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Cardamonin Inhibits the Expression of Inducible Nitric Oxide Synthase Induced by TLR2, 4, and 6 Agonists
Biomed Sci Letters 2018;24:102-107
Published online June 30, 2018;  https://doi.org/10.15616/BSL.2018.24.2.102
© 2018 The Korean Society For Biomedical Laboratory Sciences.

Ah-Yeon Kim1,§, Hyun-Jin Shim1,§, Su-Yeon Kim1, Sung-Hye Heo1, and Hyung-Sun Youn1,†

1Department of Biomedical Laboratory Science, College of Medical Sciences, SoonChunHyang University, Chungnam, Asan 31538, Korea
Correspondence to: Hyung-Sun Youn. Department of Biomedical Laboratory Science, College of Medical Sciences, SoonChunHyang University, Chungnam, Asan 31538, Korea. Tel: +82-41-530-3086, Fax: +82-41-530-3085, e-mail: hyoun@sch.ac.kr
Received April 16, 2018; Revised June 11, 2018; Accepted June 15, 2018.
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.
 Abstract

Toll-like receptors (TLRs) play an important role for host defense against invading pathogens. The activation of TLRs signaling leads to the activation of NF-κB and the expression of pro-inflammatory gene products such as cytokines and inducible nitric oxide synthase (iNOS). To evaluate the therapeutic potential of cardamonin, which is a naturally occurring chalcone from Alpinia species (zingiberaceous plant species), NF-κB activation and iNOS expression induced by MALP-2 (TLR2 and TLR6 agonist) or LPS (TLR4 agonist) were examined. Cardamonin inhibited the activation of NF-κB induced by MALP-2 or LPS. Cardamonin also suppressed the iNOS expression induced by MALP-2 or LPS. These results suggest that cardamonin has the specific mechanism for anti-inflammatory responses by regulating of TLRs signaling pathway.

Keywords : Cardamonin, Inducible nitric oxide synthase, TLR, Inflammation, LPS
꽌濡

뿼利앹 誘몄깮臾 媛먯뿼 삉뒗 議곗쭅 넀긽 썑 諛쒖깮븯뒗 蹂듭옟븳 씪젴쓽 諛섏쓳쑝濡, 二쇰줈 諛쒖쟻, 遺湲, 諛쒖뿴 諛 옣湲 넻利앹쓣 쑀諛쒗븯吏留, 쇅遺쓽 怨꾩냽맂 옄洹뱀뿉 諛섏쓳븯뿬 깮꽦릺뒗 留뚯꽦 뿼利앹 삤엳젮 뿬윭 吏덈퀝쓣 쑀룄븯뒗 寃껋쑝濡 븣젮졇 엳떎(Ferrero-Miliani et al., 2007). 뿼利앹 뿬윭 吏덈퀝怨 愿젴씠 엳뒗 蹂묓깭깮由ы븰(pathophysiology)쟻씤 쁽긽쑝濡, inducible nitric oxide synthase (iNOS) cyclooxygenase-2 (COX-2) 슚냼媛 뿼利 쑀諛쒖뿉 以묒슂븳 湲곕뒫쓣 븯뒗 寃껋쑝濡 븣젮졇 엳떎(Murakami and Ohigashi, 2007). iNOS뒗 nitric oxide(NO)瑜 깮꽦븯怨, COX-2뒗 prostaglandins (PGs)쓣 깮꽦븳떎(Moncada, 1999; Turini and DuBois, 2002). 듅엳 iNOS쓽 free radical씤 NO媛 꽭룷 룆꽦 슚怨쇰줈 쑀諛쒕릺뒗 뿼利앷낵 愿젴맂 吏덈퀝쓣 쑀룄븯뒗 寃껋쑝濡 븣젮졇 엳떎(Vallance, 2003).

