1increased continuously up to 11h. Conclusions : Though this study is only a start in the investigation of the efficasy of Samjunghwan, these results suggest that Samjunghwan has positive effects on immune responses. (native immunity)(inflammation) , (Tel , Fax jjcjh@paran.com) (endocytosis) (phagocytosis) 1-4. cytokine (native immunity) (specific immunity), 5. cytokine cytokine cytokine

2 . cytokine autocrine, paracrine, endocrine 5,6. cytokine cytokine. 7.,, (Atractylodes japonica), (Cortex lycii radicis), (Morus alba L.) 8,9. 8, cytokine. 1). 2) Fetal bovine serum (Cell-Gen, CGA15-649, German), L-glutamine (Gibco, , USA), MEM nonessential amino acids (Gibco, , USA), MEM sodium pyruvate (Gibco, , USA), 2-mercaptoethanol (Gibco, , USA), penicillin G-streptomycin (Gibco, , USA) DMEM (Hyclone, SH , USA), LPS (Sigma, L6529, USA), TRIzol reagent (Invitrogen, , USA) PCR pre-mixture (Genenmed, CAN0070, Korea), [NaCl, Tris-Cl, SDS, Triton X-100, deoxycholate, EDTA, PMSF; (Amresco, USA)], protein assay solution (Biorad , USA), enhanced chemiluminescence kit (Santacruz, USA). Mori Fructus Atractylis Rhizoma Lycii Cortex Radicis 125 g 60 g 60 g 245 Polygonati sibirici Rhizoma Lycii Fructus Eucommiae Cortex Pasoraliae Semen Juglandis Semen Zingiberis Rhizoma 150 g 150 g 300 g 100 g 100 g 100 g 62.5 g g

3 3) Anti-IL1(Santa cruz, sc-1251, USA), anti-tubulin (Santa cruz, sc-9104, USA), HRP conjugated antigoat IgG (Santa cruz, sc-2768, USA) HRP conjugated anti-goat IgG (Santa cruz, sc-2004, USA). 1) 245g 3 95% methanol g ( 8.9%) g, g. 2) S194 (mouse plasma B cell), EL4 (mouse T helper cell), RAW264.7 (mouse macrophage), BV2 (mouse microglia) 6-well tissue culture dish cells/. 10% fetal bovine serum, 2mM L-glutamine, 0.1mM MEM nonessential amino acids, 1mM MEM sodium pyruvate, 50 M 2-mercaptoethanol, 100U/ penicillin G-100 / streptomycindmem 5% CO 2, 37. 3), LPS,, 100 /, LPS1 / (FBS free DMEM media) 1hr 20hrs. 4) Total RNA TRIzol reagent chloroform isopropyl alcohol total RNA. 5) Reverse transcription Total RNA 1 oligo (dt)15 primer reverse transcription cdna 95 5 reverse transcriptase cdna PCR. 6) cdna PCR (Reverse Transcription) cdna 1primer pair (IL-1, IL-1, IL-2, IL-6, IFN-, TNF-,, inos, -actin primer) PCR pre-mixture pre-denaturation (95, 5 ), denaturation (95, 30 ), annealing (5359, 30 ), elongation (72, 30 )25 post-elongation (72, 5 ) PCR. PCR products 1.5% agarose geletbr staining.(primer sequencetable.1.) IL-1 IL-1 IL-2 IL-6 IFN TNF inos -actin Forward primer caa ctc taa gaa cta ctt cac atc agc gcc cat cct ctg tga ctc atg gtg ctc ctt gtc aac agc gc agt tgc ctt ctt ggg act gat g aac tgg caa aag gat ggt gac taa aat gtc ggc tcc agg ac gca cgt agt ctt cga tca ct ggc tgt cag agc ctc gtg gct ttg g aca ccc cag cca tgt acg ta Reverse primer tca ttt aga att aca gag act cag cac aag gcc aca ggt att ttg tcg agc atc ctg ggg agt ttc agg ttc cac gat ttc cca gag aac atg agc tca ttg aat gct tgg agg tgg att cat tcc ctt cc tct cca acc tct cct act ac ccc ttc cga agt ttc tgg cag cag c ctc ttt gat gtc acg cac ga

