中國醫藥大學機構典藏 China Medical University Repository, Taiwan:Item 310903500/41346

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(1)ύ୯ᙴᛰεᏢᔼᎦᏢ‫س‬ᅺγ੤ ᅺ γ ፕ Ў. ΒΜΒᅹϤ౎ለ(DHA)ᇨวՈ୷፦਼ϯ䁙-1 ᆶ ‫ڋ׭‬ဍዦᚯԝӢη-Į ᇨว‫ޑ‬ಒझ໔ᗹߕϩη-1 ߄౜ᜢ߯ϐ௖૸ Induction of Heme Oxygenase 1 and Inhibition of Tumor 1HFURVLV)DFWRUĮ-Induced Intercellular Adhesion Molecule 1 Expression by Docosahexaenoic Acid in EA.hy926 Cells. ࣴ‫ز‬ғǺ᚟ྼզ (Yu-Ling Wei) ࡰᏤ௲௤Ǻഋཧ໥ റγ (Haw-Wen Chen, Ph.D.). ύ๮҇୯΋ԭԃΎД July, 2011.

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(4) Ҟᒵ Ҟᒵ.................................................................................................................................i კҞᒵ.......................................................................................................................... iii ߄Ҟᒵ...........................................................................................................................iv ᕭቪ߄............................................................................................................................v ύЎᄔा......................................................................................................................vii मЎᄔा.................................................................................................................... viii ಃ΋೽ҽ........................................................................................................................1 ಃ΋ക ߻‫ق‬..........................................................................................................1 ಃΒക Ў᝘ӣ៝..................................................................................................2 ΋ǵ୏ેๆ‫ރ‬ฯϯ(atherosclerosis) .............................................................2 1. ୏ેๆ‫ރ‬ฯϯϐԋӢ.......................................................................2 2. ಒझᗹߕϩηᆶ୏ેๆ‫ރ‬ฯϯϐᜢ߯...........................................3 3. ဍዦᚯԝӢηᆶ୏ેๆ‫ރ‬ฯϯϐᜢ߯...........................................7 4. ᙯᒵӢη NF-ț% ૻ৲໺ሀၡ৩ .....................................................8 4-1. NF-ț% .................................................................................8 4-2. IKK ...................................................................................12 4-,ț% ....................................................................................13 Βǵങ‫ݨ‬ᆶЈՈᆅ੯ੰϐᜢ߯..................................................................14 1. ങ‫ཷݨ‬ॊ.........................................................................................14 2. n-3 ӭϡόႫ‫ک‬િެለ ...................................................................14 2-1. ่ᄬ .................................................................................14 2-2. ғӝԋբҔ .....................................................................15 3. DHA ‫ޑ‬ғ౛բҔ/фૈ...................................................................16 ΟǵՈ୷፦਼ϯ䁙(Heme oxygenase, HO)................................................19 1. Ո୷፦਼ϯ䁙ϐϩᜪ.....................................................................19 2. HO-1 ϐғ౛‫ف‬Յ ...........................................................................20 3. HO-1 жᖴౢ‫ނ‬ჹಒझ‫ߥޑ‬ៈբҔ ...............................................21 3-1. ᖌᆘન(biliverdin, BV)/ᖌआન(bilirubin, BR)...............21 3-2. ΋਼ϯᅹ(carbon monoxide, CO) ...................................22 3-3. ៓ᚆη(Fe2+)/៓ೈқ(ferritin).........................................23 4. ፓ௓ HO-1 ୷Ӣ߄౜ϐૻ৲໺ሀၡ৩ .........................................23 4-1. ᙯᒵӢη Nrf2.................................................................24 4-2. ‫ځ‬Ѭၡ৩(PI3K/AktǵMAPKsǵPKC) ..........................25 ಃΟക ࣴ‫ز‬Ҟ‫ޑ‬................................................................................................27 i.

(5) ಃΒ೽ҽ......................................................................................................................29 ,QGXFWLRQRI+HPH2[\JHQDVHDQG,QKLELWLRQRI7XPRU1HFURVLV)DFWRUĮ-Induced Intercellular Adhesion Molecule 1 Expression by Docosahexaenoic Acid in EA.hy926 Cells 1. Introduction......................................................................................................29 2. Materials and Methods.....................................................................................33 2.1 Chemicals...............................................................................................33 2.2 Cell cultures ...........................................................................................33 2.3 Fatty acid preparation ............................................................................34 2.4 Cell viability assay.................................................................................34 2.5 Nuclear extracts preparation ..................................................................35 2.6 Western blotting analysis .......................................................................35 2.7 RNA isolation and RT-PCR ...................................................................36 2.8 Electrophoretic mobility shift assay (EMSA)........................................37 2.9 Plasmids, transfection, and luciferase assay ..........................................38 2.10 RNA interference .................................................................................39 2.11 Peroxide measurement .........................................................................39 2.12 Monocyte adhesion assay ....................................................................40 2.13 Statistical analysis ................................................................................40 3. Results..............................................................................................................41 3.1 Cell viability...........................................................................................41 3.2 DHA inhibits TNF-Į-induced ICAM-1 expression, promoter activity and HL-60 cell adhesion ......................................................................41 3.3 DHA inhibits TNF-Į-induced NF-ț%VLJQDOLQJSDWKZD\......................41 3.4 DHA increases HO-1 expression in the presence of TNF-Į ..................42 3.5 DHA induces Nrf2 protein accumulation, nuclear translocation and the ARE-luciferase reporter activity ..........................................................43 3.6 HO-1 siRNA alleviates the inhibition of TNF-Į-induced ICAM-1 expression and p65 translocation by DHA ..........................................44 3.7 PI3K/Akt and ERK1/2 pathways are involved in DHA-induced HO-1 expression ............................................................................................45 3.8 DHA triggers early-phase ROS production in EA.hy926 cells..............45 4. Discussion ........................................................................................................60 ୖԵЎ᝘......................................................................................................................66 ߕᒵ..............................................................................................................................88. ii.

(6) კҞᒵ ಃ΋೽ҽ კ 2.1. қՈౚቻ༅բҔ..............................................................................................4. კ 2.2 TNF-ĮǵIL-ȕǵIFN-Ȗ ፓ௓ ICAM-1 ‫ޑ‬ϩη໺ሀၡ৩ ...............................7 კ 2.3 NF-ț% ಔԋࠠԄ .............................................................................................9 კ 2.4 NF-ț%ǵ,ț% Ϸ IKK ่ᄬ ............................................................................10 კ 2.5 NF-ț% ૻ৲໺ሀၡ৩ ................................................................................... 11 კ 2.6 DHA ่ᄬ......................................................................................................15 კ 2.7 ӭϡόႫ‫ک‬િެለ‫ޑ‬ғӝԋբҔ................................................................15 კ 2.8 HO-1 ϐբҔϷ‫ځ‬жᖴౢ‫ ނ‬.........................................................................23 კ 2.9 ፓ௓ HO-1 ߄౜ϐૻ৲໺ሀၡ৩ ................................................................25. ಃΒ೽ҽ Figure 1. Effect of DHA on the cell viability of EA.926 cells in the presence of TNF-Į........................................................................................................47 Figure 2. DHA decreases TNF-Į-induced ICAM-1 expression, promoter activity and HL-60 cell adhesion. ...........................................................................49 Figure 3. DHA inhibits TNF-Į-induced activation of NF-ț% ....................................52 Figure 4. DHA induces HO-1 expression in EA.hy926 cells. .....................................53 Figure 5. Effect of DHA on Nrf2/ARE signaling pathway. .........................................54 Figure 6. Effect of HO-1 siRNA on the DHA-mediated inhibition of ICAM-1 expression and p65 translocation. ..............................................................56 Figure 7. PI3K/Akt, p38 and ERK1/2 pathways are involved in DHA-induced HO-1 expression. .......................................................................................57 Figure 8. Effect of DHA on ROS generation. ..............................................................58 Figure 9. Model showing pathways that mediate the inhibition of expression of ICAM-1 and adhesion of HL-60 cells to EA.hy926 cells by DHA under inflammatory conditions. .................................................................59. iii.

(7) ߄Ҟᒵ ಃ΋೽ҽ ߄ 2.1. ୖᆶ୏ેๆ‫ރ‬ฯϯϐᗹߕϩη......................................................................5. ߄ 2.2 ߄ 2.3 ߄ 2.4. ᇨว ICAM-1 ߄౜‫ޑ‬όӕ‫ڈ‬ᐟ......................................................................6 ࢲϯ NF-țB ‫ڈޑ‬ᐟ ......................................................................................12 ‫ ڙ‬NF-țB ፓ௓‫ޑ‬ೈқ፦ ..............................................................................12. ߄ 2.5. n-3 િެለफ़եЈՈᆅ੯ੰ॥ᓀ‫ޑ‬ёૈᐒ‫ ڋ‬.............................................17. ߄ 2.6 ߄ 2.7. n-3 િެለᆶᗹߕϩηϐᜢ߯ .....................................................................17 n-3 િެለࡌ᝼ឪ‫ڗ‬ໆ .................................................................................18. iv.

(8) ᕭቪ߄ Act D AHA. actinomycin D American Heart Association. ALA. alpha-linolenic acid. AP-1 ARE ATF4. activator protein-1 antioxidant response element activating transcription factor 4. BR. bilirubin. BV C/EBP CHX CK҈ CNC-bZIP CO DHA EFA EPA EpRE HLH HO Hsp32 ICAM IFN-Ȗ IGF-1 IgSF IKK IL-1. biliverdin CCAT/enhancer binding protein cycloheximide casein kinase ҈ cap’n’Collar-basic leucine zipper carbon monoxide docosahexaenoic acid essential fatty acid eicosapentaenoic acid electrophile response element helix-loop-helix domain heme oxygenase heat shock protein 32 intercellular adhesion molecule interferon-gamma insulin-like growth factor-1 immunoglobulin superfamily ,ț%NLQDVH interleukin-1. IRE ,ț% Keap1 LA. interferon-stimulated response element LQKLELWRUț% Klech-like ECH-associated protein 1 linoleic acid. LDL LPS LZ MAPK. low-density lipoprotein lipopolysaccharide leucine zipper mitogen-activated protein kinase. MCP-1. monocyte chemotoactic protein-1 v.

(9) MCSF MMP-9. monocyte-colony-stimulating factor matrix metalloproteinase-9. NFAT. nuclear factor of activated T cells. NF-E2 NF-ț% NIK. nuclear factor-erythroid 2 nuclear factor-kappa B NF-ț%-inducing kinase. NLS. nuclear localization signal. NOS PARP PDGF PECAM PI3K PKC PPAR PUFA RHD ROS RT-PCR sGC STAT TGF-ȕ TNFR1 TNF-Į VCAM VEGF VSMC. nitric oxide synthase poly (ADP-ribose) polymerase platelet derived growth factor platelet endothelial cellular adhesion molecule phosphoinositide 3-kinase protein kinase C peroxisome proliferators activated receptor polyunsaturated fatty acid Rel homology domain reactive oxygen species reverse transcription-polymerase chain reaction soluble guanylate cyclase Janus kinases (JAK)-signal transducers and activators of transcription transforming growth factor-beta tumor necrosis factor receptor 1 tumor necrosis factor-alpha vascular cell adhesion molecule vascular endothelial growth factor vascular smooth muscle cells. vi.

(10) ύЎᄔा. ೚ӭࣴ‫ز‬ᡉҢǴว‫ݹ‬ϸᔈ‫ک‬ՈᆅϣҜಒझфૈ౦தࢂ୏ેๆኬฯϯ‫ޑ‬ख़ाଆ ‫ۈ‬ᜢᗖǶಒझ໔ᗹߕϩη 1 (ICAM-1)ࢂ΋ᅿว‫ݹ‬ғ౛ࡰ኱ǴᆶൂਡౚᗹߕԿϣ Ҝಒझ΢‫ޑ‬բҔԖᜢǶЎ᝘ࡰрǴ໯१ύឪ‫ڗ‬൤֖ n-3 ӭϡόႫ‫ک‬િެለ‫ޑ‬ങ‫ݨ‬Ǵ ‫ٯ‬ӵΒΜΒᅹϤ౎ለ (DHA)ǴёаԖਏӦ෧Ͽᑡ஻ЈՈᆅ੯ੰ‫ޑ‬॥ᓀǶӢԜҁ ჴᡍஒаဍዦᚯԝӢη-Į 71)-Į

(11) ᇨวϣҜಒझਲ਼ EA.hy926 ౢғว‫ݹ‬ϸᔈǴٰ௖ ૸ DHA ჹ‫ ܭ‬TNF-Į ‫܌‬ᇨว ICAM-1 ߄౜ϐቹៜаϷёૈୖᆶ‫ޑ‬ᐒ‫ڋ‬Ƕ่݀ว౜Ǵ '+$ ȝ0

(12) ନΑ཮‫ ڋ׭‬TNF-Į ‫܌‬ᇨว‫ ޑ‬ICAM-1 ೈқ፦ǵmRNA ߄౜ǵൔ Ꮴ୷Ӣࢲ‫܄‬ǹҭૈ຾΋‫؁‬Ӧफ़ե IKK ᕗለϯǵ,ț% ᕗለϯ‫ک‬फ़ှǵp65 ਡᙯ౽Ǵ аϷ NF-ț% ᆶ DNA ‫่ޑ‬ӝૈΚǶќ΋Бय़Ǵೀ౛ DHA ཮ᡉ๱ቚуՈ୷፦਼ϯ 䁙 1 (HO-1)‫ک‬ᙯᒵӢη Nrf2 ‫ޑ‬ೈқ፦߄౜Ǵ٠ᇨᏤ Nrf2 ᙯ౽຾ΕಒझਡϣǴӛ ΢ፓ࿯ antioxidant response element (ARE)ൔᏤ୷Ӣࢲ‫܄‬ǶӕਔǴ‫ॺך‬Ψว౜ DHA ፓ࿯ HO-1 ‫߄ޑ‬౜Ьाࢂӧᙯᒵ໘ࢤǶќѦǴճҔ siRNA υᘋ‫ ڋ׭ೌמ‬HO-1 ‫ޑ‬ ߄౜Ǵ཮೽ϩ଍ᙯ DHA ჹ‫ ܭ‬ICAM-1 ‫ڋ׭ޑ‬բҔǶᆕӝа΢่݀ள‫ޕ‬Ǵӧ EA.hy926 ಒझύǴDHA ཮ᙖҗ‫ ڋ׭‬NF-ț% ૻ৲໺ሀၡ৩Ϸቚу Nrf2-dependent HO-1 ϐ߄౜ٰफ़ե TNF-Į ‫܌‬ᇨว‫ ޑ‬ICAM-1 ߄౜ǹҁࣴ‫ز‬᛾ჴ DHA ‫ڀ‬ԖႣٛ ЈՈᆅ฻ว‫܄ݹ‬੯ੰϐወΚǶ. ᜢᗖӷǺΒΜΒᅹϤ౎ለ (DHA)ǵಒझ໔ᗹߕϩη 1 (ICAM-1)ǵဍዦᚯԝӢη-Į (TNF-Į

(13) ǵՈ୷፦਼ϯ䁙 1 (HO-1)ǵNF-ț%ǵNrf2ǵว‫ݹ‬ϸᔈ. vii.

