ၸѐЎࡰрǴୖᆶᇨᏤ HO-1 ୷Ӣ߄ϐૻ৲ሀၡ৩(signal transduction pathways)ǴЬाхࡴ MAPKs (mitogen-activated protein kinase)ǵPI3K
(phosphoinositide 3-kinase) /AktǵPKC (Owuor & Kong, 2002; Lee & Johnson,
2004 ; Xu et al., 2006; Ryter et al., 2006; Paine et al., 2010)ǹԜѦǴϩ HO-1 ୷Ӣ ޑ௴η(promoter)ୱǴวڀԖӭख़ाᙯᒵӢηޑ่ӝՏǴхࡴ NF-E2 (nuclear factor-erythroid 2)ǵAP-1ǵNF-ț% Ǵаፓ HO-1 ޑ߄(კ 2.9) (Farombi
& Surh, 2006; Alam & Cook, 2007; Gruber et al., 2010)Ƕ
4-1. ᙯᙯᒵӢη Nrf2
Nrf2 (nuclear factor erythroid 2-related factor 2)ࣁځख़ाፓޣϐǴӢ Nrf2
ᆶ࣬ᜢל਼ϯሇન୷Ӣޑࢤፓׇӈ Antioxidant Response Element (ARE)
่ӝǴࡺჴᆶ HO-1 ޑ߄ԖஏϪᜢ߯(Xu et al., 2006; Kim et al., 2007;
Johnson et al., 2009)ǶNrf2 ឦܭ Cap’n’Collar / basic leucine zipper (CNC-bZIP)ᙯᒵ ӢηৎޑঁԋǶӧؒԖڈᐟޑݩΠǴNrf2 ᙖҗᆶ Keap1 (Klech-like ECH-associated protein 1)่ӝǴ႖ᚆӧಒझ፦ύǹѿڙډࢲϯǴ೭ঁፄӝނ ஒґှǴញܫрޑ Nrf2 ளаᙯ౽ΕಒझਡϣǴԶᆶ small Maf ৎޑԋ
(i.e., MafKǵMafGǵMafF)ಔӝԋ౦፦Βᆫᡏ(Motohashi et al., 2002, 2004;
Katsuoka et al., 2005)ǶନΑ small Maf ৎޑԋǴNrf2 Ψёૈᆶ c-Jun ܈ activating transcription factor 4 (ATF4)ԋ౦፦Βᆫᡏ(heterodimers)Ǵቚம ARE/EpRE (electrophile response element)-drivenൔᏤ୷Ӣࢲ܄Ǵߦ HO-1 ޑᙯ ᒵ(Venugopal & Jaiswal, 1998; He et al., 2001; Mann et al., 2007)Ƕ
კ 2.9 ፓ HO-1 ߄ϐૻ৲ሀၡ৩(Farombi & Surh, 2006)
4-2. ځځѬၡ৩(PI3K/AktǵMAPKsǵPKC)
ςԖӭࣴزୖᆶNrf2ࢲϯޑૻ৲ሀၡ৩ǴٯӵPI3KکPKCё٬Nrf2 วғᕗለϯǴԶPI3KڋᏊ(LY-294002)܈PKCڋᏊ(Ro-32-0432)Ψёफ़եARE luciferaseൔᏤ୷Ӣޑࢲ܄ǹҗԜፕǴNrf2ϐࢲϯᆶPI3KکPKCޑࢲ܄Ԗᜢ(Lee
& Surh, 2005; Farombi & Surh, 2006; Keum et al., 2008)ǶԜѦǴMAPKsΨჴ
ୖᆶNrf2ࢲϯբҔ(Kong et al., 2001; Lee & Johnson, 2004; Xu et al., 2006)ǶҞς ޕӭᅿڀԖғࢲ܄ޑϯނ(phytochemicals)Ǵӵ౦౷⋸ለ㸰ᜪ
(isothiocyanates)ǵЇԚ(indoles)ǵΒЧ୷౷ϯނ(diallyl sulfides)ǵ✉ᜪϯӝނ (flavonoids)ᆶᖖન(curcuminoids)ǴԖߦ܈ڋNrf2่ӝԿޑ୷Ӣ promoterޑբҔ(Jeong et al., 2006)ǶӧHepG2ಒझਲ਼ኳԄΠǴᇺન(capsaicin) ёၸࢲϯPI3K/Aktૻ৲ሀၡ৩ǴቚуNrf2ᆶAREޑ่ӝǴԶ҅ӛፓHO-1
୷Ӣ߄(Joung et al., 2007)ǹCarnosolᇨวHO-1ޑ߄ΨᇡࣁᆶPI3Kૻ৲ၡ৩ Ԗᜢ(Martin et al., 2004)Ƕ
