KOREAN J. FOOD SCI. TECHNOL. Vol. 41, No. 2, pp. 220~224 (2009) 연구노트 The Korean Society of Food Science and Technology Isoliquiritigenin 의 toll-like receptor agonists 에의해서유도된 NF-κB 활성화와 cyclooxygenase-2 발현억제 박세정 1 양승주 2 윤형선 1,3 * 1 순천향대학교의료과학대학의료과학과, 2 건양대학교임상병리학과, 3 순천향대학교의료과학대학임상병리학과 NF-κB Activation and Cyclooxygenase-2 Expression Induced by Toll-Like Receptor Agonists can be Suppressed by Isoliquiritigenin Se-Jeong Park 1, Seung-Ju Yang 2, and Hyung-Sun Youn 1,3 * 1 Department of Medical Science, College of Medical Sciences, Soonchunhyang University 2 Department of Biomedical Laboratory Science, Konyang University 3 Department of Biomedical Laboratory Science, College of Medical Sciences, Soonchunhyang University Abstract Toll-like receptors(tlrs) are pattern recognition receptors(prrs) that recognize pathogen-associated molecular patterns(pamps) and regulate the activation of innate immunity. All TLR signaling pathways culminate in the activation of NF-κB, leading to the induction of inflammatory gene products such as COX-2. Licorice (Glycyrrhiza uralensis) has been used for centuries as an herbal medicine. Isoliquiritigenin(ILG), a simple chalcone-type flavonoid, is an active component present in licorice and has been used to treat many chronic diseases. However, the mechanism as to how ILG mediates health effects is still largely unknown. In the present report, we present biochemical evidence that ILG inhibits the NFκB activation induced by TLR agonists and the overexpression of downstream signaling components of TLRs, MyD88, IKKβ, and p65. ILG also inhibits TLR agonists-induced COX-2 expression. These results suggest that anti-inflammatory effects of ILG are caused by modulation of the immune responses regulated by TLR signaling pathways. Key words: toll-like receptors, isoliquiritigenin, NF-κB, COX-2, LPS 서 *Corresponding author: Hyung-Sun Youn Department of Biomedical Laboratory Science College of Medical Sciences Soonchunhyang University, Asan, Chungnam 336-745, Korea Tel: 82-41-530-3086 Fax: 82-41-530-3085 E-mail: hyoun@sch.ac.kr Received January 12, 2009; revised February 1, 2009; accepted February 3, 2009 론 오래전부터의약품으로사용되는많은식물들중에는항암, 항염증효과를가지고있다고알려져있는많은기능성소재들이포함되어있다. 그러한식물들중의하나가감초 (licorice, Glycyrrhiza uralensis) 이다. 약방의감초라는말이있듯이, 감초는한약재에널리사용되어왔다. 감초는오랫동안음식, 음료수, 담배등의맛을내는성분으로사용되었으며, 또한위궤양 (gastric ulcers), 십이지장궤양 (duodenal ulcers), 후두염 (sore throats), 감기 (coughs), 기관지염 (bronchitis), 관절염 (arthritis), 부신병 (adrenal insufficiency), 그리고알러지 (allergies) 등의치료에사용되었다 (1-4). 