Biomedical Science Letters 2019, 25(3): 267~274 https://doi.org/10.15616/bsl.2019.25.3.267 eissn : 2288-7415 Original Article Dehydrocostus Lactone Suppresses the Expression of inos Induced by TLR Agonists Su Yeon Kim *, Sunghye Heo *, Seung Han Kim *, Minji Kwon *, Sin-Aye Park ** and Hyung-Sun Youn,** Department of Biomedical Laboratory Science, College of Medical Sciences, SoonChunHyang University, Chungnam, Asan 31538, Korea Toll-like receptors (TLRs) are one of the families of pattern recognition receptors (PRR) to recognize pathogenassociated molecular patterns (PAMPs). PAMPs stimulate TLRs to initiate specific immunoactivity. The activation of TLRs signaling leads to the expression of pro-inflammatory gene products such as cytokines and inducible nitric oxide synthase (inos). To evaluate the therapeutic potential of dehydrocostus lactone (DHL), which is a natural sesquiterpene lactone derived from various medicinal plants, inos expression induced by LPS (TLR4 agonist), MALP-2 (TLR2 and TLR6 agonist), or Poly[I:C] (TLR3 agonist) were examined. DHL suppressed the inos expression induced by LPS, MALP-2, or Poly[I:C]. DHL also inhibited nitrite production induced by LPS, MALP-2, or Poly[I:C]. These results suggest that DHL can modulate TLRs signaling pathways resulting in anti-inflammatory effect. Key Words: Dehydrocostus lactone; Inducible nitric oxide synthase; TLR; Inflammation; LPS 서론만성염증 (chronic inflammation) 은박테리아나바이러스감염과같은외부자극에반응하여유도되는병리생리학 (pathophysiology) 적인현상으로, inducible nitric oxide synthase (inos) 가염증유발에중요한기능을하는것으로보고되어있다 (Murakami and Ohigashi, 2007). 산화질소 (NO) 는기체상태의간단한무기분자로, 세포내에서전달자의기능을가지고있다. NO는선천성면역체계의유도를위해서중요한역할을한다 (Lowenstein and Padalko, 2004). 활성화된대식세포는 NO를포함한다양한분자들을방출함으로써병원체복제를억제한다. NO의합성은아미노산 L-arginine 을 L-citrulline 으로변환하여, nitric oxide synthase (NOS) 로알려진효소에의해매개된다. NOS는 neuronal NOS (nnos), endothelial NOS (enos), 그리고면역반응에관여하는 inducible NOS (inos) 의세가지동위효소를가지고있다 (Knowles, 1996). nnos 와 enos 는일정하게발현되는구성 (constitutive) 효소이며, 이와는대조적으로 inos 는건강한상태에서는발현되지않고, 오히려면역반응이나미생물자극에의한세포자극에따른염증유도조건에서발현한다 (Knowles, 1996). 톨 (Toll) 유전자는처음에초파리 (Drosophila) 에서발견되었고, 배복성의 (dorsoventral) 배아발달과선천성면역기능을중재하기위하여발견되었다 (Anderson et al., 1985). 1997년인간에서 Toll 이발견되어초파리 Toll 과구별하기위하여 Toll-like receptor (TLR) 라명명되었다 (Medzhitov et al., 1997). TLR4 가최초로발견되었으며, 현재 13개의 TLRs Received: July 4, 2019 / Revised: August 8, 2019 / Accepted: August 17, 2019 * Ungraduate student, ** Professor. Corresponding author: Hyung-Sun Youn. Department of Biomedical Laboratory Science, College of Medical Sciences, SoonChunHyang University, Soonchunhyang-Ro 22, Shinchang-Myun, Asan-Si, Chungnam 31538, Korea. Tel: +82-41-530-3086, Fax: +82-41-530-3085, e-mail: hyoun@sch.ac.kr C The Korean Society for Biomedical Laboratory Sciences. All rights reserved. CC This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. - 267 -