Toll-like receptors (TLR)뒗 鍮꾪듅씠쟻 삉뒗 怨좊궃 硫댁뿭 諛⑹뼱, 듅엳 꽭洹, 諛붿씠윭뒪, 吏꾧퇏 諛 湲곗깮異⑹쓣 룷븿븳 蹂묒썝洹 移⑥엯뿉 뵲瑜 뿼利 諛섏쓳뿉 以묒슂븳 뿭븷쓣 븯뒗 蹂묒썝泥 씤떇 닔슜泥대줈 븣젮졇 엳떎(Kawai and Akira, 2010). 쁽옱 룷쑀瑜 꽭룷뿉꽌 쟻뼱룄 13媛쒖쓽 TLRs媛 諛쒓껄릺뿀쑝硫, 諛뺥뀒由ъ븘, 諛붿씠윭뒪 벑쓣 룷븿븳 떎뼇븳 蹂묒썝泥 愿젴 遺꾩옄 뙣꽩뱾쓣(PAMPs) 씤떇븳떎(Kawai and Akira, 2010; Youn, 2012). TLR1 / 2 / 4 / 5 / 6 닔슜泥대뒗 꽭룷 몴硫댁뿉 議댁옱븯뿬 誘몄깮臾 遺꾩옄 뙣꽩뱾쓣(PAMPs) 씤떇븯硫, TLR3 / 7 / 9 닔슜泥대뒗 뿏룄醫(enodosome)뿉 議댁옱븯뿬 誘몄깮臾 빑궛뱾쓣 씤떇븳떎(Kawai and Akira, 2010). TLRs 꽑泥쒖꽦 硫댁뿭怨 썑泥쒖꽦 硫댁뿭 궗씠쓽 以묒슂븳 뿰寃 怨좊━씠떎. 洹몃윭誘濡 TLRs 떊샇쟾떖 떆뒪뀥 議곗젅 湲곗쟾 洹쒕챸 뿬윭 吏덈퀝뱾쓣 삁諛⑺븯湲 쐞븯뿬 以묒슂븳 뿭븷쓣 븯寃 맆 寃껋씠떎.

븣뵾땲븘 떇臾(Alpinia, zingiberaceous plant species) 異붿텧臾 以묒쓽 븯굹씤 cardamonin (2’,4’-dihydroxy-6’-methoxychalcone) (Fig. 1) 빆뿼利 슚뒫쓣 媛吏怨 엳뒗 chalcone 援ъ“瑜 媛吏怨 엳뒗 泥쒖뿰臾쇱쭏씠떎(Goncalves et al., 2014). Cardamonin 빆뿼利(anti-inflammatory) (Chow et al., 2012), 빆醫낆뼇(antitumor) (Park et al., 2013), 빆寃고빑(antituberculosis) (Lin et al., 2002), 삁냼뙋 쓳怨 뼲젣瑜(platelet aggregation inhibitory) (Jantan et al., 2008) 룷븿븳 뿬윭 빟由ы븰쟻씤 슚뒫쓣 媛吏怨 엳뒗 寃껋쑝濡 븣젮졇 엳떎. 븯吏留 씠윭븳 cardamonin쓽 빟由ы븰쟻씤 옉슜 湲곗쟾 셿쟾엳 諛앺졇 엳吏 븡떎. 삉븳 TLRs쓽 鍮꾩젙긽쟻씤 옉룞 留롮 留뚯꽦뿼利 吏덊솚怨 愿젴씠 엳뒗 寃껋쑝濡 븣젮졇 엳떎. 洹몃윭誘濡 씠踰 뿰援ъ뿉꽌뒗 cardamonin씠 TLRs 떊샇쟾떖 떆뒪뀥쓣 議곗젅븯뿬, iNOS瑜 룷븿븳 뿼利 諛섏쓳뿉 뼱뼚븳 쁺뼢쓣 誘몄튂뒗吏瑜 븣븘蹂닿퀬옄 븳떎.

Fig. 1.

The structure of cardamonin.


옱猷 諛 諛⑸쾿

옱猷

떎뿕뿉 궗슜븳 MALP-2 (macrophage-activating lipopeptide of 2 kDa) LPS (lipopolysaccharide)뒗 Alexis Biochemical (San Diego, CA, USA)怨 List Biological Lab (San Jose, CA, USA)濡쒕꽣 媛곴컖 援ъ엯븯쑝硫, iNOS β-actin 빆泥대뒗 BD Biosciences (San Jose, CA, USA) Santa Cruz Biotechnology (Santa Cruz, CA, USA) 쉶궗濡쒕꽣 媛곴컖 援ъ엯븯떎. 洹 諛뽰쓽 떎瑜 떆빟뱾 Sigma-Aldrich 쉶궗濡쒕꽣 援ъ엯븯떎.