(14) मЎᄔा Several studies indicate that inflammation and endothelial cell dysfunction are important initiating events in atherosclerosis. Intercellular adhesion molecule 1 (ICAM-1), an inflammatory biomarker, plays a pivotal role in cardiovascular disease (CVD) progression. Dietary intake of fish oil rich in n-3 polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid (DHA), has been associated with reduced CVD risk. In this study, we investigated the effect of DHA on the tumor necrosis factor-alpha (TNF-Į

(15) -induced ICAM-1 expression in EA.hy926 cells and the possible mechanisms involved. The results showed that DHA (50 and ȝ0

(16) LQKLELWHG TNF-Į-induced ICAM-1 protein, mRNA expression, and promoter activity. In addition, TNF-Į-stimulated IKK phosphorylation, Iț% phosphorylation and degradation, p65 nuclear translocation, and NF-ț% and DNA binding activity were attenuated by pretreatment with DHA. Furthermore, DHA significantly increased the protein expression of heme oxygenase 1 (HO-1) and nuclear factor erythroid 2-related factor 2 (Nrf2), induced Nrf2 translocation to the nucleus, and up-regulated antioxidant response element (ARE)-luciferase reporter activity. HO-1 expression is primarily regulated by DHA at the transcriptional level. Transfection with HO-1 siRNA knocked down HO-1 expression and partially reversed the DHA-mediated inhibition of ICAM-1 expression. In conclusion, these results suggested that DHA inhibits TNF-Į-induced ICAM-1 expression is through attenuation of NF-ț% signaling pathway and stimulation of Nrf2-dependent HO-1 expression in EA.hy926 cells. The anti-inflammatory effects of DHA may implicate its CVD-protective potential.. Key words: DHA, HO-1, ICAM-1, inflammation, NF-ț%, Nrf2, TNF-Į viii.

(17) ಃ΋೽ҽ. ಃ΋ക ߻‫ق‬. ᔼᎦၸഭǵ‫ޥ‬औǵଯՈિǵଯᖌ‫ڰ‬ᎇǵଯՈᓸ฻ୢᚒࢂҞ߻ς໒ว୯ৎύӭ ኧΓα‫܌‬य़ᖏ‫ޑ‬ᔼᎦ࣬ᜢୢᚒǴѠ᡼ΜεԝӢ྽ύǴЈ᠌ੰǵတύ॥฻ЈՈᆅ੯ ੰ൩эΑࡐଯ‫ޑ‬К‫ٯ‬ǴЈՈᆅ੯ੰ΋‫ࢂޔ‬ӄౚ‫ޑ܄‬ख़εϦӅፁғ᝼ᚒǴ‫ځ‬ԝΫ౗ ೴ԃᚹଯǴჹ‫଼ܭ‬ந‫ޑ‬ቹៜዴჴό৒۹ຎǶ೚ӭࣴ‫ز‬ᡉҢǴ୏ેๆ‫ރ‬ฯϯ (atherosclerosis)ࢂ΋ᅿᄌ‫܄‬ว‫ݹ‬ϸᔈ(Glass & Witztum, 2001)ǴԶЪӧ୏ેฯϯ߃ යǴϣҜಒझ‫ޑ‬ว‫ݹ‬ϸᔈ‫ת‬ᄽΑཱུख़ा‫فޑ‬ՅǶ‫܌‬аǴుΕᕕှ୏ેฯϯ‫ޑ‬ԋӢ Ϸ‫ځ‬ፓ௓ᐒᙯǴԖշ‫ܭ‬ႣٛϷ‫ݯ‬ᕍ୏ેฯϯǶ. ӕਔǴεໆ‫ޑ‬ᙴᏢ᛾Ᏽว౜Ǵ௦‫ڗ‬ᑈཱུ‫ޑ‬Ⴃٛ௛ࡼёफ़եᑡ஻ЈՈᆅ੯ੰϐ ॥ᓀǶՋϡ 1971 ԃǴϏഝ‫ٿ‬ՏࣽᏢৎ੤(Bang)‫ک‬ᔎ࢙਱(Dyerberg)Ǵӧ๱ӜᙴᏢ ᚇᇞ Lancet ଞჹ n-3 όႫ‫ک‬િެለว߄ᡋΓ่݀Ǻдॺᢀჸ‫ډ‬ǴՐӧчཱུӇচ਱ ഊើ৞΢‫ޑ‬ངථ୷ነΓǴӧલЮᆘՅጫ๼‫ک‬Н݀‫ޑ‬ᕉნύǴᑡ஻ЈՈᆅ੯ੰ‫ޑ‬К ‫ࠅٯ‬К΋૓ΓեࡐӭǴЬा‫ޑ‬চӢࢂдॺ‫܌‬ឪ‫ޑڗ‬१‫ނ‬ε೽ҽࢂుੇങᜪǴӵᗵ ങǵ᜵ങ฻Ǵ໯१ύ൤֖εໆ‫ޑ‬ങ‫ݨ‬ǴӢԜࡐϿᑡ஻ЈԼఒ༞Ϸတਵ༞฻੯ੰ (Bang et al., 1971)Ƕങ‫(ݨ‬n-3 ӭϡόႫ‫ک‬િެለ)բࣁᇶշٛ‫ݯ‬ЈՈᆅ੯ੰ‫ޑ‬π‫ڀ‬ ςԖӭԃǴЪԖ೚ӭࣴ‫ز‬ൔᏤᡉҢǴံкങ‫ݨ‬ჹ‫ܭ‬फ़եЈՈᆅ੯ੰϐวғ౗Ԗ҅ य़‫ޑ‬фਏ(Wang et al., 2006; Casós et al., 2008)ǶӢԜҁࣴ‫ز‬ஒаϣҜಒझ‫߄܌‬౜ ‫ޑ‬ᗹߕϩηࣁࡰ኱Ǵ௖૸ DHA ჹ‫ځ‬ቹៜϷ࣬ᜢϐϩηᐒ‫ڋ‬Ǵයఈჹങ‫ޑݨ‬Ⴃٛ ߥ଼фਏૈԖ‫׳‬ుΕӦΑှǶ. 1.

(18) ಃΒക Ў᝘ӣ៝. ΋ǵ୏ેๆ‫ރ‬ฯϯ(atherosclerosis). 1. ୏ેๆ‫ރ‬ฯϯϐԋӢ ୏ેๆ‫ރ‬ฯϯ‫׎ޑ‬ԋၸำࣁ΋ፄᚇЪᅌ຾Ԅ‫ޑ‬ठੰၸำǴ൩ੰ౛੝ቻεठё ϩࣁ൳ঁ໘ࢤǺ(1)Ոᆅϣቫቚࠆ(intimal thickening)ǹ(2)િެદ(fatty streak)ǹ(3) ύࡋੰ‫(؞‬intermediate lesion)ǹ(4)ᠼᆢඬ༧(fibrous plaque)ǹ(5)ፄᚇ‫؞ੰ܄‬ (complicated lesion) (Hegele, 1996)Ƕ‫ځ‬Ьा‫ޑ‬ठੰᐒᙯᏢᇥࢂҗRossගр‫ޑ‬Ȩϣ Ҝಒझ‫ڙ‬໾ࡕϐϸᔈଷᇥȩ(The response to injury) (Ross, 1993)Ǵ೷ԋϣҜಒझ‫ڙ‬ ཞ‫ޑ‬চӢࡐӭǴхࡴՈనύLDL‫ޑ‬ቚуϷ೏অႬǵԾҗ୷(free radicals)ǵଯՈᓸǵ ᑗֿੰǵ‫(ࢥੰ੶ݰ‬herpesviruses)གࢉǵՊচᡏགࢉ(Chlamydia pneumoniae)฻Ǵ ࣣ཮೷ԋϣҜಒझфૈѨፓ(dysfunction)Ƕӧ୏ેฯϯ߃යǴ‫ډڙ‬໾্‫ޑ‬ϣҜಒ झфૈ‫ڙ‬ཞǴ຾Զౢғжᓭ‫܄‬ϸᔈǴ‫ׯ‬ᡂΑಒझ‫ރۓࡡޑ‬ᄊ(homeostasis)ǴϷ‫ׯ‬ ᡂϣҜቫ‫ޑ‬೯೸‫܄‬ǶϣҜቫ೯೸‫܄‬ቚуǴ٬ள΋٤εϩη‫ނ‬፦৒ܰ೯೸Ǵ‫ٯ‬ӵե ஏࡋિೈқᖌ‫ڰ‬ᎇ(low-density lipoprotein, LDL)Ǵ٠ӧՈᆅᏛ΢୴ᑈϷ೏਼ϯԋ ox-LDL (Steinberg et al., 1989)ǶԶЪ྽ϣҜಒझ‫ڈډڙ‬ᐟਔǴ཮Ꮴठ೚ӭᖿϯ‫ނ‬ ፦ϷғߏӢη(growth factor)೏ញрǴхࡴMCP-1 (monocyte chemotactic protein-1)ǵMCSF (monocyte-colony-stimulating factor)ǵTGF-ȕ WUDQVIRUPLQJJURZWK factor-beta)Ǵ཮ߦ٬Ոనൻᕉύ‫ൂޑ‬ਡౚߕ๱‫ܭ‬ՈᆅᏛǴ٠౽ՉԿϣҜΠ‫ޜ‬໔Ƕ ൂਡౚ཮‫ڙ‬MCSFቹៜԶϩϯԋѮᏘಒझ(Qiao et al., 1997)Ǵ٠տᏘၸӭ‫ޑ‬ ox-LDLǴ٬ளεໆ‫ޑ‬ᖌ‫ڰ‬ᎇǵિ፦୴ᑈӧಒझϣԶ‫׎‬ԋ‫ݣݰ‬ಒझ(foam cell)Ǵ٠ ϩ‫ݜ‬εໆ‫߻ޑ‬ว‫ݹ‬ಒझᐟન(proinflammatory cytokines)Ǵ‫ٯ‬ӵǺIL-1 (interleukin-1)ǵTNF-Į WXPRUQHFURVLVIDFWRU-alpha)ǵIFN-Ȗ LQWHUIHURQ-gamma)ǴԶ ೭٤ಒझᐟન཮‫ڐ‬ӕғߏӢηǴ‫ٯ‬ӵǺVEGF (vascular endothelial growth factor)ǵ 2.

(19) PDGF (platelet derive growth factor-1)ǵIGF-1 (insulin-like growth factor-1)‫ڈ‬ᐟѳྖ Լಒझ‫ޑ‬ቚғǴΨ཮ӛ΢ፓ࿯ᗹߕϩηϐ߄౜Ǵߦ٬ൂਡౚ‫ޑ‬ᗹߕǵ౽ՉϷϩϯǴ ቹៜϣҜಒझϷѳྖԼಒझǴԶ٬ளੰ‫׳؞‬уൾϯ(Kim et al., 2001)Ƕ. 2. ಒझᗹߕϩηᆶ୏ેๆ‫ރ‬ฯϯϐᜢ߯ ಒझᗹߕϩη(cellular adhesion molecules)ࢂ߄౜‫ܭ‬ಒझ߄य़Ǵፓ௓ಒझᆶಒ झϐ໔‫܈‬ಒझᆶಒझѦ୷፦(extracellular matrix)໔‫ޑ‬ᗹߕբҔǶӧ୏ેฯϯ‫ੰޑ‬ ౛ၸำύǴനܴᡉ‫ޑ‬੝ቻࣁ‫ڙ‬໾‫ޑ‬ϣҜಒझ߄य़཮εໆቚуқՈౚϷᗹߕϩηǴ ௗ๱཮ӢࣁϣҜቫ‫ޑ‬೯೸‫܄‬ቚуǴ٬ளқՈౚ৒ܰऀ೸ՈᆅᏛǴᆀࣁқՈౚቻ༅ բҔ(recruitment)ǴԶ೭٤ၸำӧว‫ݹ‬ϸᔈύࢂߚதख़ा‫ޑ‬Ǵ‫ځ‬ύಒझ߄य़‫ޑ‬ᗹ ߕϩη߾‫ת‬ᄽ๱ख़ा‫فޑ‬ՅǶ. қՈౚᗹߕ‫ډ‬ϣҜಒझ΢Ǵӆᙯ౽຾ΕϣҜΠ‫ޜ‬໔ǴЬाхࡴΑѤঁ໘ࢤ(კ 2.1)Ǻ(1)२ӃǴқՈౚ཮ᗹߕ‫ډ‬ϣҜಒझ΢(Capture/Tethering)ǹ(2)ௗ๱೏ᒧ᏷ન (selectins)ࢲϯࡕ཮‫ݮ‬๱ՈᆅᏛᄾ୏(rolling)ǹ(3)٠೸ၸಒझᐟન(chemokines)‫ޑ‬բ ҔǴ‫ׯ‬ᡂ᏾ӝન(integrins)‫ޑ‬ᄬࠠǴߦ٬ integrins ᆶ CAMs ຾ՉҬϕբҔǴᛙ‫ڰ‬ Ӧᗹߕ(firm adhesion)ӧϣҜಒझ΢ǹ(4)қՈౚ໒‫ۈ‬ᡂ‫׎‬Ǵऀ೸ϣҜቫ (transmigration)ԿϣҜΠ‫ޜ‬໔ǴЬाࢂҗ integrins ᆶ PECAM ٰፓ௓Ǵനࡕߦ٬ ୏ેๆ‫ރ‬ฯϯ‫׎ޑ‬ԋ(Carlos & Harlan, 1994; Blankenberg et al., 2003; Lawson & Wolf, 2009)Ƕ. 3.

(20) კ 2.1 қՈౚቻ༅բҔ(Lawson & Wolf, 2009). ೭΋ೱՍ‫ޑ‬ၸำϩձሡाόӕ‫ޑ‬ᗹߕϩηୖᆶǴ‫ځ‬ቶ‫߄ݱ‬౜Ϸϩթ‫ܭ‬Ӛᅿಒ झǴЬाёϩࣁΟᜪǺselectinsǵIgSF ‫ ک‬integrins (߄ 2.1) (Blankenberg et al., 2003)Ƕᜪխࣝౚೈқϩη(Immunoglobulin-like molecules or Immunoglobulin superfamily, IgSF)ࢂಒझጢ΢‫ޑ‬ᗐೈқௗ‫ڙ‬Ꮤ(glycoprotein receptor)Ǵҗ‫ܭ‬ಒझѦ ‫ ޑ‬Ig domain όӕǴԶԖόӕ‫ ޑ‬isoformǴ‫ٯ‬ӵ ICAMǵVCAMǵPECAMǶ. 4.