Dehydrocostous lactone Fig. 1. The structure of dehydrocostus lactone (DHL). Dehydrocostus lactone (DHL) 은 (Fig. 1) Inula helenium L. and Saussurea lappa 와같은국화과식물로부터추출되는 sesquiterpene lactone 이다. 여러연구에의하면 DHL은항염증 (Cho et al., 2000), 항괴양 (Yoshikawa et al., 1993), 항암 (Ko et al., 2004) 및면역조절 (Pandey et al., 2007) 기능을포함한여러가지약리학적효능을가지고있는것으로알려져있다. 하지만이러한 DHL의약리학적효능에대한분자적작용기전은아직까지완전히밝혀져있지않다. 그러므로본연구에서는 DHL이 TLRs 의신호전달체계를조절함으로써 inos 를포함한염증반응에미치는영향에대해알아보고자한다. 재료및방법 재료 가인간과마우스에서발견되었다 (Poltorak et al., 1998). TLR1-10 은인간에서발견되었으며, TLR1-9 과 TLR11-13 은마우스에서발견되었다. TLRs 자극제 (agonist) 는특별한면역반응을유도하기위하여 TLRs를자극하는 pathogenassociated molecular patterns (PAMPs) 이다. 가장많이연구된 TLRs 자극제는 lipopolysaccharide (LPS; TLR4 agonist), lipopeptides(tlr1, TLR2, 그리고 TLR6 agonists), flagellin (TLR5 agonist), single stranded RNA (TLR7 and TLR8 agonist), double stranded (ds) RNA (TLR3 agonist), 그리고 DNA containing the CpG motif (TLR9 agonist) 이다 (Medzhitov, 2001). 최근의연구에의하면스트레스를받거나죽은세포에서방출되는 heat shock proteins (HSP; TLR2 와 TLR4) 과 high mobility group box 1 (HMGB1; TLR2와 TLR4) 또한중요한 TLRs 자극제로알려졌다 (Asea et al., 2002; Kepp et al., 2011). TLRs 자극제가그들의수용체에결합하면둘중하나인, myeloid differential factor 88 (MyD88)- 또는 TIR domain-containing adaptor inducing interferon-β (TRIF) 를의지한신호전달체계를유도한다. MyD88 를의지한신호전달체계는 NF-κB, mitogen-activated protein kinase (MAPK) 나 cytokines, chemokines, 그리고 cytosolic 효소의전사활성화를유도하는반면에 TRIF-dependent 신호전달체계는늦은 NF-κB 나 interferon regulatory factor 3 (IRF3) 의활성을유도한다 (Kawai and Akira, 2010). 이러한전사인자의활성화는 inos와같은염증관련유전자의발현을증가시켜여러질병을초래하게된다 (Akira and Takeda, 2004). 그러므로 TLRs의신호전달체계조절은염증반응이나다양한질병들을예방할수있는것이다. 실험에사용한 LPS (lipopolysaccharide; TLR4 agonist) 는 List Biological Lab (San Jose, CA, USA) 으로부터, MALP-2 (macrophage-activating lipopeptide of 2 kda; TLR2와 6 agonist) 는 Alexis Biochemical (San Diego, CA, USA) 로부터, Polyriboinosinic polyribocytidylic acid (poly[i:c]; TLR3 agonist) 는 Amersham Biosciences (Piscataway, NJ, USA) 로부터구입하였다. inos 항체는 BD Biosciences (San Jose, CA, USA) 로부터, β-actin 항체는 Santa Cruz Biotechnology (Santa Cruz, CA, USA) 로부터구입하였다. Dehydrocostus lactone 은 Selleckchem (Houston, TX, USA) 으로부터구입하였다. 그밖의다른시약들은특별한언급이없는한 Sigma- Aldrich 로부터구입하였다. 