꽭룷 諛곗뼇

RAW264.7 꽭룷뱾(a murine monocytic cell line, ATCC TIB-71) 10% (v/v) FBS, 100 units/mL Penicillin, 100 μg/mL streptomycin씠 룷븿맂 Dulbecco’s modified Eagle’s medium (DMEM)쓣 궗슜븯뿬, 5% CO2/air, 37°C뿉꽌 諛곗뼇븯떎.

Plasmid

NF-κB iNOS 諛쒓킅 plasmid뒗 F. Mercurio (Signal Pharma-ceuticals, San Diego, CA, USA) Daniel Hwang (University of California, Davis, CA, USA)쑝濡쒕꽣 媛곴컖 젣怨듬컺븯쑝硫, Heat shock protein (HSP) 70-β-galactosidase plasmid뒗 R. Modlin (University of California, Los Angeles, CA, USA)쑝濡쒕꽣 젣怨듬컺븯떎. 쐞쓽 紐⑤뱺 DNA뒗 EndoFree Plasmid Maxi kit (Qiagen, Valencia, CA, USA)쓣 궗슜븯뿬 以鍮꾨릺뿀떎.

Transfection怨 諛쒓킅슚냼 쑀쟾옄 遺꾩꽍(luciferase reporter gene assay)

NF-κB iNOS 諛쒓킅슚냼 쑀쟾옄 遺꾩꽍 transfection 諛⑸쾿쓣 궗슜븯뿬 遺꾩꽍븯떎(Youn et al., 2005; Youn et al., 2006c). RAW264.7 꽭룷瑜 well plates뿉 洹좊벑 遺꾩<븯怨, overnight 썑 50~60% 젙룄쓽 利앹떇쓣 蹂댁씪 븣, 諛쒓킅슚냼 plasmid HSP70-β-galactosidase plasmid뒗 Superfect transfection 떆빟(Qiagen, Valencia, CA, USA)쓣 궗슜븯뿬 꽭룷 븞쑝濡 transfection 떆耳곕떎. Luciferase assay system (Promega, Madison, WI, USA)쓣 궗슜븯뿬 諛쒓킅슚냼쓽 솢꽦솕瑜 痢≪젙 븯쑝硫, β-galactosidase쓽 솢꽦솕瑜 痢≪젙븯뿬 몴以솕떆耳곕떎.

Immunoblotting

꽭룷濡쒕꽣 異붿텧맂 떒諛깆쭏뱾 SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis)뿉꽌 겕湲곗뿉 뵲씪 遺꾨━릺뿀쑝硫, 쟾湲곗쁺룞뿉 쓽빐꽌 polyvinylidene difluoride membrane쑝濡 씠룞릺뿀떎(Youn et al., 2006a; Youn et al., 2006b). Membrane 0.1% Tween 20 5% 깉吏 嫄댁“맂 슦쑀瑜 룷븿븯怨 엳뒗 phosphate-buffered saline뿉꽌 븯猷 룞븞 blocking쓣 븯쑝硫, 痢≪젙븯怨좎옄 븯뒗 떒諛깆쭏쓽 1李 빆泥대 遺숈씠怨, horseradish peroxidase媛 寃고빀맂 2李 빆泥대 遺숈씤 떎쓬뿉, iNtRON western blot detection system (Seongnam, Gyeonggi-do, Korea)쓣 궗슜븯뿬 썝븯뒗 떒諛깆쭏쓣 痢≪젙븯떎.

Nitrite assay

RAW264.7 꽭룷濡쒕꽣 nitrite 異붿텧臾쇱쓣 꽑뻾뿰援 諛⑸쾿뿉 뵲씪꽌 以鍮꾪븯떎(Lim et al., 2008). 꽭룷 諛곗뼇 뵆젅씠듃쓽 긽痢듭븸뿉 遺꾩<湲곕 궗슜븯뿬 Griess reagent瑜 泥④븯쑝硫, plate reader瑜 궗슜븯뿬 570 nm뿉꽌 씉愿묐룄瑜 痢≪젙븯떎. 몴以슜븸쑝濡 sodium nitrite瑜 궗슜븯떎.