(21) ߄ 2.1 ୖᆶ୏ેๆ‫ރ‬ฯϯϐᗹߕϩη(Blankenberg et al., 2003) Table 2.1. ಒझ໔ᗹߕϩη(Intercellular Adhesion Molecules, ICAMs)ӅԖϖᅿ٥ࠠ (subtypes)Ǵ‫ځ‬ύനத‫ ࢂޑـ‬ICAM-1 (Ξᆀ CD-54)ǴӧϣҜಒझǵ΢Ҝಒझǵᠼ ᆢ҆ಒझǵѳྖԼಒझǵқՈౚ฻೿Ԗ߄౜ǹѳਔᆢ࡭ӧࡐե‫֖ޑ‬ໆǴ྽‫߻ډڙ‬ ว‫ݹ‬ಒझᐟન(TNF-ĮǵIL-1ǵIFN-Ȗ

(22) ǵLPS (lipopolysaccharide)ǵox-LDLǵ਼ϯᓸ Κ(oxidative stress)ǵੰࢥགࢉ฻‫ڈ‬ᐟਔǴ཮εໆ߄౜Ԝᗹߕϩη(߄ 2.2) (Roebuck & Finnegan, 1999)Ǵ೸ၸᆶқՈౚ΢ϐ integrins ౢғҬϕբҔǴ٬қՈౚᆙஏᗹ ߕ(firm adhesion)‫ܭ‬ϣҜಒझ΢(Blankenberg et al., 2003)ǶճҔ‫ל‬ᡏߔᘐ ICAM-1 բҔ‫܈‬ᙖҗ ICAM-1 knockdown ኳԄ᛾ჴǺICAM-1 ӧқՈౚᗹߕᆶऀ೸ϣҜቫ ‫ޑ‬౽Չၸำύዴჴ‫ת‬ᄽ๱ख़ा‫فޑ‬Յ(Reiss & Engelhardt, 1999; Lehmann et al., 2003)ǶΓᜪ ICAM-1 ୷Ӣ‫୏௴ޑ‬η(promoter)‫ׇ‬ӈς೏ᒧ෗(clone)рٰǴ೚ӭᙯ ᒵӢη(transcription factor)཮ୖᆶፓ௓ ICAM-1 ߄౜Ǵхࡴ AP-1 (activator protein-1) (Son et al., 2006)ǵSp1 (Berendji-Grun et al., 2001)ᆶ NF-ț% QXFOHDU 5.

(23) factor-ț%

(24) (Zhou et al., 2007; Lian et al., 2010)ǵSTAT (Janus kinases (JAK)-signal transducers and activators of transcription) (Audette et al., 2001)ǶTNF-ĮǵIL-1 Ьा ࢂ೸ၸࢲϯ NF-ț% ٰፓ௓ ICAM-1Ǵ‫ٯ‬ӵǺӧа TNF-Į ᇨวΓᜪ‫ޤ‬΢Ҝಒझ (human pulmonary epithelial cells)߄౜ ICAM-1 ‫ޑ‬ኳԄύǴ᛾ჴΑ NF-ț% ‫ޑ‬ख़ा ‫(܄‬Oh et al., 2010)ǹԶ IFN-Ȗ ߾ࢂ೸ၸ STAT ໺ሀၡ৩ٰፓ௓ ICAM-1 (კ 2.2)Ƕ ICAM-1 ନΑ཮ቹៜқՈౚ‫ޑ‬౽ՉբҔϷ T ಒझ‫ࢲޑ‬ϯբҔǹӕਔǴICAM-1 Ψ ᆶ೚ӭว‫ݹ‬੯ੰԖᜢǴ‫ٯ‬ӵ਻ഹ(asthma)ǵ୏ેๆ‫ރ‬ฯϯ(atherosclerosis)ǵ࡚‫ڥ܄‬ ֎ึॐੱংဂ(acute respiratory distress syndrome)ǵલՈ‫܄‬લ਼ӆឲࢬཞ໾ (ischemia reperfusion injury)ǵԾᡏխࣝ੯ੰ(autoimmune disease)฻(Roebuck & Finnegan, 1999)Ƕ. ߄ 2.2 ᇨว ICAM-1 ߄౜‫ޑ‬όӕ‫ڈ‬ᐟ(Roebuck & Finnegan, 1999) Table 2.2. 6.

(25) კ 2.2 TNF-ĮǵIL-ȕǵIFN-Ȗ ፓ௓ ICAM-1 ‫ޑ‬ϩη໺ሀၡ৩(Roebuck & Finnegan, 1999). 3. ဍዦᚯԝӢηᆶ୏ેๆ‫ރ‬ฯϯϐᜢ߯ ဍዦᚯԝӢη(Tumor Necrosis Factor-alpha, TNF-Į

(26) ӧӚᅿғ౛Ϸੰ౛ၸำ ύ‫ת‬ᄽ๱ख़ा‫فޑ‬ՅǴхࡴಒझቚғǵϩϯǵ঒ΫϷว‫ݹ‬ϸᔈ(Gaur & Aggarwal, 2003; Liu, 2005)ǶTNF-Į ᙖҗ‫ک‬ಒझ߄य़‫ڙޑ‬Ꮤ่ӝԶౢғғ‫܄ࢲނ‬Ǵ٩Ᏽϩη ໆ‫ޑ‬όӕёஒ‫ڙ‬Ꮤϩࣁ TNFR1 (tumor necrosis factor receptor 1) (55 kDa)Ϸ TNFR2 (75 kDa)ǶTNFR1 ӧ‫܌‬Ԗᅿᜪ‫ޑ‬ಒझጢ΢೿Ԗ߄౜ǴΨࢂ TNF-Į Ьा‫ޑ‬ ‫ڙ‬ᏔǹԶ TNFR2 ߾߄౜ӧխࣝಒझϷϣҜಒझ(Gaur & Aggarwal, 2003)ǶTNF-Į ࢂ΋ᅿς‫߻ޑޕ‬ว‫ݹ‬ಒझᐟનǴ٩ಒझᅿᜪόӕϷғ౛‫ރ‬ᄊϐৡ౦ԶԖᐱ੝‫ޑ‬ғ ‫ނ‬фૈǴ೯தӧ୏ેๆ‫ރ‬ฯϯཞ໾ೀёว౜Ѭ‫ޑ‬ᙫၞ(Sana et al., 2005)ǶTNF-Į ཮‫ڈ‬ᐟϣҜಒझ߄౜ᗹߕϩη(ӵ ICAM-1ǵVCAM-1ǵE-selectin)ǴϷߦ຾қՈ ౚᗹߕբҔ(Zhou et al., 2007; Oh et al., 2010)ǴԶԜᇨวբҔࢂ،‫ܭۓ‬ᙯᒵӢη NF-ț% ‫ࢲޑ‬ϯǶӧࢌ٤௃‫ݩ‬ΠǴTNF-Į ཮೷ԋՈᆅ‫ډڙ‬໾্ǵϣҜಒझѨѐфૈǴ ӢԜத೏Ҕٰբࣁᇨวಒझౢғว‫ݹ‬ϸᔈ‫ޑ‬ኳԄǶ 7.

(27) 4. ᙯᒵӢη NF-ț% ૻ৲໺ሀၡ৩. 4-1. NF-ț% NF-ț% 1XFOHDUIDFWRU-ț%

(28) ࢂᆶว‫ݹ‬Ϸխࣝ࣬ᜢ‫ޑ‬ᙯᒵӢηǴឦ‫ ܭ‬Rel ৎ௼‫ޑ‬ ΋ᅿೈқ፦ǶҞ߻ς‫ ޕ‬NF-ț% ৎ௼җϖঁԋ঩‫܌‬ಔԋǺp65 (RelA)ǵc-RelǵRelBǵ p50/p105 (NF-ț%1)ǵp52/p100 (NF-ț%2)Ǵаӕ፦ፄӝᡏ(homodimer)‫܈‬౦፦ፄӝᡏ (heterodimer)‫ޑ‬БԄӸӧ(Jost & Ruland, 2007)Ǻε೽ϩ NF-ț% ፄӝᡏӧಒझ፦ᆶ ,ț% ೱ่ਔค‫ݤ‬຾ΕಒझਡϣǴ‫ٯ‬ӵ p50/p65 ࢂ‫ ޑࠠڂ‬NF-ț% ፄӝᡏǹԖ٤ NF-ț% ፄӝᡏǴ‫ٯ‬ӵ p52/RelBǴόᆶ ,ț% ೱ่Ǵߡૈ‫ޔ‬ௗ຾ΕಒझਡǹԶ p105 ᆶ p100 ӧ C ᆄԖ Ankyrin domain (ANK)Ǵѳਔѝૈ੮ӧಒझ፦ϣǴ྽Нှԋ p50 ᆶ p52 ਔǴω‫ڀ‬Ԗ຾Εಒझਡ‫ૈޑ‬Κ(კ 2.3ǵკ 2.4) (Perkins, 2000)Ƕ೭٤ೈқ፦೿‫ڀ‬Ԗ ΋ࢤ࣬՟‫ޑ‬ữ୷ለ‫ׇ‬ӈǴᆀϐࣁ Rel homology domain (RHD)Ǵ೭ࢤ‫ׇ‬ӈ֖Ԗ dimerization domainǵnuclear localization signal (NLS)ǵDNA-binding domainǴϩ ձё٬ NF-ț% ൂᡏ຾Չᚈᡏϯ(dimerization)ǹ྽ ,ț% ᆶ NLS ่ӝਔ཮‫ ڋ׭‬NF-ț% ‫܄ࢲޑ‬Ǵค‫ݤ‬຾ՉਡᙯՏ(nuclear translocation)ǹᆶҞ኱୷Ӣ(target genes)΢ෞ‫ޑ‬ ௴୏η(ᆀϐࣁ ț%VLWH

(29) ่ӝǴፓ௓Ҟ኱୷Ӣ‫߄ޑ‬౜(May & Ghosh, 1998; Papa et al., 2006)Ƕ NF-ț% த‫ࢲޑـ‬ϯ‫׎‬Ԅࣁ p50/p65 ౦፦ፄӝᡏǶ҅த௃‫ݩ‬ΠǴNF-ț% ࢂόࢲ ϯЪ੮ӧಒझ፦ύǴӢࣁ NF-ț% ‫ڋ׭ޑ‬ೈқ LQKLELWRUț% ,ț%

(30) Ǵ཮ᇂՐߦ٬ NF-ț% ຾Εಒझਡύ‫ޑ‬ữ୷ለ‫ׇ‬ӈ(NLS)Ǵ٬ NF-ț% ᅉ੮ӧಒझ፦ύǶ྽ಒझ‫ډڙ‬ว‫ݹ‬ ϟ፦‫ڈ‬ᐟਔ(߄ 2.3)Ǵ,ț% ཮‫ډڙ‬΢ෞೈқ ,ț%NLQDVH ,..

(31) ᕗለϯǴௗ๱ ,ț% ཮ ೏‫ݱ‬નϯ(ubiquitinated)٠຾Εೈқ䁙ᡏ(proteasome)ύϩှǴ຾Զ೷ԋ NF-ț% ‫ޑ‬ ࢲϯǴ٬‫ځ‬຾Εಒझਡϣ‫ ک‬DNA ่ӝǴ௴୏ว‫୷ݹ‬Ӣ‫߄ޑ‬౜Ǵ‫ٯ‬ӵಒझᐟનǵ ᗹߕϩη฻(კ 2.5ǵ߄ 2.4) (Barnes et al., 1997; Ghosh & Karin, 2002; Hayden et al., 2006; Sun & Karin, 2008; Rahman & McFadden, 2011)Ƕࣴ‫ࡰز‬рǴӧΓᜪ‫ݰޤ‬΢ 8.

(32) Ҝಒझ(human alveolar epithelial cell)ύǴTNF-Į ཮࿶җࢲϯೈқᐟ䁙 C (protein kinase C, PKC)Ǵࢲϯ c-SrcǴࢲϯ‫ ޑ‬c-Src ཮࿶җᕗለϯ IKK Զࢲϯ IKKǹќ΋ Бय़ǴIKK Ψ཮࿶җ NF-ț%-inducing kinase (NIK)೭చ໺ሀၡ৩‫ࢲډڙ‬ϯ(Huang et al., 2003)Ƕ. კ 2.3 NF-ț% ಔԋࠠԄ(Perkins, 2000). 9.

(33) Figure 2.4. კ 2.4 NF-ț%ǵ,ț% Ϸ IKK ่ᄬ(Jost & Ruland, 2007) 10.

(34) კ 2.5 NF-ț% ૻ৲໺ሀၡ৩(Rahman & McFadden, 2011). 11.

(35) ߄ 2.3 ࢲϯ NF-țB ‫ڈޑ‬ᐟ(Barnes et al., 1997). ߄ 2.4 ‫ ڙ‬NF-țB ፓ௓‫ޑ‬ೈқ፦(Barnes et al., 1997) Table 2.4. Table 2.3. 4-2. IKK NF-țB ૻ৲໺ሀၸำࢂҗ΢ෞ‫ ޑ‬NIK ᕗለϯ IKKǴIKK complex ࢂҗ IțB kinase Į (IKKĮ)ǵIțB kinase ȕ (IKKȕ)‫ ک‬IțB kinase Ȗ (IKKȖǴΞᆀࣁ NEMO, NF-țB essential modulator)Ϸ casein kinase ҈ (CK҈)‫܌‬ಔԋǶ,..Į,..ȕ ‫ ک‬,..Ȗ ‫׎‬ԋ ፄӝ‫ނ‬Ǵ,..Į ‫ ک‬,..ȕ ឦ‫ ܭ‬serine-specific kinaseǴϩηໆϩձࣁ 85 kDa Ϸ 87 kDaǴЪ‫ڀ‬Ԗ 52ʘϐ࣬՟‫(܄‬homology)Ƕ,..Į җ 745 ঁữ୷ለ‫܌‬ಔԋǴ,..ȕ ߾ җ 756 ঁữ୷ለ‫܌‬ಔԋǴ‫่ځ‬ᄬࣁ N ᆄ Kinase domainǵLeucine zipper (LZ) region Ϸ C ᆄ Helix-loop-helix domain (HLH) (კ 2.4)Ƕӧ NF-ț% ࢲϯၸำύǴ,..Į ,..ȕ ‫ ک‬,..Ȗ ፄӝ‫཮ނ‬٬ ,ț%Į ‫ ܭ‬N ᆄ serine 32 ‫ ک‬36 ‫ޑ‬Տ࿼ᕗለϯǴᏤठ ,ț%Į ᄬࠠ‫ׯ‬ᡂ٠ᆽᑈ‫ૈޑ୼ى‬ໆǴᇨᏤ 26S-proteasome ‫ ܭ‬,ț%Į ‫ ޑ‬lysine 21 ᆶ 22 ຾ Չफ़ှբҔǹԶ ,ț%ȕ ߾‫ ܭ‬ser-19 ‫ ک‬ser-23 Տ࿼೏ᕗለϯǴՠ ,ț%ȕ ‫ޑ‬ϩှೲࡋၨ ,ț%Į ጗ᄌǹӢԜǴ,ț%Į ჹ NF-ț% ࢲϯࢂ‫ז‬ೲԶอኩ‫ޑ‬ǴԶ ,ț%ȕ ߾ၨૈ୼ᆢ࡭ 12.