세포배양 RAW264.7 세포들은 (murine monocytic cells, ATCC TIB-71) Dulbecco's modified Eagle's medium (DMEM) (10% (v/v) FBS, 100 units/ml Penicillin, 100 μg/ml streptomycin) 을사용하여 5% CO 2 /air, 37 에서배양하였다. Plasmid inos-luciferase plasmid는 Daniel Hwang (University of California, Davis, CA, USA) 으로부터제공받았다. Heat shock protein (HSP) 70-β-galactosidase plasmid 는 R. Modlin (University of California, Los Angeles, CA, USA) 으로부터제공받았다. 위의모든 DNA는 EndoFree Plasmid Maxi kit (Qiagen, Valencia, CA, USA) 을사용하여준비되었다. - 268 -
Cell viability test 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium (MTT) 사용하여세포생존율을측정하였다 (Kim et al., 2018b). RAW264.7 세포를 96 well plate 에분주하고 4시간동안 MTT 약물을처리한후, well plate 에 CellTiter 96 AQueous One Solution Reagent (Promega, Madison, WI, USA) 를첨가하였으며, 4시간동안배양하였다. 그후 96 well plate reader 를사용하여 490 nm에서흡광도를측정하여세포생존율을측정하였다. Transfection 과 luciferase 를이용한유전자발현분석 (luciferase reporter gene assay) inos 유전자발현은 luciferase assay를사용하여분석하였다 (Kim et al., 2018a). RAW264.7 세포를 48 well plates 에균등분주하여배양하였다, 하루뒤에 50~60% 정도세포증식을보일때, inos-luciferase plasmid 와 HSP70-βgalactosidase plasmid는 Superfect transfection 시약 (Qiagen, Valencia, CA, USA) 을사용하여세포안으로 transfection 시켰다. 10 또는 20 μm DHL을 pretreat 한후 1시간동안배양하였다. 그리고 agonist 를처리한후 8시간동안배양하였다. Luciferase assay system (Promega, Madison, WI, USA) 을사용하여 luciferase 의활성을 luminometer (Berthold, Bad- Wildbad, Germany) 로측정하였으며, β-galactosidase 의활성화정도를확인하여표준화시켰다. Western blotting TLR agonist 와 DHL을처리한세포로부터추출된단백질들은 SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) 에서크기에따라분리되었다. 이후분리된단백질들은 immunoblotting 방법을사용하여 polyvinylidene difluoride membrane 으로이동되었다 (Youn et al., 2006a; Youn et al., 2006b). Membrane 은 0.1% Tween 20와 5% 탈지건조된우유를포함하고있는 phosphate-buffered saline에서 blocking 되었다. Membrane 에 1차항체 (inos 또는 β-actin) 를붙이고 horseradish peroxidase 가결합된 2차항체를붙인후, intron western blot detection system (Seongnam, Gyeonggi-do, Korea) 을사용하여단백질의발현을확인하였다. Nitrite assay DHL의 inos에대한효과를측정하기위하여 inos에 Fig. 2. Cells viability assay of dehydrocostus lactone. RAW264.7 cells were treated with DHL (10, 20, 50 μm) for 4 h. Twenty microliters of the CellTiter 96 AQ ueous One Solution Reagent was added directly to culture wells. The plate was incubated at 37 for 4 h in a humidified 5% CO 2 atmosphere. The absorbance was recorded at 490 nm with a 96-well plate reader. Veh, vehicle; DHL, dehydrocostus lactone. 의해서만들어지는 nitrite (NO 2 ) 를측정하였다 (Lim et al., 2008). NO 는매우불안정한물질로측정이쉽지않아서 대신 NO 의산화과정에의해서만들어지는 nitrite (NO 2 ) 를 대신측정하였다. 세포배양 well plate 의상층액에분주기 를사용하여 Griess reagent 를첨가하였으며, plate reader 를 사용하여 570 nm 에서흡광도를측정하였다. 표준물질로 sodium nitrite 를사용하였다. 데이타분석 각각의데이타값은서로다른세개의값을정량하여 Student's t-test 로얻어졌으며, mean ± standard error mean (SEM) 으로표현되었다. 결과및고찰 Dehydrocostus lactone 의세포독성효과 첫번째로, RAW264.7 세포에서 DHL 의독성효과를평 가하기위해 MTT assay 를사용하여세포생존율을측정 하였다. 세포들은 DHL 20 μm 의농도에서 100.0%, 50 μm 농도에서 92.1% 생존율을보였다 (Fig. 2). 이후의모든실 험은 DHL 20 μm 농도까지사용하였다. - 269 -