뜲씠 遺꾩꽍

媛곴컖쓽 뜲씠 媛믪 꽭 媛쒓 븳 踰뚯쓽 떎뿕쑝濡 뼸뼱議뚯쑝硫, mean ± standard error mean (SEM)쑝濡 몴쁽릺뿀떎.

寃곌낵 諛 怨좎같

Cardamonin TLR2 TLR6 삉뒗 TLR4 agonists뿉 쓽빐꽌 쑀룄맂 NF-κB 솢꽦솕瑜 뼲젣븯떎.

TLRs뒗 쟾궗씤옄 NF-κB媛 룷븿맂 downstream 떊샇쟾떖泥닿퀎瑜 솢꽦솕떆궓떎(Akira and Takeda, 2004). 뿬윭 蹂묒썝泥대뱾뿉 쓽빐 솢꽦솕맂 떊샇뱾 NF-κB 뼲젣젣 IκBα瑜 씤궛솕(phosphorylation) 떆궎硫, 씤궛솕맂 IκBα뒗 26S proteasome뿉 쓽빐꽌 遺꾪빐릺뼱 NF-κB瑜 솢꽦솕떆궓떎. 솢꽦솕맂 NF-κB뒗 빑(nucleus) 븞쑝濡 쟾醫(translocation)릺뼱 源 쑀쟾옄쓽 DNA뿉 寃고빀븯뿬 쟾궗(transcription)瑜 쑀룄빐 뿬윭 떒諛깆쭏쓣 留뚮뱾寃 맂떎(Pahl, 1999: Ghosh and Karin, 2002). 슦由щ뒗 NF-κB 諛쒓킅슚냼 쑀쟾옄 遺꾩꽍떎뿕쓣 넻빐 NF-κB 솢꽦뿉 븳 Cardamonin쓽 슚뒫쓣 痢≪젙븯떎. Cardamonin MALP-2 (TLR2 TLR6 agonist) 삉뒗 LPS (TLR4 agonist)뿉 쓽빐 쑀룄맂 NF-κB 솢꽦쓣 뼲젣븯떎(Figs. 2A, 2B).

Fig. 2.

Cardamonin inhibits NF-觀B activation induced by MALP-2 or LPS. (A) RAW 264.7 cells were transfected with NF-κB-luciferase reporter plasmid and pre-treated with cardamonin (30, 50 μM) for 1 h and then treated with MALP-2 (10 ng/mL) (A) or LPS (10 ng/mL) (B) for an additional 8 h. 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 β-galactosidase activity. Values are mean ± SEM (n=3). *, Significantly different from MALP-2 alone, P<0.01 (**) (A). +, Significantly different from LPS alone, P<0.01 (++) (B). Veh, vehicle; CAR, cardamonin.


Cardamonin TLR2 TLR6 삉뒗 TLR4 agonists뿉 쓽빐꽌 쑀룄맂 iNOS쓽 諛쒗쁽쓣 뼲젣븯떎.

떎쓬 떎뿕쑝濡 cardamonin씠 NF-κB 솢꽦솕뿉 쓽빐꽌 쑀룄릺뒗 쑀쟾옄씤 iNOS쓽 깮꽦뿉 뼱뼚븳 쁺뼢쓣 誘몄튂뒗吏 븣븘蹂댁븯떎. 癒쇱 iNOS 諛쒓킅슚냼 쑀쟾옄 遺꾩꽍踰뺤뿉 쓽븯硫 cardamonin MALP-2 삉뒗 LPS뿉 쓽빐 쑀룄맂 iNOS쓽 怨쇰컻쁽쓣 뼲젣떆耳곕떎(Figs. 3A, 3B). Cardamonin MALP-2 삉뒗 LPS뿉 쓽빐 쑀룄맂 iNOS 떒諛깆쭏 怨쇰컻쁽쓣 뼲젣떆궎뒗 寃껋쓣 Western blotting 諛⑸쾿쓣 넻븯뿬 솗씤븯떎(Figs. 3C, 3D). 삉븳 Cardamonin MALP-2 삉뒗 LPS뿉 쓽빐꽌 쑀룄맂 iNOS쓽 깮꽦臾쇱씤 nitrite쓽 깮꽦쓣 뼲젣떆耳곕떎(Figs. 4A, 4B). 씠윭븳 寃곌낵뱾 cardamonin씠 TLR agonists뿉 쓽빐꽌 쑀룄맂 iNOS瑜 議곗젅븷 닔 엳떎뒗 寃껋쓣 蹂댁뿬二쇰뒗 寃곌낵씪 븷 닔 엳寃좊떎.