(36) ߏΦǶ,..Į,..ȕ ό཮ჹ‫ځ‬Ѭ ,ț%LVRIRUP ,ț%İ

(37) ϐ serine resides ຾ՉᕗለϯǶ casein kinase ҈ (CK҈)ёஒ ,ț% ೈқ፦ C ᆄ PEST ‫ׇ‬ӈᕗለϯǴ΋٤Ꮲ‫ޣ‬ᇡࣁǺ Ѹ໪ӃԖ ,ț% ೈқ፦ C ᆄ࡭ុ‫܄‬ᕗለϯǴωૈᇨว ,ț% ೈқ፦ N ᆄᕗለϯ(May & Ghosh, 1998; Karin & Delhase, 2000; Jost & Ruland, 2007)Ƕ. 4-,ț% ,ț% ೈқ፦‫ޑ‬ԋ঩хࡴ ,ț%Įǵ,ț%ȕǵ,ț%Ȗǵ,ț%İ ‫ ک‬Bcl-3(‫܈‬ᆀ IKAP) (კ 2.4)Ǵ ,ț% ೈқ፦ᙖҗ Ankyrin domain ‫ ک‬C ᆄ PEST (Proine-, Glutamio acid-, Serine- and Threonine-rich)‫ׇ‬ӈᆶ NF-ț% ‫ ޑ‬RHD ่ӝǶᗨฅ೭٤ೈқ፦೿ૈߔЗ NF-ț% ຾ ΕಒझਡǴՠࢂόӕ‫ ޑ‬,ț% Ԗ‫ځ‬੝‫ۓ‬ϐբҔჹຝǴ‫ٯ‬ӵǺ,ț%Į ёа੝‫่ۓ‬ӝ‫ډ‬ p50/p65 ‫ ک‬p65/p65ǴՠόૈԖਏӦ่ӝ‫ ډ‬p50/p50ǹԶ ,ț%ȕ ཮஑΋‫่ޑ܄‬ӝ‫ډ‬ p50/c-Rel ౦፦ፄӝᡏǹ,ț%İ ߾཮่ӝ‫ ډ‬p65 Ϸ c-Rel ‫ޑ‬ӕ፦ፄӝᡏǹ,ț%Ȗ ‫ ک‬Bcl-3 ߾ࢂ஑΋‫่܄‬ӝ‫ ډ‬p50 Ϸ p52 ӕ፦ፄӝᡏ΢Ƕ൩ NF-ț% ӸӧࠠԄК‫ٯ‬നଯ‫ޑ‬ p50/p65 ౦፦ፄӝᡏԶ‫ق‬Ǵ,ț%Į ่ӝ‫ ډ‬p50/p65 ‫ޑ‬ᒃ‫ک‬Κ(affinity)К่ӝ‫ ډ‬p65/p65 ‫ޑ‬ᒃ‫ک‬Κଯ 27 ७ǴК่ӝ‫ ډ‬p50/p50 ‫ޑ‬ᒃ‫ک‬Κଯ 60 ७Ƕ,ț%Į ୷Ӣ‫୏௴ޑ‬η΢‫ڀ‬ Ԗ ț%VLWH ፓ௓‫ׇ‬ӈǴӢԜ NF-ț% ࢲϯஒᏤठ ,ț%Į εໆӝԋǴ߃ӝԋ‫ ޑ‬,ț%Į ཮ ຾ΕಒझਡύǴ่ӝ NF-ț% ஒ‫ځ‬஥ӣಒझ፦Ƕՠ ,ț%ȕ ٠ό‫ࢲڙ‬ϯ‫ ޑ‬NF-ț% ‫܌‬ፓ ௓ǴӢԜ ,ț%ȕ ϐफ़ှёа๏ϒ NF-ț% ၨߏ‫ࢲޑ‬ϯਔ໔(May & Ghosh, 1998; Christman et al., 2000; Wertz & Dixit, 2010)Ƕ. 13.

(38) Βǵങ‫ݨ‬ᆶЈՈᆅ੯ੰϐᜢ߯. 1. ങ‫ཷݨ‬ॊ ੇࢩੌෞғ‫֖ނ‬Ԗ n-3 ‫س‬ӈ‫ ޑ‬$/$ Į-linolenic acidǴԛ٥ഞ‫ݨ‬ለ)ǵEPA (eicosapentaenoic acidǴΒΜᅹϖ౎ለ)Ϸ DHA (docosahexaenoic acidǴΒΜΒᅹϤ ౎ለ)ǴҗλࠠങᜪӞΠࡕǴӆ೏εࠠങ‫ਂ܌‬१Ǵӧ‫׎‬ԋ१‫ނ‬᜘‫ޑ‬ၸำύǴ೏ങ ឪ‫ ޑڗ‬ALA ཮ӆᙯᡂԋ‫ځ‬д n-3 ‫س‬ӈ‫׎ޑ‬ԄǴᑈӸӧങᡏϣǴջࢂങ‫ޑݨ‬Ԗਏ ԋҽǶԶങ‫ݨ‬ύ‫ ޑ‬n-3 િެለǴЬाࣁ EPA ‫ ک‬DHAǶᗨฅ෌‫ݨނ‬ύ‫ ޑ‬ALA ҭё ӧΓᡏϣᙯඤԋ‫ځ‬д n-3 િެለǴՠᙯඤК‫ٯ‬λ‫ܭ‬Μϩϐ΋(Siddiqui et al., 2008)ǶϺฅ१‫ނ‬ύ‫ޑ‬ుੇങᜪǴӵᗵങǵᗴങǵ᜵ങ฻Ǵ‫ݨځ‬િ֖Ԗၨଯໆ‫ޑ‬ EPA Ϸ DHAǴ‫܌‬а΋૓ӵ݀ाံк EPA Ϸ DHAǴᗋࢂаങ‫ࣁݨ‬ന٫ٰྍǶ. 2. n-3 ӭϡόႫ‫ک‬િެለ ӭϡόႫ‫ک‬િެለ(polyunsaturated fatty acids, PUFAs)ࡪ n ጓဦ‫س‬಍ǴਥᏵಃ ΋ঁᚈᗖ‫܌‬ӧ‫ޑ‬Տ࿼ёஒόႫ‫ک‬િެለϩࣁѤᅿᜪࠠǴջ n-3ǵn-6ǵn-7 ‫ ک‬n-9 ‫س‬ӈǴՠ‫ڀ‬Ԗख़ाғ‫ނ‬Ꮲཀက‫ ࢂޑ‬n-3 ‫ ک‬n-6 PUFAsǶΓᜪค‫ݤ‬ԾՉӝԋ n-3 ‫ک‬ n-6 όႫ‫ک‬િެለǴѸ໪வ१‫ނ‬ύ‫ޔ‬ௗឪ‫ڗ‬ǴӢԜΞ೏ᆀࣁѸሡિެለǴЀ‫ࢂځ‬ Į-ԛ٥ഞ‫ݨ‬ለ(n-Į-linolenic acid, ALA)‫ک‬٥ഞ‫ݨ‬ለ(n-6, linoleic acid, LA) (SanGiovanni & Chew, 2005)ǶҞ߻ၨදၹ‫ ޑ‬n-3 િެለԖ ALAǵEPA ‫ ک‬DHA Ο ᅿǴନΑ ALAǴEPA ‫ ک‬DHA ೯தӸӧ‫ܭ‬ుੇങ‫ݨ‬ύǶ. 2-1. ่ᄬ n-3 ӭϡόႫ‫ک‬િެለ(n-3 PUFAs)ࢂх֖ኧঁа΢όႫ‫ک‬ᗖ‫ޑ‬િެለǴӢࣁ ಃ΋ঁᚈᗖр౜ӧᅹ᜘ຯҘ୷ᆄ‫ޑ‬ಃΟঁᅹচη΢Ǵ‫܌‬аᆀϐࣁ n-3 િެለǴΨ ћբ Ȧ-3 િެለǶவკ 2.6 ύёа࣮‫ډ‬ǴDHA ԖϤঁᚈᗖǵΒΜΒঁᅹচηǶ. 14.

(39) კ 2.6 DHA ่ᄬ(SanGiovanni & Chew, 2005). 2-2. ғӝԋբҔ Į-ԛ٥ഞ‫ݨ‬ለ Į-linolenic acid, ALA)ߡࢂ n-3 િެለ‫ࠠڂޑ‬ж߄ǴѬԖΟঁ ᚈᗖǶALA ࢂჹΓᡏ଼நߚதख़ा‫ޑ‬΋ᅿિެለǴՠΓᡏόૈ҅தӝԋǴӢԶ ೏ຎࣁࢂ΋ᅿѸሡિެለ(EFA)ǶALA ёа೸ၸѐႫ‫ک‬䁙(desaturase)‫ک‬ᅹ᜘‫ߏۯ‬ 䁙(elongase)‫ޑ‬໽ϯբҔǴനࡕӝԋ EPA ‫ ک‬DHAǴ಍ᆀࣁ n-3 ‫س‬ӈિެለǶ. კ 2.7 ӭϡόႫ‫ک‬િެለ‫ޑ‬ғӝԋբҔ(De Caterina & Basta, 2001). 15.

(40) 3. DHA ‫ޑ‬ғ౛բҔ/фૈ ങ‫ݨ‬ύ൤֖ n-3 ӭϡόႫ‫ک‬િެለ(n-3 PUFAs)Ǵ‫ٯ‬ӵ EPA ‫ ک‬DHAǴࣁങ‫ݨ‬ ‫܄ࢲޑ‬ԋϩǴ‫ڀ‬ԖӚᅿғ‫ނ‬фૈǴ཮ቹៜΓᜪ଼ந‫ک‬੯ੰ‫ޑ‬วғǴ‫ڀ‬Ԗ‫ۯ‬጗୏ે ๆ‫ރ‬ฯϯǴ‫ל‬ЈࡓѨதϷ‫ׯ‬๓Ոనࢬ୏฻ӭᅿЈՈᆅਏᔈ(Kris-Etherton et al., 2002)Ƕࣴ‫ز‬ᡉҢǴቚу n-3 PUFAs ‫ޑ‬ឪ‫ڗ‬ёа೸ၸаΠ೼৩फ़եЈՈᆅ੯ੰ‫ޑ‬ วғ౗Ǻ(1)෧Ͽᖌ‫ڰ‬ᎇ୴ᑈǴ๤጗୏ેๆ‫ރ‬ฯϯඬ‫ޑ‬ғߏǹ(2)फ़եՈమΟለҒ ‫✊ݨ‬ᐚࡋǴ٠ૈ‫ׯ‬๓жᖴੱংဂ‫ޑ‬௃‫(ݩ‬Schmidt et al., 1992; Jiménez-Gómez et al., 2010)ǹ(3)ႣٛՈਵ‫׎ޑ‬ԋǴफ़եՈనᗹ࿨ࡋǴ‫ڀ‬Ԗ‫ڋ׭‬Ոλ݈Ꮙ໣฻բҔǴឪ Ε፾྽Ꮚໆ‫ޑ‬ങ‫ݨ‬ё٬ TXA2 ѳᑽӛԖճБӛᙯᡂ(Umemura et al., 1995)ǹ(4)Ⴃ ٛЈࡓό᏾‫ޑ‬วғǴឪ‫ڗ‬ങ‫ݨ‬ёफ़եЈ᠌₽ԝ‫ޑ‬॥ᓀ(Bucher et al., 2002; Leaf et al., 2003)ǹ(5)फ़եՈᓸǺDHA ёа೸ၸቹៜՈᆅ่ᄬவԶౢғफ़եՈᓸ‫ޑ‬բҔ (Diep et al., 2000)ǹ(6)ፓ࿯ว‫ݹ‬ϸᔈǴёૈхࡴǺķቹៜΒΜᅹ₧ᜪϯӝ‫ނ‬ (eicosanoids)‫ޑ‬ӝԋǴ‫ٯ‬ӵ n-6 PUFAs ឪΕၸӭ཮ғԋ߻ӈဏન(PGI2)ǵқΟ౎ન (LTB4)‫ک‬Ոਵન(TXA2)Ǵᕴᡏ߄౜ࣁၨம‫ޑ‬Ոλ݈Ꮙ໣‫ک܄‬ว‫ݹ‬ϸᔈǴவԶЇ ଆᜢ࿯‫ݹ‬฻Ǵӵ݀ቚуᑧ१ύ‫ ޑ‬n-3 PUFAs ߾ૈ‫ڋ׭‬೭٤࣬ᜢ‫ޑ‬ว‫ݹ‬ϸᔈ(Calder, 2006)Ƕĸ٬ጢԋϩวғ‫ׯ‬ᡂǴቹៜጢࢬ୏‫(܄‬Chen et al., 2007; Chapkin et al., 2008)ǶĹቹៜಒझᐟન‫ޑ‬ϩ‫(ݜ‬von Schacky, 2007)Ƕĺፓ௓୷Ӣ‫߄ޑ‬ၲǺPUFAs ёа‫ޔ‬ௗ຾Εಒझਡᆶਡ‫ڙ‬ᡏ(nuclear receptor)‫܈‬ᙯᒵӢη่ӝǴ຾Զቹៜ೚ӭ ว‫࣬ݹ‬ᜢ୷Ӣ‫߄ޑ‬౜ǹ‫ٯ‬ӵǺn®3 PUFAs ё‫ ڋ׭‬NF-ț% ࢲ‫܄‬Ǵҗ‫ ܭ‬NF-ț% ‫ޔ‬ௗ ‫܈‬໔ௗፓ௓΋٤ว‫ݹ‬ϸᔈǴ෧Ͽᗹߕϩη‫ޑ‬ғԋ(SanGiovanni & Chew, 2005; Chen et al., 2005; Chapkin et al., 2009)ǹn®3 PUFAs Ψࢂ peroxisome proliferators activated receptors (PPARs)‫ޑ‬ԾฅଛՏη(ligands)ǴPPAR Ψ཮೸ၸቹៜ NF-ț% ၡ ৩Զ‫ڋ׭‬ว‫ݹ‬ϸᔈ(Delerive et al., 2000, 2001; Moraes et al., 2006)ǴӢԜ n®3 PUFAs ჹ‫ܭ‬ፓ࿯ NF-ț% ૻ৲ၡ৩‫ת‬ᄽ๱ख़ा‫فޑ‬ՅǶĻፓ௓ࢌ٤ሇનࢲ‫܄‬Ǻ൤ ֖ n-3 PUFAs ϐങ‫ݨ‬ё೸ၸᇨว‫਼ל‬ϯሇનϐ߄౜Ǵٰ‫ ڋ׭‬ApoE ߹ନλႵЈ᠌ Ոᆅύ୏ેๆ‫ރ‬ฯϯඬϐ‫׎‬ԋ(Wang et al., 2004)Ƕ(7)‫ׯ‬๓ՈᆅϣҜಒझфૈ(De 16.