감초는의학적으로효과를가지고있는 glycyrrhizin, glabridin, 그리고 isoliquiritigenin 과같은성분들을포함하고있다고많은연구에의해서알려졌다 (5-7). 그중에서도 chalcone 구조를가지고있는 flavonoid 중의하나인 isoliquiritigenin(ilg)(fig. 1) 은 vasorelaxant (8), anti-platelet aggregation(9) 그리고 anti-allergic activity(10) 를가지고있다. 또한 ILG 는 RAW 264.7 cells 에서 LPS(TLR4 agonist) 에의해서유도된 inducible nitric oxide(inos) 와 cyclooxygenase-2(cox-2) 의발현을억제하여항염증효과를가지고있다 (2). Toll-like receptors(tlrs) 는 pathogen-associated molecular patterns(pamps) 을인식하는 pattern recognition receptors(prr) 이며, 선천성면역 (innate immunity) 반응을유도하고, 뒤이어후천성면역 (acquired immunity) 반응을유도하는중요한역할을한다 (11-14). 현재까지최소한열세개의 TLRs 가포유동물세포안에서발견되었으며, 각각의 TLRs 는병원체들이가지고있는각각의독특한구조를인식한다 (11). TLR1, TLR2, TLR4, TLR5, 그리고 TLR6 는세포막에존재하며박테리아의구성성분들을인식하는반면에, TLR3, TLR7, TLR8, 그리고 TLR9 은 endosome 과같은세포질에있는성분들의막에존재하며바이러스핵산들을인식한다 (Fig. 2)(15). TLR9 은또한박테리아핵산을인식한다. TLRs 에 agonists 가붙으면 MyD88(myeloid differential factor 88) 또는 TRIF(Toll/IL-1R domain-containing adaptor inducing IFN-β) 를통 Fig. 1. The structure of isoliquiritigenin. 220
Isoliquiritigenin 에의한 cyclooxygenase-2 발현억제 221 재료및방법 재료실험에사용한 isoliquiritigenin 은 Sigma-Aldrich(St. Louis, MO, USA) 로부터구입하였다. MALP-2(Macrophage-activating lipopeptide of 2 kda), LPS(lipopolysaccharide) 그리고 poly[i:c](polyriboinosinic polyribocytidylic acid) 는 Alexis Biochemical(San Diego, CA, USA), List Biological Lab(San Jose, CA, USA) 그리고 Amersham Biosciences(Piscataway, NJ, USA) 회사로부터각각구입하였다. COX- 2 와 actin 항체는 Santa Cruz Biotechnology(Santa Cruz, CA, USA) 회사로부터구입하였다. 그밖의다른시약들은 Sigma-Aldrich(St. Louis, MO, USA) 회사로부터구입하였다. 세포배양 RAW 264.7 cells(a murine monocytic cell line, ATCC TIB-71) 과 293T cells(human embryonic kidney) 은 10%(v/v) FBS, 100 units/ml Penicillin, 100 mg/ml streptomycin을포함하고있는 Dulbecco's modified Eagle's medium(dmem) 에서배양하였으며, 세포들은 5% CO 2 /air를포함하고있는 37 o C 배양기안에서배양하였다. Fig. 2. Toll-like receptor signaling pathways. TLRs have two major downstream signaling pathways; MyD88- and TRIFdependent pathways leading to the activation of NF-κB and IRF3. LPS, lipopolysaccharide; MyD88, myeloid differential factor 88; TRIF, TIR domain-containing adapter inducing IFNβ; IFN, interferon; TIRAP, TIR domain-containing adapter protein; TRAM, TRIF-related adapter molecule; IκBα, inhibitor of kappa B alpha; IKK, IκB kinase; IRAK, IL-1 receptorassociate kinase; TBK1, TANK-binding kinase 1; RIP1, receptor interacting protein 1; IRF3, IFN-regulatory factor 3; NF-κB, nuclear factor kb. 한신호전달체계를통하여전사요소 nuclear factor κb(nf-κb) 를활성화시킨다 (11). NF-κB 의활성화는결국 cytokine 이나 (COX- 2) 와같은염증을유발하는유전체들을유도한다 (11). 오늘날염증을치료하기위한많은약들이개발되었다. 