Dehydrocostus lactone은 TLR agonists에의해유도된 inos 발현을억제다음실험으로 DHL이여러 TLR agonists 에의해서유도된 inos에어떠한영향을미치는지알아보았다. DHL 은 LPS (TLR4 agonist), MALP-2 (TLR2와 TLR6 agonist), 그리고 Poly[I:C] (TLR3 agonist) 에의해유도된 inos의과발현을억제하는것을 inos-luciferase assay를활용하여측정하였다 (Figs. 3A-C). 또한 DHL이 LPS, MALP-2, 그리고 Poly[I:C] 에의해유도된 inos 단백질의과발현을저해하는것을 Western blotting 방법을통하여확인하였다 (Figs. 4A-C). 또한 DHL이 inos의생성물인 nitrite (NO 2 ) 의생성에어떠한영향을미치는지확인해보았다. Nitrite (NO 2 ) 는 NO의산화과정에의해서만들어지는 NO의생리학적인저장풀로여겨진다. DHL은 LPS, MALP-2, 그리고 Poly[I:C] 에의해서유도된 nitrite (NO 2 ) 의생성을억제하였다 (Figs. 5A-C). 이러한결과들을종합해보면 DHL은 TLR 자극제에의해유도되는 inos의발현과그생성물인 nitrite (NO 2 ) 의생성을조절하여염증반응을조절할수있다는것을예측할수있다. NOS는 nnos, enos, inos 등세개의이성질체로구성되어있다 (Lind et al., 2017). nnos 는중추신경계와말초신경계모두의신경조직에서 NO를생성한다 (Lind et al., 2017). nnos는또한세포의사소통에서역할을수행하며 A B C Fig. 3. Dehydrocostus lactone inhibits inos expression induced by TLRs agonists. (A-C) RAW264.7 cells were transfected with inos luciferase reporter plasmid and pretreated with 10 or 20 μm DHL for 1h and then treated with LPS (10 ng/ml) (A), MALP-2 (10 ng/ml) (B), or Poly[I:C] (10 μg/ml) (C) for an additional 8 h. Cell lysates were prepared and luciferase enzyme activities were determined. Values represent the mean ± SEM (n=3). #, Significantly different from LPS alone, P<0.01 (##) (A). *, Significantly different from MALP-2 alone, P<0.05 (*) (B). +, Significantly different from Poly[I:C] alone, P<0.01 (++) (C). Veh, vehicle; DHL, dehydrocostus lactone: RLA, relative luciferase activity. A B C Fig. 4. Dehydrocostus lactone inhibits inos protein induced by TLRs agonists. (A-C) RAW264.7 cells were pretreated with 10 or 20 μm DHL for 1 h and then further stimulated with LPS (10 ng/ml) (A), MALP-2 (10 ng/ml) (B), or Poly[I:C] (10 μg/ml) (C) for an additional 8 h. Cell lysates were analyzed for inos and β-actin protein by immunoblots. Veh, vehicle; DHL, dehydrocostus lactone. - 270 -
A B C Fig. 5. Dehydrocostus lactone inhibits nitrite production induced by TLRs agonists. (A-C) RAW 264.7 cells were pretreated with 10 or 20 μm DHL for 1 h and then treated with LPS (10 ng/ml) (A), MALP-2 (10 ng/ml) (B), or Poly[I:C] (10 μg/ml) (C) for an additional 20 h. The amounts of nitrite in supernatant were measured using Griess reagent. Values represent the mean ± SEM (n=3). #, Significantly different from LPS alone, P<0.01 (##) (A). *, Significantly different from MALP-2 alone, P<0.01 (**) (B). +, Significantly different from Poly[I:C] alone, P<0.01 (++) (C). Veh, vehicle; DHL, dehydrocostus lactone. 