(41) Caterina et al., 2000; Brown & Hu, 2001)Ƕ. ߄ 2.5 n-3 િެለफ़եЈՈᆅ੯ੰ॥ᓀ‫ޑ‬ёૈᐒ‫(ڋ‬Kris-Etherton et al., 2002) Table 2.5. ߄ 2.6 n-3 િެለᆶᗹߕϩηϐᜢ߯(Brown & Hu, 2001) Table 2.6. ऍ୯Ј᠌Ꮲ཮(American Heart Association)ࡌ᝼ԋԃΓᔈ၀ឪ‫ڗ‬፾ໆങᜪǶࠖ ՕᙴᏢଣᏢ‫ޣ‬ΨᇡࣁǴ‫ຼ؂‬ԿϿ‫ٿ‬ԛឪΕങԺǴӵǺᗴങǵߎᄳങǵ؅΍ങ฻ૈ ߔЗँว‫₽܄‬ԝǴӢࣁങԺύ‫ޑ‬όႫ‫ک‬િެለё෧ϿЈࡓό᏾ǵႣٛЈ᠌ੰൺ วǵ‫ۯ‬጗Ј᠌ੰ஻ಒझԴϯ(Kris-Etherton et al., 2002; Hu et al., 2002; Farzaneh-Far et al., 2010)Ƕ. 17.

(42) ߄ 2.7 n-3 િެለࡌ᝼ឪ‫ڗ‬ໆ(Kris-Etherton et al., 2002) Table 2.7. 18.

(43) ΟǵՈ୷፦਼ϯ 䁙(Heme oxygenase, HO). 1. Ո୷፦਼ϯ䁙ϐϩᜪ Ҟ߻ς‫ޕ‬Ո୷፦਼ϯ䁙(Heme oxygenase, HO)ԖΟᅿӕф౦ᄬ䁙Ǵϩձࣁಃ ΋ࠠՈ୷፦਼ϯ䁙(HO-1)Ǵϩηໆऊࣁ 32 kDaǹಃΒࠠՈ୷፦਼ϯ䁙(HO-2)Ǵϩ ηໆऊࣁ 36 kDaǹаϷಃΟࠠՈ୷፦਼ϯ 䁙(HO-3)Ǵϩηໆऊࣁ 33 kDa (Farombi & Surh, 2006)ǶHO-1 ឦ‫ܭ‬ᇨᏤ߄౜ࠠ‫ޑ‬ሇનǴ཮‫ډڙ‬೚ӭόӕ‫߼ޑ‬ᐟӢન‫܌‬ᇨ วǴӵว‫ݹ‬ϸᔈǵֽ೽લՈ(ischemia)ǵၸ਼ੱ(hyperoxia)ǵલ਼(hypoxia)ǵଯ዗ (hyperthermia)‫ࢌک‬٤ख़ߎឦ(ӵᙿǵልǵઈ) (Farombi & Surh, 2006; Idriss et al., 2008)฻ǶHO-1 ቶ‫ݱ‬ϩթ‫ܭ‬๠᠌ǵ‫᠌ط‬Ϸᆛ‫ރ‬ϣҜ‫س‬಍ύǹHO-2 ‫ ک‬HO-3 ߾ࢂ ࡭ុ߄౜‫ܭ‬ӚಔᙃಒझύǴӵတǵઓ࿶‫س‬಍ǵ‫᠌ط‬ǵ๠᠌ǵ⢀ΤаϷЈՈᆅಔᙃ ฻(Siow et al., 1999)ǶHO-2 ࢂғ౛‫ރ‬ᄊΠ‫ޑ‬ЬाӸӧࠠԄǴૈ೏๝΢ဏҜ፦ન‫܌‬ ፓ࿯(Raju et al., 1997)Ǵՠ HO-3 ࣁൂ΋ᙯᒵౢ‫ނ‬Ǵ‫܄ࢲځ‬ᡉ๱ե‫ ܭ‬HO-1 ‫ ک‬HO-2 (Hayashi et al., 2004)ǴҞ߻ჹ‫ ܭ‬HO-3 ࣬ᜢ‫زࣴޑ‬٠όӭǴ‫ځ‬ӧғ౛΢‫ת܌‬ᄽ‫ޑ‬ ‫ف‬Յۘ҂మཱǶHO-1 ᆶ HO-2 ‫ޑ‬ữ୷ለ‫ׇ‬ӈԖ 43%‫࣬ޑ‬՟‫܄‬ǴԶ HO-3 ࢂ΋ᅿ ᆶ HO-2 ߚத߈՟‫ޑ‬ӕф䁙Ǵ‫ځ‬ữ୷ለ‫ׇ‬ӈᆶ HO-2 ऊԖ 90%‫࣬ޑ‬՟‫(܄‬Siow et al., 1999; Hayashi et al., 2004)Ƕ. ಃ΋ࠠՈ୷፦਼ϯ䁙(Heme oxygenase-1, HO-1)ࢂᡏϣख़ा‫਼לޑ‬ϯሇનǶ HO-1 നԐӧ 1964 ԃҗ Wise ฻Γவಒझύஒ‫ځ‬ϩᚆ٠ว౜ѬёӧᡏѦஒՈ୷፦ (heme)ϩှౢғᖌᆘન(biliverdin)Ƕ1968 ԃ Tenhunen ฻Γ‫ܭ‬εႵ‫᠌ط‬ǵ๠ǵ๝‫ޑ‬ ༾ಈᡏ(microsome)ύ᛾ჴΑՈ୷፦਼ϯ䁙‫ޑ‬ӸӧǶHO-1 ӧ҅தғ౛‫ރ‬ᄊΠѝԖ Ͽໆ߄ၲǴΓᜪ HO-1 ୷Ӣ(hmox-1)Տ‫ࢉܭ‬Յᡏ 22q12 Տ࿼Ǵ୷Ӣӄߏ 6.8 kb (Lavrovsky, 1993)ǴЬाё‫਼ډڙ‬ϯᓸΚǵว‫ݹ‬ᐟનǵख़ߎឦ฻‫ڈ‬ᐟǴ೸ၸፓ௓ antioxidant response element (ARE)Զᇨว‫ځ‬εໆ߄౜Ǵҗ‫ ܭ‬HO-1 ё‫ډڙ‬዗Ҷլ. 19.

(44) ϸᔈࢲϯǴӢԜΞᆀࣁ዗Ҷլೈқ 32 (heat shock protein 32, Hsp32)Ǵࢂ΋ᅿϣྍ ‫ߥ܄‬ៈೈқ፦(Farombi & Surh, 2006)Ƕ. 2. HO-1 ϐғ౛‫ف‬Յ HO-1 ୷Ӣ೏ᇡࣁࢂ΋ᅿӢᔈᡏϣᕉნᡂϯ‫୷ޑ‬ӢǴΨࢂҞ߻ว౜‫ډڙ‬നӭ ӢનᇨᏤ‫ޑ‬ᓸΚϸᔈೈқǴHO-1 ୷Ӣ߄ၲ‫ޑ‬ፓ௓Ьाวғӧᙯᒵ໘ࢤ(Alam & Cook, 2003)Ƕ೚ӭࣴ‫ࡰز‬рǴ྽ಔᙃ‫܈‬ಒझೀ‫਼ܭ‬ϯᓸΚ‫܈‬ཞ໾฻௃‫ݩ‬ਔǴ֡ё ᇨᏤ HO-1 ߄౜Ǵ೭ࢂ‫ي‬ᡏ‫ޑ‬΋ᅿٛፁ‫܄‬ϸᔈǴፓ௓ಔᙃ‫܈‬ಒझٰӢᔈғ౛ᡂϯ аᆢ࡭‫୏ځ‬ᄊѳᑽ‫ޑ‬ᜢᗖ(Gruber et al., 2010)ǶќԖЎ᝘ࡰрǴϺฅӸӧ‫ޑ‬෌ϯ ‫(ނ‬ӵ quercetinǵresveratrolǵcurcumin ᆶ sulforaphane ฻)ҭ཮೸ၸፓ௓ HO-1 ߄ ౜Ǵ‫ׯ‬๓ಒझϣ‫਼ޑ‬ϯᓸΚ(Balogun et al., 2003; Lin et al., 2004; Juan et al., 2005; Farghali et al., 2009)Ƕ. HO-1 ‫ڀ‬Ԗ࣬྽ӭ‫ޑ‬ғ౛բҔǴς‫ޕ‬Ԗ(1)‫ל‬ว‫ݹ‬Ǻቚу HO-1 ߄౜ς೏᛾ჴ ᆶफ़եว‫ݹ‬ϸᔈԖᜢ(Takahashi et al., 2007; Kim et al., 2007; Lee et al., 2009)Ǵ೸ ၸफ़եϣҜಒझϐᗹߕϩηᆶᖿϯ‫ނ‬፦‫߄ޑ‬౜Ǵ‫ޔ‬ௗ‫܈‬໔ௗ‫ڋ׭‬ว‫ݹ‬ϸᔈ (Vachharajani et al., 2000; Soares et al., 2004; Lin et al., 2005; Yu et al., 2010)Ƕ೭ঁ ཷ‫ۺ‬ё࿶җ‫ٿ‬໨ᒪ໺ว౜ٰև౜Ǵಃ΋ǺલЮ HO-1 ϐλႵ཮ቚу‫ځ‬ว‫ݩރݹ‬ (Kapturczak et al., 2004; Tracz et al., 2007)ǴಃΒǺHO-1 લЮ஻‫ޑޣ‬ว‫ࢂރੱݹ‬೷ ԋ‫ځ‬ԝΫ‫ޑ‬চӢϐ΋(Kawashima et al., 2002; Koizumi, 2007)Ƕ(2)‫ל‬ಒझ঒ΫǺӧ λႵ߃ж‫ط‬ಒझཞ໾ኳԄΠǴቚу HO-1 ߄౜Ԗշ‫ڋ׭ܭ‬ಒझ঒Ϋ(Zuckerbraun et al., 2003)Ƕ(3)‫ל‬ಒझቚғ(Morse & Choi, 2002)Ƕ(4)բࣁЈՈᆅ੯ੰ‫ݯ‬ᕍϐ኱‫ޑ‬Ǻ HO-1 ‫ޑ‬ౢ‫ ނ‬CO ёૈ೸ၸफ़ե p38 MAPK ᕗለϯ߄౜Ǵ٠‫ ڋ׭‬calcineurin/NFAT ೼৩‫ࢲޑ‬ϯٰ෧ϿЈԼ‫ޥ‬ε‫ޑ‬วғ(Tongers et al., 2004)ǹIshikawa ฻Γ(2001)ว౜ ӧ୏ેๆ‫ރ‬ฯϯՈᆅύǴᇨᏤ HO-1 ཮‫ڋ׭‬Ոዀύિ፦ၸ਼ϯ‫ޑނ‬ғԋǴᇥܴଯ િՈੱᇨᏤ‫ ޑ‬HO-1 ჹ୏ેๆ‫ރ‬ฯϯ‫׎ޑ‬ԋ‫ڀ‬ԖߥៈբҔǴ٠ёૈ೸ၸቹៜ NO 20.

(45) ೼৩ٰวචբҔǶ(5)ᗉխᏔ‫۔‬౽෌௨ѾϸᔈǺቚу HO-1 ࢲ‫܄‬ёٛЗЈՈᆅ‫ډڙ‬ ֽ೽લՈ/ӆឲࢬ(ischemia/reperfusion)‫ޑ‬໾্Ǵ‫ٯ‬ӵ HO-1 ёа೸ၸፓ࿯ NOS (nitric oxide synthase)߄౜Ϸࢲ‫ٰ܄‬फ़եᑗֿੰεႵЈԼલՈӆឲࢬϐཞ໾ (L'Abbate et al., 2007; Abraham & Kappas, 2008)Ƕ(6)ፓ௓ಒझຼය(cell cycle)Ǻ HO-1 ཮‫ڋ׭‬ՈᆅѳྖԼಒझ(vascular smooth muscle cells, VSMC)‫ޑ‬ಒझຼයǴ ೷ԋಒझଶᅉӧ G1/S යǴӕਔΨ཮ፓ௓ cyclin kinase inhibitor p21Cip (Duckers et al., 2001)ǹӧ VSMC ύ CO ‫ޑ‬ቚуǴ཮‫ ڋ׭‬E2F-1 ‫ޑ‬ғԋǴE2F-1 ӧಒझຼයύ ‫ת‬ᄽፓ࿯ c-mycǵcyclin ‫ ک‬DNA polymerase ‫فޑ‬Յ(Morita & Kourembanas, 1995)Ƕ(7)‫ڋ׭‬ᕎಒझߟ᠍‫ک‬ᙯ౽Ǻࣴ‫ز‬᛾ჴ HO-1 ૈ‫ڋ׭‬٢ᕎಒझߟ᠍‫ک‬ᙯ౽ ‫ૈޑ‬ΚǴ‫ځ‬բҔᐒᙯᆶ‫ ڋ׭‬MMP-9 (Matrix metalloproteinase-9)୷ӢࢲϯԖᜢ (Lin et al., 2008)Ƕ(8)ନΑ΢ॊғ౛фૈϐѦǴന‫ݙډڙ‬Ҟ‫਼לځࢂ߾ޑ‬ϯૈΚǴ ᙖҗፓ௓ HO-1 ߄౜Ϸ‫ځ‬жᖴౢ‫ނ‬ϐբҔǴᆢ࡭ᡏϣ਼ϯᗋচ‫ރ‬ᄊϐࡡ‫ۓ‬Ǵफ़ե ಒझ਼ϯᓸΚ‫ک‬ፓ࿯ӭᅿಒझߥៈբҔǴӧ೚ӭ੯ੰว৖ၸำύ‫ת‬ᄽख़ा‫فޑ‬Յ (Slebos et al., 2003; Hwang & Jeong, 2008; Lee et al., 2009)Ƕ. 3. HO-1 жᖴౢ‫ނ‬ჹಒझ‫ߥޑ‬ៈբҔ HO-1 ӧ਼ϩη(O2)ǵNADPHǵಒझՅન P450 ᗋচ䁙(cytochrome P450 reductase)‫ୖޑ‬ᆶΠǴё໽ϯՈ୷፦(heme)फ़ှࣁᖌᆘન(biliverdin)ǵ΋਼ϯᅹ (carbon monoxide, CO)ǵෞᚆ៓(Fe2+)ǴࢂՈ୷፦жᖴၸำύ‫ޑ‬ೲ౗ज़‫ڋ‬ሇનǴѬ ቶ‫ݱ‬ϩթ‫ܭ‬ғ‫ނ‬ᡏϣӚᅿಔᙃ‫ک‬Ꮤ‫۔‬ǴԖ๱ख़ा‫ޑ‬ғ౛фૈ(კ 2.8) (Farombi & Surh, 2006)Ƕ೚ӭࣴ‫ز‬ว౜ǴHO-1 Ϸ‫ځ‬жᖴ࣬ᜢౢ‫ނ‬ёӅӕวච‫ל‬ว‫ݹ‬ǵ‫਼ל‬ ϯǵ‫ڋ׭‬ಒझ঒Ϋ‫ׯک‬๓ಔᙃ༾ൻᕉ฻բҔ(Ryter et al., 2007)ǶHO-1 жᖴ࣬ᜢౢ ‫ޑނ‬ғ౛фૈӵΠǺ. 3-1. ᖌᆘન(biliverdin, BV)/ᖌआન(bilirubin, BR) HO-1 ‫ޑ‬жᖴౢ‫ނ‬ᖌᆘન(biliverdin)ё຾΋‫؁‬࿶ᖌᆘનᗋচ䁙(biliverdin 21.