Fig. 3.

Cardamonin inhibits iNOS expression induced by MALP-2 or LPS. (A, B) RAW 264.7 cells were transfected with iNOS luciferase reporter plasmid and pretreated with 30 or 50 μM cardamonin for 1 h and then treated with MALP-2 (10 ng/mL) (A) or LPS (10 ng/mL) (B) 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 MALP-2 alone, P<0.01 (**) (A). +, Significantly different from LPS alone, P<0.01 (++) (B). (C, D) RAW 264.7 cells were pretreated with 30 or 50 μM cardamonin for 1 h and then further stimulated with MALP-2 (10 ng/mL) (C) or LPS (10 ng/mL) (D) for 8 h. Cell lysates were analyzed for iNOS and β-actin protein by immunoblots. Veh, vehicle; CAR, cardamonin.


Fig. 4.

Cardamonin inhibits nitrite production induced by MALP-2 or LPS. (A, B) RAW 264.7 cells were pretreated with 30 or 50 μM cardamonin for 1 h and then treated with MALP-2 (10 ng/mL) (A) or LPS (10 ng/mL) (B) 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 MALP-2 alone, P<0.01 (A). +, Significantly different from LPS alone, P<0.01 (++) (B). Veh, vehicle; CAR, cardamonin.


怨듦린以묒쓽 媛옣 뭾遺븳 吏덉냼 궛냼 몢 湲곗껜쓽 1:1 議고빀臾쇱씤 NO뒗 1987뀈源뚯 쟾湲고솕븰諛섏쓳쓽 遺궛臾쇰줈 뿬寃⑥졇 솕떎(Hanafy et al., 2001). 븯吏留 吏湲 NO뒗 룞臾 꽭룷뿉꽌 L-arginine 븘誘몃끂궛쑝濡쒕꽣 留뚮뱾뼱吏怨, 씠 NO뒗 꽭룷뿉꽌 떎瑜 꽭룷 궗씠쓽 떊샇쟾떖쓣 쐞빐 궗슜븯뒗 寃껋쑝濡 븣젮졇 엳떎(Hanafy et al., 2001). NO뒗 endothelial NOS (eNOS), neuronal NOS (nNOS) inducible NOS (iNOS)씤 3媛쒖쓽 NOSs뿉 쓽빐꽌 留뚮뱾뼱吏꾨떎(Vallance, 2003). eNOS nNOS뒗 씪젙븯寃 諛쒗쁽릺뼱 엳뒗 슚냼씤뜲 諛섑빐꽌, iNOS뒗 媛먯뿼씠굹 뿼利앹꽦 씤옄(proinflammatory stimuli)뿉 쓽빐꽌 쑀룄릺뒗 슚냼씠떎(Vallance, 2003). 듅엳 NOS isoforms 以묒뿉꽌, iNOS쓽 怨쇰컻쁽씠 룞臾쇱쓽 蹂묓깭깮由ы븰怨 媛옣 愿怨꾧 엳뒗 寃껋쑝濡 븣젮졇 엳떎(Green et al., 1990). 씪諛섏쟻쑝濡 떇 꽭룷뿉꽌 iNOS뒗 諛쒗쁽릺吏 븡吏留, LPS瑜 룷븿븳 떎뼇븳 뿼利 옄洹뱀젣뿉 쓽빐꽌 iNOS 쑀쟾옄媛 솢꽦솕릺뼱, iNOS 떒諛깆쭏씠 留뚮뱾뼱吏꾨떎(Palmer et al., 1993). iNOS뿉 쓽빐꽌 깮꽦릺뒗 遺궛臾쇱씤 NO뒗 peroxynitrite瑜 삎꽦븯湲 쐞븯뿬 O2 寃고빀뻽쓣 븣, 뿬윭 슚냼 떊샇쟾떖泥닿퀎뿉 쁺뼢쓣 誘몄튂뒗 寃껋쑝濡 븣젮졇 엳떎(Vallance, 2003).