특히 COX 에의한통증을완화시키기위해서 Non-steroidal anti-inflammatory drugs(nsaids) 들이개발되었다. NSAIDs 의대표적인약들로는 aspirin, ibuprofen 과 naproxen 등이있다 (16). 하지만 NSAIDs 에의한 gastric problems 때문에오늘날많은연구자들은염증치료를위해서자연에서얻어진물질에보다높은관심을가지기시작하였다 (17). 항염증효과를가지고있는음식들은대부분이색깔을가지고있는과일과채소, 생선기름 (fish oil), 견과류 (nuts), 씨앗 (seeds), 허브 (herbs), 그리고향신료 (spices) 등이있다 (18). 이번연구에서는자연에존재하는물질의항염증효능을알아보기위해서한약제로널리사용되고있는감초안에포함되어있는천연물질을선택하였다. 특히 ILG 가면역시스템조절을위해서중요한역할을하는 TLRs 신호전달체계를어떻게조절하여항염증효과를가지고있는지알아보았다. ILG 는여러 TLR agonists 에의해서유도된 COX-2 를억제하였으며, 이러한결과들은우리가매일먹는식품들이어떻게여러병원균들로부터유도되는염증반응이나만성적인질병들을조절할수있는지를보여주는중요한결과라할수있겠다. 트랜스펙션 (transfection) 과발광효소유전자분석 (luciferase reporter gene assay) NF-κB발광 plasmid, heat shock protein(hsp) 70-β-galactosidase plasmid 등 transfection을위한모든 DNA는 EndoFree Plasmid Maxi kit(qiagen, Valencia, CA, USA) 을사용하여준비되었다. NF-κB발광효소유전자분석은선행연구에서사용한방법에의하여분석하였다 (19, 20). 발광효소 plasmid와 HSP70-β-galactosidase plasmid는 Superfect transfection 시약 (Qiagen) 을사용하여세포안으로 transfection 시켰다. 발광효소의활성화는 luciferase assay system(promega, Madison, WI, USA) 을사용하여측정하였다. 발광효소의활성화는 β-galactosidase의활성화를측정하여표준화시켰다. 면역압 (immunoblotting) 방법 Western blotting 은선행연구의방법에의하여분석하였다 (21, 22). 단백질추출물들은 SDS-PAGE(sodium dodecyl sulfate-polyacrylamide gel electrophoresis) 에서분리되어 polyvinylidene difluoride membrane 으로전기영동에의해서이전되었으며, Membrane 은 0.1% Tween 20 그리고 5% 탈지건조된우유를포함하고있는 phosphate-buffered saline 을가지고 blocking 하였다. Membrane 1 차항체를가지고 blotting 하고, horseradish peroxidase 와복합된 2 차항체에노출시킨다음, ECL western blot detection 시약 (Amersham Biosciences, Arlington Heights, IL, USA) 을사용하여단백질을규명하였다. 데이타분석각각의데이타값은세개가한벌의실험으로얻어졌으며, mean ± standard error mean(sem) 으로표현되었다. 결과및고찰 ILG 의 TLR agonists 에의해유도된 NF-κB 활성화와 COX-2 발현의억제먼저우리는감초에포함되어있는 phytochemicals 중의하나
222 한국식품과학회지제 41 권제 2 호 (2009) 인 ILG 가여러 TLR agonists 에의해서유도된 NF-κB 활성화와 COX-2 발현을억제하는지를알아보았다. ILG 는 MALP-2(TLR2 and TLR6 agonist), poly[i:c](tlr3 agonist) 그리고 LPS(TLR4 agonist) 에의해서유도된 NF-κB 활성화를억제하였다 (Fig. 3). 다음실험으로발광효소유전자분석과 western blotting 방법을사용하여 NF-κB 활성화에의해서조절되는유전자인 COX-2 발현이 ILG 에의해서억제되는지알아보았다. ILG 는 MALP-2, poly[i:c] 그리고 LPS 에의해서유도된 COX-2 발현을억제시켰다 (Fig. 4). 이러한결과는 ILG 가여러 TLR agonists 에의해서유도된신호전달체계를억제한다는것을의미한다. ILG의 TLRs downstream 분자에의해서유도된 NF-κB 활성화억제마지막으로우리는 ILG가 TLRs의 downstream 분자에의해서유도된 NF-κB 활성화를억제하는지알아보았다. ILG는 MyD88, IKKβ 또는 p65에의해서유도된 NF-κB 활성화를억제시켰다 (Fig. 5). 많은식물들에는항염증효과를가지고있는 phytochemicals들이포함되어있다. 이러한 phytochemicals 중에 Michael addition 에의해서 sulfhydryl 그룹에반응할수있는 α,β-unsaturated carbonyl 그룹을가지고있는폴리페놀이나 sesquiterpene lactone 등이많이있다 (23, 24). 