(46) reductase)‫ޑ‬բҔǴᙯᡂࣁᖌआન(bilirubin)Ƕ߈ԃ‫زࣴޑ‬᛾ჴǴBR ࢂ΋ᅿख़ा‫ޑ‬ ϣྍ‫਼ל܄‬ϯϩηǴԖமε‫਼לޑ‬ϯૈΚǴૈԖਏӦమନ਼Ծҗ୷Ǵफ़ե਼ϯᓸ Κ(Baranano et al., 2002)ǹߥៈЈՈᆅխ‫ֽܭ‬೽લ਼‫ޑ‬໾্(Clark et al., 2000; Ollinger et al., 2007)ǹ‫ ڋ׭‬LPS ᇨวᗹߕϩη‫ޑ‬բҔǴफ़եว‫ݹ‬ϸᔈ(Vachharajani et al., 2000)ǹӧа LPS ᇨวεႵҶլ‫ޑ‬ኳԄΠǴว౜ BV ёफ़եՈమ߻ว‫ݹ‬ಒझ ᐟનᐚࡋ(Sarady-Andrews et al., 2005)ǹBR Ψё೸ၸ‫ ڋ׭‬E-selectin ‫ ک‬VCAM-1 ߄౜ٰफ़եϣҜಒझ‫ࢲޑ‬ϯբҔ(Soares et al., 2004)ǹќѦǴBV/BR ‫לޑ‬ว‫ݹ‬բ ҔёૈᆶѬॺᏤठ NF-ț% ѨࢲԖᜢ(Soares et al., 2004; Sarady-Andrews et al., 2005)Ƕ. 3-2. ΋਼ϯᅹ(carbon monoxide, CO) ೚ӭࣴ‫ࡰز‬рǴϣྍ‫܄‬΋਼ϯᅹ(CO)ёբࣁ΋ᅿૻ৲໺ሀϩηǴࢲϯёྋ‫܄‬ ച㧿ለᕉϯ䁙(soluble guanylate cyclase, sGC)ǴsGC ཮຾΋‫؁‬ஒ GTP ࢲϯԶ‫׎‬ԋ ᕉᕗለച㧿(cGMP) (Morita et al., 1995)ǴᝩԶวචቶ‫ޑݱ‬ғ౛ፓ࿯фૈǴхࡴፓ ࿯Ոᆅ๤஭ǵЍ਻ᆅᘉ஭ǵ‫ڋ׭‬Ոλ݈Ꮙ໣ǵ෧ϿՈਵ‫׎‬ԋǵ෧ᇸલՈӆឲ‫ݙ‬ཞ ໾Ϸ‫ڋ׭‬ՈᆅѳྖԼಒझቚғ฻բҔ(Slebos et al., 2003; Piantadosi, 2008)Ϸᆢ࡭ ༾Ոᆅൻᕉ‫ޑ‬ѳᑽ(Suematsu & Ishimura, 2000)ǶCO ೏ᇡࣁࢂॄೢ HO-1 ε೽ϩ ‫ל‬ว‫ݹ‬բҔ‫ޑ‬Ӣη(Ryter et al., 2006)ǺӧѮᏘಒझύǴCO ೸ၸፓ௓ p38 ٰ‫ڋ׭‬ ߻ว‫ݹ‬ಒझᐟન TNF-Į ‫ޑ‬ౢғ(Otterbein et al., 2000)ǹҭԖЎ᝘ࡰрǴCO ё೸ၸ MAPK ೼৩(Otterbein et al., 2003)‫ڋ׭‬ᠼᆢ҆ಒझ(fibroblast)‫܈‬ϣҜಒझ (endothelial cells)঒ΫǴಒझ঒Ϋ཮уቃว‫ݹ‬ϸᔈǴЀ‫ࢂځ‬঒Ϋ‫ޑ‬ՈᆅϣҜಒझ ཮‫ڈ‬ᐟՈਵ‫׎‬ԋǴӢԜ CO ‫לޑ‬঒ΫբҔӧಒझߥៈբҔύΜϩख़ाǶӧΓᜪ T ಒझύǴCO ೸ၸ‫ ڋ׭‬ERK ၡ৩ٰफ़ե IL-2 ϩ‫ݜ‬Ϸಒझεໆቚғ(Pae et al., 2004)ǹӧѮᏘಒझύǴCO ཮೸ၸፓ௓ C/EBP ‫ ک‬NF-ț% ٰ‫߻ڋ׭‬ว‫ݹ‬ሇન iNOS ‫ ک‬COX-2 ‫߄ޑ‬౜(Suh et al., 2006)ǹӧΓᜪ่ဉ΢ҜಒझǴCO ೸ၸፓ௓ NF-ț%ǵ AP-1ǵC/EBPǵ‫ ک‬MAPK ၡ৩ٰ‫ ڋ׭‬iNOS ߄౜аϷ IL-6 ϩ‫(ݜ‬Megías et al., 2007)Ƕ 22.

(47) 3-3. ៓ᚆη(Fe2+)/៓ೈқ(ferritin) ៓ೈқ(ferritin)ᆶ HO-1 ‫߄ޑ‬౜ቚу‫ڀ‬Ԗ΋ठ‫(܄‬Balla et al., 2005)Ǵᗨฅۘ҂ ֹӄ᛾ჴ៓ೈқࢂցᆶ HO-1 ‫לޑ‬ว‫ݹ‬բҔ࣬ᜢǴՠࢂ៓ೈқ‫ޑ‬ዴࢂ΋ᅿԖਏ‫ޑ‬ ‫਼ל‬ϯϩη(Arosio et al., 2009)ǶΒሽ៓ࢂ΋ᅿ਼ܰϯ‫ߎޑ‬ឦᚆηǴ৒ܰЇଆว‫ݹ‬ ϸᔈǹԶ྽ HO-1 жᖴՈ୷፦ਔ཮ញрΒሽ៓ǴΒሽ៓΋ѿ೏ញрǴ཮೏៓ೈқ ‫ז‬ೲӦௗԏǴቚу៓ᓯӸ‫ޑ‬ਏ౗ǴӢԜε൯Ӧज़‫ڋ‬Ѭ‫਼ߦޑ‬ϯ/߻ว‫ૈݹ‬ΚǴа ᆢ࡭ಒझϣ៓ᚆηᐚࡋϐࡡ‫ۓ‬Ǵࡺಒझϣ៓ೈқ֖ໆගଯਔё‫਼לܢ‬ϯ໾্Ǵ٠ ౢғᓸΚፓ፾(stress adaptation)ၲ‫ߥډ‬ៈಒझϐբҔ(Balla et al., 2005)ǹਥᏵၗ਑ ᡉҢǴᇨว HO-1 ߄౜཮ቚу៓ೈқӝԋǴ‫ڋ׭‬ว‫ݹ‬ϸᔈౢғ(Schaer et al., 2006)Ƕ. კ 2.8 HO-1 ϐբҔϷ‫ځ‬жᖴౢ‫(ނ‬Farombi & Surh, 2006). 4. ፓ௓ HO-1 ୷Ӣ߄౜ϐૻ৲໺ሀၡ৩ ၸѐЎ᝘ࡰрǴୖᆶᇨᏤ HO-1 ୷Ӣ߄౜ϐૻ৲໺ሀၡ৩(signal transduction pathways)ǴЬाхࡴ MAPKs (mitogen-activated protein kinase)ǵPI3K (phosphoinositide 3-kinase) /AktǵPKC ฻(Owuor & Kong, 2002; Lee & Johnson,. 23.

(48) 2004 ; Xu et al., 2006; Ryter et al., 2006; Paine et al., 2010)ǹԜѦǴϩ‫ ݋‬HO-1 ୷Ӣ ‫୏௴ޑ‬η(promoter)୔ୱǴว౜‫ڀ‬Ԗ೚ӭख़ाᙯᒵӢη‫่ޑ‬ӝՏ࿼Ǵхࡴ NF-E2 (nuclear factor-erythroid 2)ǵAP-1ǵNF-ț% ฻Ǵаፓ௓ HO-1 ‫߄ޑ‬౜(კ 2.9) (Farombi & Surh, 2006; Alam & Cook, 2007; Gruber et al., 2010)Ƕ. 4-1. ᙯᒵӢη Nrf2 Nrf2 (nuclear factor erythroid 2-related factor 2)ࣁ‫ځ‬ख़ाፓ௓‫ޣ‬ϐ΋ǴӢ Nrf2 ཮ᆶ࣬ᜢ‫਼ל‬ϯሇન୷Ӣ΢‫ޑ‬΋ࢤፓ௓‫ׇ‬ӈ Antioxidant Response Element (ARE) ่ӝǴࡺ೏᛾ჴᆶ HO-1 ‫߄ޑ‬౜ԖஏϪᜢ߯(Xu et al., 2006; Kim et al., 2007; Johnson et al., 2009)ǶNrf2 ឦ‫ ܭ‬Cap’n’Collar / basic leucine zipper (CNC-bZIP)ᙯᒵ Ӣηৎ௼‫ޑ‬΋ঁԋ঩ǶӧؒԖ‫ڈ‬ᐟ‫ޑ‬௃‫ݩ‬ΠǴNrf2 ᙖҗᆶ Keap1 (Klech-like ECH-associated protein 1)่ӝǴ೏႖ᚆӧಒझ፦ύǹ΋ѿ‫ࢲډڙ‬ϯǴ೭ঁፄӝ‫ނ‬ ஒ೏ґှǴ೏ញ‫ܫ‬р‫ ޑ‬Nrf2 ளаᙯ౽຾ΕಒझਡϣǴ຾Զᆶ small Maf ৎ௼‫ޑ‬ԋ ঩(i.e., MafKǵMafGǵMafF)ಔӝԋ౦፦Βᆫᡏ(Motohashi et al., 2002, 2004; Katsuoka et al., 2005)ǶନΑ small Maf ৎ௼‫ޑ‬ԋ঩ǴNrf2 Ψёૈᆶ c-Jun ‫܈‬ activating transcription factor 4 (ATF4)‫׎‬ԋ౦፦Βᆫᡏ(heterodimers)Ǵቚம ARE/EpRE (electrophile response element)-driven ൔᏤ୷Ӣࢲ‫܄‬Ǵߦ຾ HO-1 ‫ޑ‬ᙯ ᒵ(Venugopal & Jaiswal, 1998; He et al., 2001; Mann et al., 2007)Ƕ. 24.

(49) Figure 2.9. კ 2.9 ፓ௓ HO-1 ߄౜ϐૻ৲໺ሀၡ৩(Farombi & Surh, 2006). 4-2. ‫ځ‬Ѭၡ৩(PI3K/AktǵMAPKsǵPKC) ςԖ೚ӭࣴ‫ز‬௖૸ୖᆶNrf2ࢲϯ‫ૻޑ‬৲໺ሀၡ৩Ǵ‫ٯ‬ӵPI3K‫ک‬PKCё٬Nrf2 วғᕗለϯǴԶPI3K‫ڋ׭‬Ꮚ(LY-294002)‫܈‬PKC‫ڋ׭‬Ꮚ(Ro-32-0432)Ψёफ़եARE luciferaseൔᏤ୷Ӣ‫܄ࢲޑ‬ǹҗԜ௢ፕǴNrf2ϐࢲϯᆶPI3K‫ک‬PKC‫܄ࢲޑ‬Ԗᜢ(Lee & Surh, 2005; Farombi & Surh, 2006; Keum et al., 2008)ǶԜѦǴMAPKsΨ೏᛾ჴ ୖᆶNrf2ࢲϯբҔ(Kong et al., 2001; Lee & Johnson, 2004; Xu et al., 2006)ǶҞ߻ς ‫ޕ‬ӭᅿ‫ڀ‬Ԗғ౛ࢲ‫ޑ܄‬෌ϯ‫(ނ‬phytochemicals)Ǵӵ౦౷⋸ለ㸰ᜪ (isothiocyanates)ǵЇԚ(indoles)ǵΒ౎Ч୷౷ϯ‫(ނ‬diallyl sulfides)ǵ໳✉ᜪϯӝ‫ނ‬ (flavonoids)ᆶᖖ໳ન(curcuminoids)฻Ǵ೿Ԗߦ຾‫ڋ׭܈‬Nrf2่ӝԿ኱‫୷ޑ‬Ӣ promoter΢‫ޑ‬բҔ(Jeong et al., 2006)ǶӧHepG2ಒझਲ਼ኳԄΠǴᇺ෍ન(capsaicin) ё೸ၸࢲϯPI3K/Aktૻ৲໺ሀၡ৩ǴቚуNrf2ᆶARE‫่ޑ‬ӝǴ຾Զ҅ӛፓ௓HO-1 ୷Ӣ߄౜(Joung et al., 2007)ǹCarnosolᇨวHO-1‫߄ޑ‬౜Ψ೏ᇡࣁᆶPI3Kૻ৲ၡ৩ Ԗᜢ(Martin et al., 2004)Ƕ. 25.