TLRs뒗 媛곴컖 꽌濡 떎瑜 옄洹뱀젣瑜 씤떇븯뿬 떊샇쟾떖 떆뒪뀥쓣 쑀룄븯뒗 寃껋쑝濡 븣젮졇 엳떎(Kawai and Akira, 2010). TLRs媛 옄洹뱀젣瑜 씤떇븯硫 理쒖큹 諛섏쓳쑝濡 TLRs媛 룞醫낆씠빀泥대 삎꽦븳떎. TLRs쓽 룞醫낆씠빀泥 삎꽦 뼱뙌꽣 遺꾩옄(adaptor molecule)씤 myeloid differential factor 88 (MyD88) 삉뒗 TIR domain-containing adaptor inducing interferon-β (TRIF)瑜 쑀룄븯硫, 寃곌뎅 쟾궗슂냼 NF-κB굹 interferon regulatory factor 3 (IRF3)쓽 솢꽦솕瑜 쑀룄븳떎(Kawai and Akira, 2010). 쟾궗슂냼쓽 솢꽦솕뒗 寃곌뎅 cytokine, iNOS 媛숈 inflammatory gene products瑜 쑀룄븯뿬 뿬윭 吏덈퀝쓣 쑀룄븯寃 맂떎(Akira and Takeda, 2004). 洹몃윭誘濡 TLRs쓽 떊샇쟾떖 떆뒪뀥쓣 議곗젅븷 닔 엳떎硫 뿬윭 蹂묒썝洹좊뱾濡쒕꽣 쑀룄릺뒗 뿼利 諛섏쓳씠굹 留뚯꽦쟻씤 吏덈퀝뱾쓣 留됯굅굹 移섏쑀븷 닔 엳寃 릺뒗 寃껋씠떎.

슦由щ뒗 씠踰 뿰援ъ뿉꽌 cardamonin씠 TLRs 옄洹뱀젣씤 MALP-2 LPS뿉 쓽빐꽌 쑀룄맂 쟾궗 슂냼 NF-κB 솢꽦솕 諛 洹멸쾬뿉 쓽빐꽌 쑀룄릺뒗 iNOS 떒諛깆쭏 諛쒗쁽뿉 뼱뼡 쁺뼢쓣 誘몄튂뒗吏 븣븘蹂댁븯떎. Cardamonin MALP-2 LPS뿉 쓽빐꽌 쑀룄맂 NF-κB 솢꽦솕 iNOS쓽 怨쇰컻쁽쓣 뼲젣떆耳곕떎. 씠윭븳 寃곌낵뒗 cardamonin씠 TLRs 떊샇쟾떖 떆뒪뀥쓣 議곗젅븯뿬 뿼利 諛섏쓳怨 媛숈 留뚯꽦쟻씤 吏덈퀝뱾쓣 移섏쑀븷 닔 엳떎뒗 寃껋쓣 蹂댁뿬二쇰뒗 以묒슂븳 寃곌낵씪 븷 닔 엳寃좊떎. 씠윭븳 뿰援щ뒗 븵쑝濡 뿼利 옉슜 湲곗쟾 洹쒕챸 諛 빆뿼利앹젣 媛쒕컻뿉 以묒슂븳 뿭븷쓣 븷 寃껋쑝濡 湲곕븳떎.

ACKNOWLEDGEMENTS

蹂 뿰援щ뒗 닚泥쒗뼢븰援 븰닠뿰援щ퉬쓽 씪遺 吏썝怨 2017뀈룄 븳援뿰援ъ옱떒쓽 吏썝쓣 諛쏆븘 닔뻾맂 湲곗큹뿰援ъ궗뾽(2017R1D1A1B03031534)쓽 吏썝쓣 諛쏆븘 닔뻾맂 뿰援 寃곌낵臾쇰줈 洹 吏썝뿉 媛먯궗 뱶由쎈땲떎.

CONFLICT OF INTEREST

The authors have no conflicts of interest to disclose.

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