많은 α,β-unsaturated carbonyl 그룹을가지고있는 phytochemicals 은 IKK에있는 cysteine의 sulfhydryl(-sh) 그룹에붙어서 IKK 인산화효소의효과를억제시키는것으로알 Fig. 4. Isoliquiritigenin suppressed the COX-2 expression induced by MALP-2, poly[i:c] or LPS. A) RAW264.7 cells were transfected with COX-2 luciferase reporter plasmid and pre-treated with isoliquiritigenin (50, 100 µm) for 1 hr and then treated with MALP- 2 (10 ng/ml), poly[i:c] (10 µg/ml) or LPS (10 ng/ml) for an additional 8 hrs. Cell lysates were prepared and luciferase and β- galactosidase enzyme activities were measured as described in Materials and Methods Relative luciferase activity (RLA) was normalized with b-galactosidase activity. Values are mean±sem (n = 3). **, Significantly different from MALP-2 alone, p<0.01. +, Significantly different from poly[i:c] alone, p<0.05. #, Significantly different from LPS alone, p <0.05. B) RAW264.7 cells were pretreated with isoliquiritigenin (50, 100 µ M) for 1 hr and then further stimulated with MALP-2 (10 µ g/ml), poly[i:c] (10 mg/ml) or LPS (10 ng/ml) for 8 hrs. Cell lysates were analyzed for COX-2 and b- actin protein by immunoblots. Veh, vehicle; ILG, isoliquiritigenin. Fig. 3. Isoliquiritigenin suppressed the NF-κB activation induced by MALP-2, poly[i:c] or LPS. A-C) RAW264.7 cells were transfected with NF-κB luciferase reporter plasmid and pre-treated with isoliquiritigenin (50, 100 µm) for 1 hr and then treated with MALP- 2 (10 µg/ml) (A), poly[i:c] (10 mg/ml) or (B), LPS (10 ng/ml) (C) for an additional 8 hrs. Cell lysates were prepared and luciferase and β-galactosidase enzyme activities were measured as described in Materials and Methods. Relative luciferase activity (RLA) was normalized with b-galactosidase activity. Values are mean±sem (n = 3). *, Significantly different from MALP-2 alone, p <0.05.+, Significantly different from poly[i:c] alone, p < 0.05. ##, Significantly different from LPS alone, p < 0.01. Veh, vehicle; ILG, isoliquiritigenin. 려져있다 (25). Sesquiterpene lactone 중의하나인 helenalin 은 p65 안에있는두개의 cysteine(cys38, Cys120) 에붙어서전사요소 NF-κB 의 DNA 결합을억제시키는것으로알려져있다 (26, 27). 또한선행연구에의하면 α,β-unsaturated carbonyl 그룹을가지고있는 curcumin, helenalin, 그리고 cinnamaldehyde 는 LPS(TLR4 agonist) 에의해서유도된 TLR4 의동종이합체의형성을억제하여 NF-κB 활성화나 COX-2 발현을억제시키는것을알아내었다 (19, 28). ILG 또한 α,β-unsaturated carbonyl 그룹을가지고있다 (Fig. 1). 이러한구조는 ILG 가 IKK 나 TLR 의 cysteine 의 thiol 그룹에붙어서 NF-κB 활성화나 COX-2 발현을억제시킬것이라는것을예측할수있다. 앞으로 kinase assay 나이합체형성실험을통해서 ILG 가어떻게 NF-κB 활성화나 COX-2 발현을억제하는지정확한분자학적인타깃이밝혀질것이다. Kumar 등 (29) 은 ILG 가 IkB kinase 를억제하여서 NF-κB 활성화를억제한다고보고하였다. 또한 Kim 등 (2) 은 ILG 가 RAW 264.7 cells 안에서 NF-κB 활성화의억제를통하여 LPS 에의해서유도된 inos 와 COX-2 를억제한다고보고하였다. 하지만이번실험에서는앞선두그룹에서발견하지못한다른중요한결과를발견하였다. TLRs 는커다랗게두개 (MyD88 과 TRIF-dependent) 의신호전달체계를통하여 NF-κB 를활성화한다 (Fig. 2). MyD88-dependent pathway 는 IKKβ 를통한 carnonical pathway 를통하여 NF-κB 를활성화시키지만, TRIF-dependent pathway 는 IKKβ 대신에 RIP-1 을통하여 NF-κB 를활성화시킨다고알려져있다. MALP-2(TLR2 와 TLR6 agonist) 와 LPS(TLR4 agonist) 는 IkB kinase 를통하여 NF-κB 를활성화하지만, poly[i:c](tlr3