(50) ᗨฅࡐӭ‫ ޑ‬phytochemicals ೏᛾ჴёаᇨว HO-1 ߄౜Ъ‫ڀ‬ԖߥៈಒझբҔ (cytoprotection)ǴՠҞ߻ࣁЗǴDHA ᇨว HO-1 ߄౜‫࣬زࣴޑ‬ჹၨϿǶࣴ‫ز‬ว౜ ങ‫ݨ‬ύख़ाԋϩ DHA ёᇨว BV-2 microglia ߄౜ HO-1 (Lu et al., 2010)Ǵ‫ځ‬ᐒ‫ڋ‬ ёૈᆶ Akt ‫ ک‬ERK ԖᜢǶќѦǴDHA ё೸ၸ Nrf2-dependent ૻ৲໺ሀٰᇨว mouse peritoneal macrophages ߄౜ HO-1Ǵ຾Զ‫ ڋ׭‬LPS ᇨว‫ޑ‬ว‫ݹ‬ϸᔈ(Wang et al., 2010)ǶGao ฻Γ(2007)ว౜ DHA ሡ࿶ၸ਼ϯբҔࡕ‫܌‬ౢғ‫ޑ‬ౢ‫ނ‬ω‫ڀ‬Ԗᇨว Nrf2 ߄౜‫ࢲک‬ϯ ARE ‫ׇ‬ӈ‫ޑ‬բҔǴԶЪдॺ௢ෳ DHA ਼ϯౢ‫ނ‬Ψёૈ཮ᆶ Keap1 բҔ຾Զࢲϯ Nrf2Ƕ. 26.

(51) ಃΟക ࣴ‫ز‬Ҟ‫ޑ‬. ਥᏵፁғ࿿‫ޑ‬಍ीǴЈՈᆅ੯ੰ΋‫ޔ‬՞ۚ୯ϣΜεԝӢ‫߻ޑ‬൳ӜǴ୏ેๆ‫ރ‬ ฯϯࢂЈՈᆅ੯ੰ‫ޑ‬΋ᅿǶᖏ‫׉‬ᙴᏢ᛾ᏵᡉҢǴ୏ેๆ‫ރ‬ฯϯࢂ΋ᅿᄌ‫܄‬ว‫ݹ‬੯ ੰǴ཮೷ԋՈᆅϣᏛિެ୴ᑈϷᠼᆢඬ༧‫׎ޑ‬ԋǹԜѦǴࣴ‫ز‬ว౜ಒझᗹߕϩη ‫ޑ‬ғԋჹ‫ે୏ܭ‬ๆ‫ރ‬ฯϯ੯ੰ‫ޑ‬ว৖‫ת‬ᄽख़ा‫فޑ‬ՅǴᗹߕϩηϐ΋ ICAM-1 ё բࣁ΋ᅿว‫ޑݹ‬ғ౛ࡰ኱ǴٰႣෳқՈౚӧϣҜಒझύ‫ޑ‬ᗹߕ௃‫׎‬ǶTNF-Į ࢂ΋ ᅿς‫߻ޑޕ‬ว‫ݹ‬ಒझᐟનǴ཮೸ၸࢲϯᙯᒵӢη NF-ț% ٰ‫ڈ‬ᐟᗹߕϩη‫߄ޑ‬౜Ǵ ‫܌‬аத೏Ҕٰբࣁᇨวಒझౢғว‫ݹ‬ϸᔈ‫ޑ‬ኳԄǶ. Αှ୏ેๆ‫ރ‬ฯϯ‫׎ޑ‬ԋ٠уаႣٛࢂҞ߻ႣٛᙴᏢ‫ޑ‬ख़ाፐᚒϐ΃Ǵ೚ӭ Ў᝘ࡰрങ‫ݨ‬Ϸ‫ځ‬ख़ाࢲ‫܄‬ԋϩ DHA ‫ڀ‬Ԗ‫ל‬ว‫ݹ‬բҔǴ٠Ъ཮೸ၸ೚ӭ೼৩ٰ फ़եЈՈᆅ੯ੰ‫ޑ‬วғ౗ǹԶ HO-1 ࢂᡏϣख़ा‫਼לޑ‬ϯሇનǴЬा཮‫਼ډڙ‬ϯ ᓸΚǵว‫ݹ‬ǵϯᏢ‫ނ‬ǵख़ߎឦ฻‫ڈ‬ᐟᇨวԶεໆ߄౜Ǵ೭ࢂ΋ᅿٛፁ‫܄‬ϸᔈǴፓ ௓ಔᙃ‫܈‬ಒझٰӢᔈғ౛ᡂϯаᆢ࡭‫୏ځ‬ᄊѳᑽ‫ޑ‬ᜢᗖǶҁჴᡍ࠻Ӄ߻ࣴ‫ز‬ว౜ аऀЈጪϣ✊Ⴃೀ౛ HUVECs Ϸ EA.926 ಒझǴёа‫ ڋ׭‬TNF-Į ‫܌‬ᇨว‫ ޑ‬ICAM-1 ߄౜(Chao et al., 2011)ǶӢԜҁჴᡍஒճҔ TNF-Į ᇨวϣҜಒझ EA.hy926 ౢғ ว‫ݹ‬ϸᔈ‫ޑ‬ኳԄǴ௖૸ DHA ࢂցёаᙖҗቹៜ NF-ț% ૻ৲໺ሀၡ৩ٰ‫ڋ׭‬ TNF-Į ‫܌‬ᇨว‫ ޑ‬ICAM-1 ߄౜Ǵ٠Ъ௖૸ DHA ‫܌‬ᇨว‫ ޑ‬HO-1 ࢂցୖᆶ‫ڋ׭‬ว ‫ޑݹ‬ᐒ‫ڋ‬Ǵ຾Զၲ‫ډ‬Ⴃٛว‫ݹ‬੯ੰ‫ޑ‬фਏǶ. 27.

(52) 28. ჴᡍࢎᄬ.

(53) ಃΒ೽ҽ Induction of Heme Oxygenase 1 and Inhibition of 7XPRU1HFURVLV)DFWRUĮ-Induced Intercellular Adhesion Molecule 1 Expression by Docosahexaenoic Acid in EA.hy926 Cells. 1. Introduction. Fish oils, rich in long-chain n-3 polyunsaturated fatty acids (n-3 PUFAs), especially eicosapentanoic acid (EPA, 20:5) and docosahexanoic acid (DHA, 22:6), are well known for their anti-inÀDPPDWRU\ Mullen et al., 2010), immunoregulatory (Simopoulos, 2002), anti-aging (Jicha et al., 2010), and anti-tumor (Ghosh-Choudhury et al., 2009) properties. Additionally, EPA and DHA were shown to possess anti-arrhythmic effect (Leaf et al., 2005). Epidemiological studies have provided evidence indicating that n-3 PUFAs supplementation regulates inflammation partially via improvement of endothelial functions (Brown & Hu, 2001). DHA was shown to significantly decrease the cytokine-induced adhesion molecule expression (Chen et al., 2003), diminish the adhesion of leukocytes to the activated endothelial cells (De Caterina et al., 2000; Mayer et al., 2002), and inhibit production of cytokines by endothelial cells (Novak et al., 2003; von Schacky, 2007). It has been demonstrated that treatment with n-3 PUFAs suppressed ICAM-1 and VCAM-1 expressions in TNF-Į,/-1, and VEGF-stimulated endothelial cells (Chen et al., 2005), with DHA being more potent than EPA (Weldon et al., 2007). It is reported that DHA affects several target genes via inhibition of the NF-kB activation (Chapkin et al., 2009; Wang et al., 2011). Dietary intake of n-3 PUFAs is associated with a reduced risk of. 29.

(54) atherosclerosis (Kris-Etherton et al., 2002; Paulo et al., 2008), and this is considered to play a pivotal role in the prevention of cardiovascular disease (CVD).. In recent years, it has been recognized that inflammation is a major contributing factor to many cardiovascular events (Blake, 2001). Atherosclerosis, a chronic inflammatory disease of the vasculature, is characterized by infiltration of leucocytes (Blankenberg et al., 2003), deposition of lipids and thickening of the vascular wall in response to cytokines (Ross, 1999; Lusis, 2000), and it increasingly threatens human health worldwide (Hansson & Libby, 2006). Leukocyte recruitment is a multistep process and this process is predominantly mediated by cellular adhesion molecules, such as intracellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1) and selectins, which are expressed on the surface of epithelial and endothelial cells in response to several inflammatory stimuli, including oxidized LDL, free radical species, lipopolysaccharide (LPS), and cytokines, such as tumor necrosis factor- alpha (TNF-Į

(55) LQWHUOHXNLQ-ȕ ,/-ȕ

(56) DQGLQWHUIHURQ-gamma (INF-Ȗ

(57) (Roebuck & Finnegan, 1999; Blankenberg et al., 2003). Studies have shown that TNF-ĮWKHSUR-inflammatory cytokine, is commonly found in atherosclerotic lesions and can induce expression of ICAM-1 and VCAM-1, which are critically dependent on the activation of nuclear factor-ț% 1)-ț%

(58) Liu, 2005; Oh et al., 2010). NF-ț%LV an important transcription factor regulating the expression of many inflammatory response genes such as adhesion molecules and cytokines (Luo et al., 2005). In quiescent cells, NF-ț%LVVHTXHVWHUHGLQWKHF\WRSODVPWKURXJKLWVLQWHUDFWLRQZLWKWKH LQKLELWRU\NDSSD% ,ț%

(59) IDPLO\ 6XQ .DULQ

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(61) FRPSOH[ 0D\ Ghosh, 1998; Karin & Delhase, 2000) and subsequently degraded by the ATP-dependent 26S proteasome complex (Chen et al., 1995; Wertz & Dixit, 2010). 30.

(62) ,ț%GHJUDGDWLRQIUHHV1)-ț%DQGDOORZV1)-ț%WUDQVORFDWLRn to the nucleus, where it FDQELQGWRWKHț%HOHPHQWRISURPRWHURIWDUJHWJHQHV 5DKPDQ& McFadden, 2011).. Heme oxygenase (HO)-1 is an inducible enzyme responsible for the rate-limiting step of heme degradation and produces carbon monoxide (CO), free iron and biliverdin (BV), which is further converted into bilirubin (BR) via biliverdin reductase (Farombi & Surh, 2006; Abraham & Kappas, 2008). HO-1 can be triggered by a variety of stress-related cellular stimuli, including its substrate heme, heavy metals, oxidative stress, UV radiation, inflammatory cytokines, hypoxia, and ischemia-reperfusion (Farombi & Surh, 2006; Idriss et al., 2008). The physiological relevance of the HO-1 expression has been reported in several pathological states such as atherosclerosis and inÀDPPDWLRQZKHUHLQLWFRQIHUVF\WRSURWHFWLRQ 0RULWD Idriss et al., 2008; Lee et al., 2009; Paine et al., 2010; Kim et al., 2010). HO-1 induction reduces atherosclerotic lesion size in Watanabe heritable hyperlipidemic rabbits (Ishikawa et al., 2001a) and in LDL-receptor knockout mice (Ishikawa et al., 2001b). Moreover, transgenic mice deficient in HO-1 of an apolipoprotein E null background (Yet et al., 2003) exhibited accelerated and more advanced atherosclerotic lesion formation in response to a Western diet. Nevertheless, recent evidence suggests that by-products of HO-1, alone or in concert, mediate the protective effects of HO-1 (Kirkby & Adin, 2006; Ryter et al., 2006, 2007). Bilirubin is an endogenous radical scavenger with recently recognized antioxidant, anti-inflammatory, anti-proliferative properties (Ollinger et al., 2007). The release of free iron is rapidly sequestered into the iron storage protein, ferritin, leading to additional antioxidant and anti-apoptotic effects (Arosio et al., 2009). CO exerts several biological functions, including anti-apoptotic, anti-inflammatory, and vasodilatory effects (Kirkby & Adin, 2006; Ryter et al., 2006, 2007). HO-1 expression is primarily regulated at the transcriptional 31.

(63) level (Alam & Cook, 2003), and its inducibility by diverse inducers is linked to the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf-2) (Shan et al., 2006; Kim et al., 2007). Under basal conditions, Nrf2 is sequestered in the cytoplasm by binding to Kelch-like ECH-associated protein 1 (Keap1) (Itoh et al., 2004; Kaspar et al., 2009). When disrupted by electrophilic antioxidants, Nrf2 is released from Keap1 and translocates to the nucleus, dimerizes with Maf, and activates transcription of genes containing the antioxidant response element (ARE) sequences in the promoter regions (Owuor & Kong, 2002; Katsuoka et al., 2005; Kobayashi & Yamamoto, 2005; Kensler et al., 2007).. Although anti-inflammatory effect of DHA (n-3, 22:6) has been studied before, the molecular mechanism underlying DHA-mediated inhibition of TNF-Į-induced ICAM-1 expression in human vascular endothelial cells still remains unclear. The aim of this study was to evaluate the effect of DHA on the adhesion of monocytes to TNF-Į-activated endothelial cells which is mediated by adhesion molecules such as ICAM-1, as well as the molecular mechanisms underlying DHA inhibition of ICAM-1 expression.. 32.

(64) 2. Materials and Methods. 2.1 Chemicals Dulbecco’s modified Eagle medium (DMEM), RPMI 1640, RPMI-1640 (without phenol red), OPTI-MEM, and penicillin/streptomycin were from GIBCO/BRL (Grand Island, NY); 0.25% trypsin-EDTA was from BioWest (Miami, FL); fetal bovine serum (FBS) was from HyClone (Logan, UT); docosahexaenoic acid (DHA) was from Cayman Chemical (Ann Arbor, MI); 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT), sodium bicarbonate, human tumor necrosis factor-alpha (TNF-Į

(65) DQGDQWL-ȕ-actin antibody were from Sigma-Aldrich (St. Louis, MO); Z-Leu-Leu-Leu-CHO (MG-132) was from Boston Biochem (Cambridge, MA); 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF-AM) was from Molecular Probes (Eugene, OR); H2DCFDA and TRIzol reagent were from Invitrogen (Carlsbad, CA); antibody against HO-1 was obtained from Calbiochem (Darmstadt, Germany); antibodies against Nrf2, IțBĮ, IKKĮ/IKKȕ-1.SKRVSKR-JNK, ERK, and p38 were from Santa Cruz Biotechnology (Santa Cruz, CA); antibodies against ICAM-1, phospho-IțBĮ (Ser32/36), phospho-IKKĮ(Ser180)/IKKȕ(Ser181), PARP, phospho-ERK, and phospho-p38 were from Cell Signaling Technology (Boston, MA); antibody against p65 was from BD Bioscience (San Jose, CA).. 2.2 Cell cultures The human endothelial cell line EA.hy926 was a kind gift from Dr. T. S. Wang, Chung Shan Medical University, Taichung, Taiwan, and was cultured in DMEM supplemented with 3.7 g/L NaHCO3, 10% FBS, 100 units/mL penicillin, and 100 ȝJP/VWUHSWRP\FLQDWoC in a 5% CO2 humidified incubator. Human leukemia promyelocytic cells (HL-60) were obtained from Bioresources Collection and 33.