플라즈마막과연관되어있다. 질소산화물 3 (NOS3) 이라고도하는 endothelial NOS (enos) 는혈관에 NO를발생시키며혈관기능을조절하는데관여한다. 혈관내피에서 enos에의해생성된 NO는혈관색조 (vascular tone), 세포증식 (cellular proliferation), 백혈구부착 (leukocyte adhesion), 및혈소판응집 (platelet aggregation) 을조절하는데중요한역할을한다 (Forstermann and Munzel, 2006). 그러므로 enos는건강한심혈관을유지하기위하여필수적인요소이다 (Lind et al., 2017). 구성 NOS인 nnos 및 enos와다르게, inos는다양한생리학적조건 ( 혈압조절, 상처치유, 호스트방어메커니즘등 ) 과병리생리학 ( 염증, 감염, 신가소성질환, 간경화증, 당뇨병등 ) 에서중요한역할을담당한다 (Lechner et al., 2005). inos 는 nitric oxide (NO) 를생성하고, 생성된 NO는세포독성효과가있어이와관련된염증및질병을유도하는것으로알려져있다 (Vallance, 2003) inos는 NF-κB 경로를통한염증성 cytokines 에의해유도되는것으로알려져있다. Gochman et al. (2012) 은 NF-κB 가대장염에관여하는것을조사할때, 인체대장염조직에서 inos 발현이증가함을입증하여염증성질환에대한기여와, 가능한치료법으로서 inos의억제를제안하였다 (Gochman et al., 2012). inos 유전자를없앤마우스에서대동맥과중원동맥 (mesenteric arteries) 에대한박테리아 lipopolysaccharide 의영향을평가할때, Chauhan et al. 등은 lipopolysaccharide 에의해유도된내피기능장애 (endothelial dysfunction) 가 inos 유도에의지한다는것을입증하였다 (Chauhan et al., 2003). Mungrue et al. 연구진은마우스모델의심근세포에서 inos의과발현은서맥성부정맥 (bradyarrythmia), 심근병 (cardiomyopathy) 및갑작스런심장사망과관련이있다고제안하였다 (Mungrue et al., 2002). 따라서, 이러한연구들에서보여지는증거는염증기간동안 inos가질병의손상효과에기여하므로, inos 억제가질병예방에효과가있다는것을보여주는결과이다. 선천성면역체계는병원균에대한숙주의빠른반응이다. 대식세포, 자연살인세포등선천성면역체계를유도하는세포들은 pattern recognition receptor (PRR) 를이용하여병원균과관련된 PAMPs 를인식한다 (Medzhitov, 2001). PRR 역할을하는대표적인수용체가 TLRs이다. TLRs는일반적으로 MyD88 과 TRIF에의지한두개의신호전달체계를가지고있다 (Fitzgerald et al., 2003; Hultmark, 1994). MyD88은 TLR3를제외한모든포유동물에서발견되는 TLRs의 TIR (Toll/IL-1R) 도메인에붙는즉각적인어댑터 (adaptor) 분자이며, 전사요소 NF-κB 의활성화를유도한다. MyD88 신호전달체계를통한 NF-κB 활성은 cytokines 등염증유도물질들의발현을증가시켜다양한질병을유발하는것으로알려져있다 (Takeda and Akira, 2005). 한편, TLR3의활성은 MyD88 대신에어댑터분자인 TRIF를의지한신호전달체계를통하여전사인자 IRF3의 - 271 -
활성화를유도한다 (Fitzgerald et al., 2003). TLR4는 MyD88 과 TRIF 를의지한양쪽신호전달체계를활용한다. 활성화된 IRF3는 type I IFN 유전자들의발현을유도한다 (Sato et al., 2003). 따라서병원균들이숙주안으로들어오면 TLRs가 PAMPs 을인식하여 MyD88 또는 TRIF를의지한신호전달체계를활용하여신호를아래로전달하고, 여러단백질들의활성화를통해 cytokines 등염증유전자물질들의발현을증가시키지만, 숙주는후천성면역반응을활성화시켜여러질병으로부터숙주를보호하는것으로알려져있다. 하지만계속된 PAMPs 의자극에의해숙주의면역시스템에과부하가걸리게되면, 오히려염증유도물질들에의해각종질병이발생되게된다. 그러므로, PAMPs 에의해유도된 TLRs 의신호전달체계의활성을억제하는것은만성염증과같은여러질병을예방하는데있어매우중요하다고할수있다 (Takeda and Akira, 2005). 오랜역사를가지고있는전통한의학에따르면 3,000 여종의식물이암치료에효과적으로알려져있다. 이전의연구에따르면 DHL은항염증 (Cho et al., 2000), 항우울제 (Yoshikawa et al., 1993), 면역조절 (Pandey et al., 2007), 및항암 (Ko et al., 2004) 효과를가지고있다. 