4 7) Western blot LPS(RAW264.7) ice-cold PBS RIPA buffer (150mM NaCl, 10mM Tris-Cl (ph 7.2), 0.1% SDS, 1% Triton X-100, 1% deoxycholate, 5mM EDTA, 1mM PMSF) lysis 1.5ml tube ml tube 14,000 rpm ml tube protein assay solution (Biorad ) 30ug 12% acrylamide gel nitrocellulose membranetransfer. 10% skim milk membrane 1 blocking 1 anti-il1 anti-tubulin 1, 2HRP conjugated antirabbit IgG HRP conjugated anti-goat IgG 1 ECL (Enhanced chemiluminescence) kit. 8) ELISA IL1-indirect ELISA 11. LPSRAW ul96well4 12 TBS-T 10% skim milk well blocking TBS-T 3. 1 anti-il1 1 TBS-T 3 2 HRP conjugated anti-goat IgG. TBS-T 3TBS 2 50ul ECL solution luminometer. RAW264.7 (mouse macrophage) (100 / )RT-PCR Cytokine. Negative controlnon-treated. (100 / ) (100 / ). Positive control LPS (1 / ). LPS Positive control IL-1, IL-1, IL-6, TNF- Cytokine,, inos. Negative control (Non-treated), Cytokine, inos. LPS IL-1 IL-1 IL-6 TNFα inos N I S C LPS α β

5 . IL-1 (Fig.1). -actin Loading control. S194 (mouse plasma B cell), EL4 (mouse T helper cell) (100 / ) Negative controlnon-treated. (100 / ) (100 / )5 cytokine. B IL-1, IL-1T helper IL-2, IFN- (Fig. 2). BV2 (microglia) NIH3T3 (fibroblast) (100 / ) negative controlnon-treated. (100 / ) (100 / ) 5 cytokine. BV2 (A) NIH3T3 (B) IL-1, (Fig. 3). A. N I S C A. B. IL-2 INFγ N I S C N I S C B. N I S C (100 / ) 15, 4, 1IL-1,. (100 / ) (100 / )

6 . IL IL-1 IL-1. IL-1 IL-1 (Fig 4). IL-6 TNFα 1 hr S/L L IL-6 TNFα inos Samjunghwan N 1h 4h 15h inos α β α β α β α β LPS (1 / ), (100 / ), LPS (1 / ) (100 / )1 cytokine, inos RT-PCR. LPS 1 LPSIL-1 (Fig. 5). IL-1 Western blot. β-tubulin LPS S (100μg/ ml ) N 11hrs 1hr 5hrs 11hrs β β β β

IL-1., negative controlnon-treated IL-1 positive controllps IL-1 (Fig. 6). IL-1 indirect ELISA negative control. luminometer control(mock). IL-1. 5 1 1.

7 IL-1., negative controlnon-treated IL-1 positive controllps IL-1 (Fig. 6). IL-1 indirect ELISA negative control. luminometer control(mock). IL IL-1, 111 3IL β β 7-9.,,, 12-4, 15.,,,, 12-4, 3T3-L , 17. 8,9, cytokine 19.. cytokine. cytokine IL-1 T helper NK B. ICAM-1

8 ,, hepatocytes 5. IL-1 (, ).. IL-1 IL-1, IL-1 20,21. BV2 (mouse microglia). IL-1. IL-1. IL-1 IL-1 feedback inhibition IL-1. LPS LPS IL-1. IL-1. RNA, Western blotelisa. Western blot IL-1, mrna translation, ELISA IL-1. in vitro IL-1 RNA. IL-1 Receptor Cell signaling pathway, in vivo. cytokine RNA. cytokine IL-1, cytokine. IL-1, LPS IL-1. IL-1,. 1. Incani, R. N. and McLaren, D. J. Histopathological and ultrastructural studies of cutaneous reactions elicited in naive and chronically infected mice by invading schistosomula of Schistosoma

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Supplemental file 3. All 306 mapped IDs collected by IPA program. Supplemental file 6. The functions and main focused genes in each network.

LIST OF SUPPLEMENTAL FILES Supplemental file 1. Primer sets used for qrt-pcr. Supplemental file 2. All 1305 differentially expressed genes. Supplemental file 3. All 306 mapped IDs collected by IPA program.