(66) Research Center (BCRC, Hsinchu, Taiwan). The HL-60 cells were cultured in T-75 tissue culture flasks in RPMI-1640 medium supplemented with 10% FBS, 100 units/mL penicillin, and 100 mg/L streptomycin at 37oC in a 5% CO2 humidified incubator.. 2.3 Fatty acid preparation DHA samples were prepared and complexed with fatty acid-free bovine serum albumin at a 6:1 molar ratio before addition to the culture medium. At the same time, EXW\ODWHGK\GUR[\WROXHQHDQGȝ0Į-tocopheryl succinate were added to the culture medium to prevent lipid peroxidation.. 2.4 Cell viability assay Cell viability was assessed by the MTT assay. The MTT assay measures the ability of viable cells to reduce a yellow 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide to a purple formazan by mitochondrial succinate dehydrogenase. EA.hy926 cells were grown to 70-80% confluence and were then treated with different concentrations of DHA (0-10ȝ0

(67) IRUKIROORZHGE\ incubation with 1 ng/mL TNF-Į for an additional 6 h. Finally, the medium was removed, and the cells were washed with PBS. The cells were then incubated with MTT (0.5 mg/mL) in DMEM medium at 37oC for an additional 3 h. The medium was removed, and 2-propanol was added to dissolve the formazan. After centrifugation at 14,000×g for 5 min, the supernatant of each sample was transferred to 96-well plates, and the absorbance was read at 570 nm in an ELISA reader. The absorbance in control group was regarded as 100% cell viability.. 34.

(68) 2.5 Nuclear extracts preparation After each experiment, cells were washed twice with cold PBS and were then scraped from the dishes with 1,ȝ/RI3%6&HOOKRPRJHQDWHVZHUHFHQWULIXJHGDW 2,000×g for 5 min. The supernatant was discarded, and the cell pellet was allowed to VZHOORQLFHIRUPLQDIWHUWKHDGGLWLRQRIȝ/RIK\SRWRQLFEXIIHU P0 HEPES, 10 mM KCl, 1 mM MgCl2, 1 mM EDTA, 0.5 mM DTT, 0.5% NP-40, 4 ȝJP/OHXSHSWLQȝJP/DSURWLQLQDQGP0306)

(69) $IWHUFHQWULIXJDWLRQDW 6,000×g for 15 min, pellets containing crude nuclei were resuspended in 50 ȝ/RI hypertonic buffer (10 mM HEPES, 400 mM KCl, 1 mM MgCl2, 0.2 mM EDTA, 0.5 P0'77ȝJP/OHXSHSWLQȝJP/DSURWLQLQP0306)DQGJO\FHURO

(70) at 4oC for 30min. The samples were then centrifuged at 10,000×g for 15min. The supernatant containing the nuclear proteins was collected and stored at -80oC until the Western blotting and electrophoretic mobility shift assays.. 2.6 Western blotting analysis After each experiment, cells were washed twice with cold PBS and were KDUYHVWHGLQȝ/RIO\VLVEXIIHU P07ULV-HCl, pH 8, 0.1% Triton X-100, 320 mM sucrose, 5 mM EDTA, 1 mM PMSF, 1 mg/L leupeptin, 1 mg/L aprotinin, and 2 mM dithiothreitol). Cell homogenates were centrifuged at 14,000×g for 20 min at 4oC. The resulting supernatant was used as a cellular protein for Western blotting analysis. The total protein was analyzed by use of the Coomassie Plus protein assay reagent kit (Pierce Biotechnology, Rockford, IL). Equal amounts of cellular proteins were electrophoresed in a sodium dodecyl sulfate (SDS)-polyacrylamide gel, and proteins were then transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA). Nonspecific binding sites on the membranes were blocked with 5% nonfat milk in 15 mM Tris/150 mM NaCl buffer (pH 7.4) at room temperature for 2 h. 35.

(71) Membranes were probed with antibodies. The membranes were then probed with the secondary antibody labeled with horseradish peroxidase. The bands were visualized by using an enhanced chemiluminescence kit (PerkinElmer Life Science, Boston, MA) and scanned by a luminescent image analyzer (LAS-4000, FUJIFILM, Japan). The bands were quantitated with ImageGauge software (FUJIFILM).. 2.7 RNA isolation and RT-PCR Total RNA of EA.hy926 cells was extracted by using TRIzol reagent. After WUHDWPHQWFHOOVZHUHZDVKHGWZLFHZLWKFROG3%6DQGVFUDSHGZLWKȝ/Rf TRIzol UHDJHQW&HOOVDPSOHVZHUHPL[HGZLWKȝ/RIFKORURIRUPDQGFHQWULIXJHGDW 11,000×g for 15 min. The supernatant was collected and mixed with 250 ȝ/RI isopropyl alcohol. After centrifuged at 11,000×g for 15 min, the supernatant was discarded and the cell pellet was stored in 70% ethanol or dissolved in deionized ZDWHUIRUTXDQWLILFDWLRQ:HXVHGȝJRIWRWDO51$IRUWKHV\QWKHVLVRIILUVW-strand cDNA by using Moloney murine leukemia virus reverse transcriptase (Promega) in a final volume of 2ȝ/FRQWDLQLQJQJRIROLJR-dT and 40 units of RNase inhibitor. 3&5ZDVFRQGXFWHGLQDWKHUPRF\FOHULQDUHDFWLRQYROXPHRIȝ/FRQWDLQLQJȝ/ RIF'1$%LR7DT3&5EXIIHUȝPRORIHDFKGHR[\ULERQXFOHRWLGHWULSKRVSKDWH 1.25 mmol/L MgCl2, and 1 unit of BioTaq DNA polymerase (BioLine). Oligonucleotide primers of ICAM-1 (forward, 5’-TGAAGGCCACCCCAGAGGACAAC-3’; reverse, 5’-CCCATTATGACTGCGGCTGCTGCTACC-3’), HO-1 (forward, 5’-CTGAGTTCATGAGGAACTTTCAGAAG-3’; reverse, 5’-TGGTACAGGGAGGCCATCAC-3’), and glyceraldehyde-3-phosphate dehydrogenase (forward, 5’-CCATCACCATCTTCCAGGAG-3’; reverse, 5’-CCTGCTTCACCACCTTCTTG-3’) were designed on the basis of published 36.

(72) sequences (Meagher et al., 1994). Amplification of ICAM-1 and GAPDH were achieved when samples were heated to 95oC for 5 min and then immediately cycling 32 times through a 1-min denaturing step at 94oC, a 1-min annealing step at 56oC, and a 1-min elongation step at 72oC. Amplification of HO-1 and GAPDH were achieved when samples were heated to 95oC for 5 min and then immediately cycling 39 times through a 1-min denaturing step at 95oC, a 1-min annealing step at 55oC, and a 2-min elongation step at 72oC. The glyceraldehyde-3- phosphate dehydrogenase cDNA level was used as the internal standard. PCR products were resolved in a 1% agarose gel and were scanned by a Digital Image Analyzer (Alpha Innotech) and quantitated with ImageGauge software.. 2.8 Electrophoretic mobility shift assay (EMSA) EMSA was performed according to our previous study (Cheng et al., 2004). The LightShift Chemiluminescent EMSA Kit and synthetic biotin-labeled double-stranded NF-țB consensus oligonucleotides (forward, 5’-AGTTGAGGGGACTTTCCCAGGC -3’; reverse, 5’-GCCTGGGAAAGTCCCCTCAACT-3’) were used to measure the NF-țB nuclear protein-'1$ELQGLQJDFWLYLW\1XFOHDUH[WUDFW ȝJ

(73) SRO\ G,-dC), and biotin-labeled double-stranded NF-țB oligonucleotides were mixed with the ELQGLQJEXIIHU WRDILQDOYROXPHRIȝ/

(74) DQGZHUHLQFXEDWHGDWoC for 30 min. In addition, the unlabeled and mutant double-stranded NF-țB oligonucleotides (5’-AGTTGAGGCGACTTTCCCAGGC-3’) were used to confirm the protein binding specificity, respectively. These oligonucleotide primers were synthesized by MDBio Inc. (Taipei, Taiwan). The nuclear protein-DNA complex was separated by electrophoresis on a 6% TBE-polyacrylamide gel and then were transferred to Hybond-N+ nylon membranes (Amersham Pharmacia Biotech, Inc., Pisscataway, NJ). Next, the membrane were cross-linked by UV light for 10 min and treated with 37.

(75) streptavidin-horseradish peroxidase, and the nuclear protein-DNA bands were developed with Chemiluminescent Substrate (Pierce Biotechnology, Rockford, IL). The bands were scanned by a luminescent image analyzer.. 2.9 Plasmids, transfection, and luciferase assay A p2xARE/Luc fragment containing tandem repeats of double-stranded oligonucleotides spanning the Nrf2 binding site, 5’-TGACTCAGCA-3’, as described by Kataoka et al. (2001) was introduced into the pGL3 promoter plasmid. The ICAM-1 promoter-luciferase construct (pIC339, -339 to 0) was a gift from Dr. P. T. van der Saag (Hubrecht Laboratory, Utrecht, The Netherlands). pIC339 contains NF-țB (-187/-178), AP-1 (-84/-279), AP-1 (-48/-41), and Sp1 (-59/-53, -206/-201) binding sites (van de Stolpe et al., 1994). All subsequent transfection experiments were performed by using nanofection reagent (PAA, Pasching, Austria) according to the manufacturer’s instructions. EA.hy926 cells were transiently transfected with 0.4 ȝJRIS,&RUpGL3 SODVPLGDQGȝJRIȕ-JDODFWRVLGDVHSODVPLGE\XVLQJȝ/ of nanofectin in OPTI-MEM medium for 8 h. After transfection, cells were changed to DMEM medium and treated with DHA for 16 h before being challenged with TNF-ĮIRUDQDGGLWLRQDOK&HOOVZHUHWKHQZDVKHGWZLFHZLWKFROG3%6 scraped with lysis buffer, and centrifuged at 14,000×g for 3 min. The supernatant was collected IRUWKHPHDVXUHPHQWRIOXFLIHUDVHDQGȕ-galactosidase activities by using a Luciferase Assay Kit (Promega, Madison, WI) according to the manufacturer’s instructions, and the luciferase activity was measured by a microplate luminometer (TROPIX TR- 717, Applied Biosystems). The luciferase activity of each sample was FRUUHFWHGRQWKHEDVLVRIȕ-galactosidase activity, which was measured at 420 nm with O-nitrophenyl-beta-D-galactopyranoside as a substrate.. 38.

(76) 2.10 RNA interference by small interfering RNA of HO-1 and Nrf2 Predesigned small interfering RNA (siRNA) against human HO-1, Nrf2, and non-targeting control-pool siRNA were purchased from Dharmacon Inc. (Lafayette, CO). EA.hy926 cells were transfected with HO-1 and Nrf2 siRNA SMARTpool by using DharmaFECT1 transfection reagent (Thermo) according to the manufacturer’s instructions. The four siRNAs against the human HO-1 gene are (1) AUGCUGAGUUCAUGAGGAA, (2) ACACUCAGCUUUCUGGUGG, (3) CAGUUGCUGGUAGGGCUUUA, and (4) AGAUUGAGCGCAACAAGGA. The 4 siRNAs against the human Nrf2 gene are (1) UAAAGUGGCUGCUCAGAAU, (2) GAGUUACAGUGUCUUAAUA, (3) UGGAGUAAGUCGAGAAGUA, and (4) CACCUUAUAUCUCGAAGUU. Non-targeting siRNA construct (NC) was used as negative control. Specific silencing was confirmed by at least three independent Western blotting assays with cellular extracts 8 h after transfection.. 2.11 Peroxide measurement Detection of intracellular oxidative states was performed by using the probe 2,7-dichlorofluorescin diacetate (H2DCF-DA) (Molecular Probes Inc., Eugene, OR) (Bae et al., 1997). Briefly, cells were grown to 60-70% confluence and then serum-starved in DMEM supplemented with 0.5% (v/v) FBS for an additional 2 days. The cells were then stabilized in serum-free DMEM without phenol red for at least 30 min before exposure to DHA or TNF-Į for the indicated time periods. Cells were then incubated for 10 min with the ROS-sensitive fluorophore H2DCF-'$ ȝ0

(77) Cells were immediately observed under a laser-scanning Confocal microscope (Leica TCS SP2). DCF fluorescence was excited at 488 nm using an argon laser, and the evoked emission was filtered with a 515-nm long pass filter.. 39.

(78) 2.12 Monocyte adhesion assay EA.hy926 cells in 12-well plates were allowed to grow to 80% confluence and were then pretreated with DQGȝ0'+$IRUKfollowed by incubation with 1 ng/mL TNF-Į for an additional 6 h. The human monocytic HL-60 cells cultured in RPMI-PHGLXPZLWK)%6ZHUHODEHOHGZLWKȝM 2,7-bis(2-carboxyethyl)5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM). At the end of the DHA and TNF-Įtreatment, a total of 1×106 BCECF-AM-labeled HL-60 cells were added to each well, and the cells were co-incubated with EA.hy926 cells at 37oC for 30 min. The wells were washed and filled with cell culture medium, and the plates were sealed, inverted, and centrifuged at 100×g for 5 min to remove nonadherent HL-60 cells. Bound HL-60 cells were lysed in a 1% SDS solution, and the fluorescence intensity was determined in a fluoroscan ELISA plate reader (FLX800, Bio-Tek, Winooski, VT) with an excitation wavelength of 480 nm and an emission wavelength of 520 nm. A control study showed that fluorescence is a linear function of HL-60 cell density in the range of 3,000-80,000 cells/well. The results are reported on the basis of the standard curve obtained.. 2.13 Statistical analysis Data were analyzed by using analysis of variance (SAS Institute, Cary, NC). The significance of the difference among mean values was determined by one-way analysis of variance followed by Tukey’s test and the difference between mean values was determined by student’s t-test; P values <0.05 were taken to be statistically significant.. 40.