Isoliquiritigenin 에의한 cyclooxygenase-2 발현억제 223 에서, 옛날부터지금까지전통적인약재로써질병치료에다양하게쓰이고있는감초의뿌리에서추출한성분중의하나인 ILG 가 NF-κB 활성과 COX 발현을어떻게조절하여항염증효과를가지고있는지알아보았다. ILG 는 TLR agonists 인 MALP-2, Poly[I:C], 그리고 LPS 에의해유도된 NF-κB 활성화와 COX-2 발현을억제시켰다. 또한 ILG 는리간드 (ligand) 에독립적인 TLRs signaling downstream molecules 인 MyD88, IKKβ, 그리고 p65 에의해서유도된 NF-κB 활성을억제시켰다. 이러한결과는한약재로서많이이용되는감초가단지한약의쓴맛을줄이기위함이아니라 TLRs 신호전달체계를조절하여항염증효과를가지고있다는것을보여주는것이라할수있겠다. 문 헌 Fig. 5. Isoliquiritigenin inhibits the activation of NF-κB induced by the overexpression of MyD88, IKKβ, or p65. A-C) 293T cells were co-transfected with NF-κB-luciferase reporter plasmid and an expression plasmid for MyD88, IKKβ, or p65. pcdna was used as a vector control for MyD88, IKKb and p65. After 3 hrs, cells were treated with isoliquiritigenin (50, 100 µm) for 18 hrs. Relative luciferase activity (RLA) was determined by normalization with β-galactosidase activity. Values are mean±sem (n = 3). *,Significantly different from MyD88 plus vehicle, p < 0.05. +, Significantly different from IKKb plus vehicle, p < 0.01. #, Significantly different from p65 plus vehicle, p < 0.05. Veh, vehicle; ILG, isoliquiritigenin. agonist) 는 IkB kinase 를통하지않고 NF-κB 를활성화한다 (11, 30). 실험결과에의하면, ILG 가 MALP-2 와 LPS 에의해서유도된 NFκB 를억제하는것은 IKKβ 의억제에기인한다고할수있겠지만, ILG 가 poly[i:c] 에의해서유도된 NF-κB 를억제하는것은 ILG 가 IKKβ 아닌다른곳을분자학적인타깃으로하고있다는것을제시한다고할수있다. 기존의연구들은 ILG 가 IKKβ 를타깃으로삼아 MyD88-dependent pathway 를통한 NF-κB 활성화를억제시킨다는것을밝혀내었지만, 우리연구에서는 ILG 가 MyD88- dependent pathway 뿐만아니라 TRIF-dependent pathway 를통한 NF-κB 활성화도억제시킨다는것을알아내었다. 그러므로앞으로의연구에서 ILG 가어떻게 TRIF-dependent pathway 를통한 TLRs 신호전달체계를조절하는지정확한분자학적인타깃이밝혀질것으로기대한다. 우리는이번연구를통해서감초추출물인 ILG 가여러 TLR agonists 에의해서유도된 NF-κB 활성화와 COX-2 발현을억제시키는것을밝혀내었다. 이러한발견은한약재로오랫동안사용되어온감초가 TLRs 에의해서조절되는염증반응이나또는뒤이어발생하는만성적인질병들을조절할수있다는중요한가능성을제시해준다고할수있겠다. 요 선천성면역반응을위해중요한역할을하는 TLRs 가외부병원성물질에자극을받게되면 NF-κB 를활성화시키며, 그결과로염증을유도하는 COX 와같은유전자를발현한다. 이번연구 약 1. Fintelmann V. Modern phytotherapy and its uses in gastrointestinal conditions. Planta Med. 57: S48-S52 (1991) 2. Kim JY, Park SJ, Yun KJ, Cho YW, Park HJ, Lee KT. Isoliquiritigenin isolated from the roots of Glycyrrhiza uralensis inhibits LPS-induced inos and COX-2 expression via the attenuation of NF-kappaB in RAW 264.7 macrophages. Eur. J. Pharmacol. 