최근에는백혈병 (Butturini et al., 2011), 폐암 (Hung et al., 2010), 유방암 (Pitchai et al., 2014), 간암 (Hsu et al., 2009), 난소암 (Sun et al., 2003), 전립샘암 (Kim et al., 2012), 방광암 (Rasul et al., 2013), 대장암 (Sun et al., 2015) 등다양한종류의암에대한 DHL 의잠재적인항암활동으로인해연구원들에게매우높은관심을불러일으켰다. 그러나 DHL의정확한항염증 항암효과와작용기전이아직설명되지않았으며, DHL의표적단백질이충분히규명되지는않고있다. 본연구에서는 DHL의항염증효능을규명하기위하여 TLRs 자극제인 LPS, MALP-2, 그리고 Poly[I:C] 에의해유도된 inos의발현에미치는영향에대해확인하였다. DHL은 TLRs 의 MyD88 를의지한신호전달체계자극제인 MALP-2 (TLR2 and TLR6 agonist) 에의해서유도된 inos 의과발현을억제하였다. DHL은 TLRs의 TRIF를의지한신호전달자극제인 Poly[I:C] (TLR3 agonist) 에의해서유도된 inos의과발현을억제하였다. 또한 DHL은 TLRs의 MyD88 과 TRIF 양쪽신호전달체계를사용하는 LPS (TLR4 agonist) 에의해유도된 inos의과발현을억제하였다. 이러한결과는 DHL이 TLRs 의 MyD88 과 TRIF 양쪽신호전달체계를조절할수있다는것을보여주는결과이다. 따라서본연구의결과는 DHL이 TLRs의신호전달체계를 조절하여다양한만성질환치료및치료제개발에있어서중요한역할을할것으로기대된다. ACKNOWLEDGEMENT 본연구는순천향대학교학술연구비의일부지원과 2017년도한국연구재단의지원을받아수행된기초연구사업 (2017R1D1A1B03031534) 의지원을받아수행된연구결과물로그지원에감사드립니다. CONFLICT OF INTEREST No potential conflict of interest relevant to this article was reported. REFERENCES Akira S, Takeda K. Toll-like receptor signalling. Nat Rev Immunol. 2004. 4: 499-511. Anderson KV, Bokla L, Nusslein-Volhard C. Establishment of dorsal-ventral polarity in the Drosophila embryo: the induction of polarity by the Toll gene product. Cell. 1985. 42: 791-798. Asea A, Rehli M, Kabingu E, Boch JA, Bare O, Auron PE, Stevenson MA, Calderwood SK. Novel signal transduction pathway utilized by extracellular HSP70: role of toll-like receptor (TLR) 2 and TLR4. J Biol Chem. 2002. 277: 15028-15034. Butturini E, Cavalieri E, de Prati AC, Darra E, Rigo A, Shoji K, Murayama N, Yamazaki H, Watanabe Y, Suzuki H, et al. Two naturally occurring terpenes, dehydrocostuslactone and costunolide, decrease intracellular GSH content and inhibit STAT3 activation. PLoS One. 2011. 6: e20174. Chauhan SD, Seggara G, Vo PA, Macallister RJ, Hobbs AJ, Ahluwalia A. Protection against lipopolysaccharide-induced endothelial dysfunction in resistance and conduit vasculature of inos knockout mice. FASEB J. 2003. 17: 773-775. Cho JY, Baik KU, Jung JH, Park MH. In vitro anti-inflammatory effects of cynaropicrin, a sesquiterpene lactone, from Saussurea lappa. Eur J Pharmacol. 2000. 398: 399-407. Fitzgerald KA, McWhirter SM, Faia KL, Rowe DC, Latz E, Golenbock DT, Coyle AJ, Liao SM, Maniatis T. IKKepsilon and TBK1 are essential components of the IRF3 signaling pathway. Nat Immunol. 2003. 4: 491-496. Forstermann U, Munzel T. Endothelial nitric oxide synthase in vascular disease: from marvel to menace. Circulation. 2006. 113: 1708-1714. - 272 -
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