Figure 2.9
ᗨฅࡐӭޑ phytochemicals ჴёаᇨว HO-1 ߄ЪڀԖߥៈಒझբҔ (cytoprotection)ǴՠҞࣁЗǴDHA ᇨว HO-1 ߄ޑࣴز࣬ჹၨϿǶࣴزว
ങݨύख़ाԋϩ DHA ёᇨว BV-2 microglia ߄ HO-1 (Lu et al., 2010)Ǵځᐒڋ ёૈᆶ Akt ک ERK ԖᜢǶќѦǴDHA ёၸ Nrf2-dependent ૻ৲ሀٰᇨว mouse peritoneal macrophages߄ HO-1ǴԶڋ LPS ᇨวޑวݹϸᔈ(Wang et
al., 2010)ǶGao Γ(2007)ว DHA ሡၸ਼ϯբҔࡕ܌ౢғޑౢނωڀԖᇨว
Nrf2߄کࢲϯ ARE ׇӈޑբҔǴԶЪдॺෳ DHA ਼ϯౢނΨёૈᆶ
Keap1բҔԶࢲϯ Nrf2Ƕ
ಃ
ಃΟക ࣴزҞޑ
ਥᏵፁғޑीǴЈՈᆅ੯ੰޔ՞ۚ୯ϣΜεԝӢޑ൳ӜǴેๆރ ฯϯࢂЈՈᆅ੯ੰޑᅿǶᖏᙴᏢᏵᡉҢǴેๆރฯϯࢂᅿᄌ܄วݹ੯
ੰǴԋՈᆅϣᏛિެ୴ᑈϷᠼᆢඬ༧ޑԋǹԜѦǴࣴزวಒझᗹߕϩη ޑғԋჹܭેๆރฯϯ੯ੰޑวתᄽख़ाޑفՅǴᗹߕϩηϐ ICAM-1 ё
բࣁᅿวݹޑғࡰǴٰႣෳқՈౚӧϣҜಒझύޑᗹߕǶTNF-Į ࢂ
ᅿςޕޑวݹಒझᐟનǴၸࢲϯᙯᒵӢη NF-ț% ٰڈᐟᗹߕϩηޑ߄Ǵ
܌аதҔٰբࣁᇨวಒझౢғวݹϸᔈޑኳԄǶ
Αှેๆރฯϯޑԋ٠уаႣٛࢂҞႣٛᙴᏢޑख़ाፐᚒϐǴӭ ЎࡰрങݨϷځख़ाࢲ܄ԋϩ DHA ڀԖלวݹբҔǴ٠Ъၸӭ৩ٰ
फ़եЈՈᆅ੯ੰޑวғǹԶ HO-1 ࢂᡏϣख़ाޑל਼ϯሇનǴЬाڙډ਼ϯ ᓸΚǵวݹǵϯᏢނǵख़ߎឦڈᐟᇨวԶεໆ߄Ǵ೭ࢂᅿٛፁ܄ϸᔈǴፓ
ಔᙃ܈ಒझٰӢᔈғᡂϯаᆢځᄊѳᑽޑᜢᗖǶҁჴᡍ࠻Ӄࣴزว
аऀЈጪϣ✊Ⴃೀ HUVECs Ϸ EA.926 ಒझǴёаڋ TNF-Į ܌ᇨวޑ ICAM-1 ߄(Chao et al., 2011)ǶӢԜҁჴᡍஒճҔ TNF-Į ᇨวϣҜಒझ EA.hy926 ౢғ
วݹϸᔈޑኳԄǴ DHA ࢂցёаᙖҗቹៜ NF-ț% ૻ৲ሀၡ৩ٰڋ
TNF-Į ܌ᇨวޑ ICAM-1 ߄Ǵ٠Ъ DHA ܌ᇨวޑ HO-1 ࢂցୖᆶڋว
ݹޑᐒڋǴԶၲډႣٛวݹ੯ੰޑфਏǶ
28
ჴჴ ᡍ ࢎ ᄬ
ಃ
ಃΒҽ
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 (Chapkinet al., 2009;
Wang et al., 2011). Dietary intake of n-3 PUFAs is associated with a reduced risk of
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 LQWHUOHXNLQ-ȕ ,/-ȕ DQGLQWHUIHURQ-gamma (INF-Ȗ
(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% 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% IDPLO\ 6XQ .DULQ ,QUHVSRQVHWRVWLPXODWLRQ
,ț%-ĮLVSKRVSKRU\ODWHGDW6HUDQGE\WKH,ț%NLQDVH ,.. FRPSOH[ 0D\ Ghosh, 1998; Karin & Delhase, 2000) and subsequently degraded by the
ATP-dependent 26S proteasome complex (Chen et al., 1995; Wertz & Dixit, 2010).
,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;
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.