584: 175-184 (2008) 3. Kamei J, Saitoh A, Asano T, Nakamura R, Ichiki H, Iiduka A, Kubo M. Pharmacokinetic and pharmacodynamic profiles of the antitussive principles of Glycyrrhizae radix (licorice), a main component of the Kampo preparation Bakumondo-to (Mai-mendong-tang). Eur. J. Pharmacol. 507: 163-168 (2005) 4. Haggag EG, Abou-Moustafa MA, Boucher W, Theoharides TC. The effect of a herbal water-extract on histamine release from mast cells and on allergic asthma. J. Herb. Pharmacother. 3: 41-54 (2003) 5. Fukai T, Satoh K, Nomura T, Sakagami H. Preliminary evaluation of antinephritis and radical scavenging activities of glabridin from Glycyrrhiza glabra. Fitoterapia 74: 624-629 (2003) 6. Yokota T, Nishio H, Kubota Y, Mizoguchi M. The inhibitory effect of glabridin from licorice extracts on melanogenesis and inflammation. Pigm. Cell Res. 11: 355-361 (1998) 7. Zhou S, Koh HL, Gao Y, Gong ZY, Lee EJ. Herbal bioactivation: the good, the bad and the ugly. Life Sci. 74: 935-968 (2004) 8. Yu SM, Kuo SC. Vasorelaxant effect of isoliquiritigenin, a novel soluble guanylate cyclase activator, in rat aorta. Brt. J. Pharmacol. 114: 1587-1594 (1995) 9. Tawata M, Aida K, Noguchi T, Ozaki Y, Kume S, Sasaki H, Chin M, Onaya T. Anti-platelet action of isoliquiritigenin, an aldose reductase inhibitor in licorice. Eur. J. Pharmacol. 212: 87-92 (1992) 10. Kakegawa H, Matsumoto H, Satoh T. Inhibitory effects of some natural products on the activation of hyaluronidase and their antiallergic actions. Chem. Pharm. Bull. 40: 1439-1442 (1992) 11. Takeda K, Akira S. Toll-like receptors in innate immunity. Int. Immunol. 17: 1-14 (2005) 12. Medzhitov R. Toll-like receptors and innate immunity. Nat. Rev. Immunol. 1: 135-145 (2001) 13. O'Neill LA. TLRs: Professor Mechnikov, sit on your hat. Trends Immunol. 25: 687-693 (2004) 14. Vogel SN, Fitzgerald KA, Fenton MJ. TLRs: Differential adapter utilization by toll-like receptors mediates TLR-specific patterns of gene expression. Mol. Interv. 3: 466-477 (2003) 15. Kawai T, Akira S. Toll-like receptor and RIG-I-like receptor signaling. Ann. NY Acad. Sci. 1143: 1-20 (2008) 16. Harris RE, Beebe-Donk J, Doss H, Burr Doss D. Aspirin, ibuprofen, and other non-steroidal anti-inflammatory drugs in cancer prevention: A critical review of non-selective COX-2 blockade (review). Oncol. Rep. 13: 559-583 (2005) 17. Naesdal J, Brown K. NSAID-associated adverse effects and acid control aids to prevent them: A review of current treatment options. Drug Safety 29: 119-132 (2006) 18. Surh YJ. Cancer chemoprevention with dietary phytochemicals.
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