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Assessment of the Cardioprotection Offered by Fisetin in H2O2-induced Zebrafish (Danio rerio)-Tg (cmlc2: egfp)
Biomed Sci Letters 2018;24:130-133
Published online June 30, 2018;  https://doi.org/10.15616/BSL.2018.24.2.130
© 2018 The Korean Society For Biomedical Laboratory Sciences.

Jeong-Soo Lee1, Eun-Seok Park2,†, and In-Sik Kim1,3,†

1Department of Senior Healthcare, BK21 Plus Program, Graduate School, Eulji University, Daejeon 34824, Korea,
2Department of Biomedical Laboratory Science, Kyungbok University, Gyeonggi-do 12051, Korea,
3Department of Biomedical Laboratory Science, School of Medicine, Eulji University, Daejeon 34824, Korea
Correspondence to: In-Sik Kim. Department of Biomedical Laboratory Science School of Medicine, Eulji University 77, Gyeryoung-ro 771 beon-gil, Jung-Gu, Daejeon 34824, Korea. Tel: +82-42-259-1753, Fax: +82-42-259-1759, e-mail: orientree@eulji.ac.kr
Eun-Seok Park. Department of Biomedical Laboratory Science, Kyungbok University, 425, Kyeongbokdae-ro, Jinjeop-eup, Namyangju-si, Gyeonggi-do 12051, Korea. Tel: +82-31-539-5430, Fax: +82-31-570-9929, e-mail: eunspark@kbu.ac.kr
Received December 21, 2017; Revised May 29, 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

The aim of this study was to evaluate the protective function of fisetin, a natural flavonoid in zebrafish heart for the treatment of myocardial infarction in coronary and ischemic heart disease. For this purpose, we induced oxidative stress zebrafish (Danio rerio)-Tg (cmlc2: egfp) by H2O2 and then administered fisetin, the protective effect of fisetin was determined by measuring the heart rate following fisetin administration. After testing the toxicity of fisetin, we found that the heartt increased in a concentration-dependent manner, however there was no difference between the heart rates of embryos and adults. The improved heart rate demonstrated the cardioprotective effect of fisetin. The result showed that fisetin, at concentration of 3and 5 μM, significantly increased heart rate compared with the heart with H2O2 alone. This indicates that fisetin plays an important role in the prevention of heart damage and treatment of cardiovascular diseases caused by oxidative stress due to ischemia / reperfusion.

Keywords : Fisetin, Zebrafish (Danio rerio)-Tg (cmlc2: egfp), Cardiovascular disease, H2O2
Body

떖삁愿怨꾩쭏솚 쟾꽭怨꾩쟻쑝濡 궗留앸쪧 1쐞瑜 李⑥븯怨 엳뒗 吏덊솚씠떎(Bots et al., 2017). 떖삁愿怨꾩쭏솚뿉뒗 떖洹쇨꼍깋, 怨좏삁븬, 遺젙留, 룞留κ꼍솕뿉 쓽븳 뿀삁꽦 떖옣吏덊솚 벑씠 二쇱슂 떖옣吏덊솚씠硫, 뇤議몄쨷怨 留먯큹삁愿吏덊솚 벑씠 삁愿吏덊솚씠떎(Kuller et al., 2017). 떖옣뿉 궛냼 쁺뼇쓣 怨듦툒븯뒗 愿긽룞留μ씠 醫곸븘吏嫄곕굹 留됲 諛쒖깮븯뒗 떖洹쇨꼍깋怨 삊떖利앹쓣 쑀諛쒗븯怨, 쐞뿕 슂씤뿉뒗 怨좏삁븬, 떦눊, 씉뿰 洹몃━怨 怨좎삁利, 鍮꾨쭔, 슫룞遺議, 뒪듃젅뒪, 뿬꽦쓽 룓寃, 洹몃━怨 쑀쟾쟻 슂씤 벑씠 떖洹 꽭룷쓽 꽭룷 궗硫몄뿉 以묒슂븳 뿭븷쓣 愿뿬븳떎뒗 寃껋쓣 蹂닿퀬븯떎(Wei et al., 2017). 떖洹 꽭룷 궗硫 利앷뒗 떖洹 뿀삁/옱愿瑜(I/R)濡 꽭룷쓽 뼇쓽 媛먯냼맖뿉 뵲씪 떖옣 湲곕뒫쓣 媛먯냼떆궎怨 떖遺쟾利앹쓣 珥됱쭊떆궓떎(DeBerge et al., 2017). 뵲씪꽌 떖옣 삁愿怨 떖옣洹쇱쑁꽭룷 궗硫몄뿉 珥덉젏쓣 留욎텣 뿰援щ뒗 떖삁愿吏덊솚 삁諛⑹뿉꽌 留ㅼ슦 以묒슂븳 遺遺꾩씠떎(Sara et al., 2017). 理쒓렐 紐 뀈媛꾩쓽 뿰援ъ뿉 뵲瑜대㈃ 뿀삁 / 옱愿瑜 넀긽쓣 룷븿븳 留롮 슂씤뱾씠 떖洹 꽭룷뿉꽌 꽭룷 궗硫몄쓣 씪쑝궗 닔 엳쓬씠 諛앺議뚮떎(Andrienko et al., 2017). 뵲씪꽌 留롮 뿰援щ 넻빐 궛솕 뒪듃젅뒪 媛숈 꽭룷 룆꽦 옄洹뱀쑝濡쒕꽣 떖洹 꽭룷瑜 蹂댄샇븯뒗 諛⑸쾿뿉 以묒젏쓣 몢뿀떎(Park et al., 2017). I / R 넀긽 吏곸젒쟻쑝濡 삉뒗 媛꾩젒쟻쑝濡 利앷맂 꽭룷吏 移쇱뒛 냽룄瑜 쑀룄븯뒗 궛솕 뒪듃젅뒪濡 씤븳 誘명넗肄섎뱶由ъ븘 湲곕뒫 옣븷濡 굹궃떎(Swerdlow, 2017). Myocardial I / R 넀긽쑝濡 씤븳 誘명넗肄섎뱶由ъ븘 湲곕뒫 옣븷뒗 mitochondrial permeability transition pore쓽 媛쒕갑쓣 쑀諛쒗븯뿬 諛섏쓳꽦 궛냼 醫(reactive oxygen species, ROS) 쑀諛 꽭룷 궗硫몄쓽 以묐븳 媛쒖떆 떒怨꾩씤 떆넗겕濡 c쓽 꽭룷吏덈줈쓽 諛⑹텧쓣 珥덈옒븳떎(Yang et al., 2017). 怨쇱궛솕닔냼 H2O2, superoxide radical, hydroxyl radical, peroxynitrite瑜 룷븿븯뒗 ROS뒗 利앷븯怨 떖洹 I / R 넀긽쓽 蹂묒씤뿉 愿뿬븳떎怨 蹂몃떎(Khaper et al., 2017).

Fisetin (tetrahydroxyflavone) 빆궛솕 옉슜쓣 븯뒗 뵆씪蹂대끂씠뱶怨꾨줈꽌 옄쑀 씪뵒移쇱쓣 젣嫄고븷 닔 엳怨, 꽭룷 궡뿉꽌 냼닔꽦 솕빀臾쇰줈꽌 꽭룷留됱뿉 遺숈뼱 빆궛솕 슚怨쇰 諛쒗쐶븳떎(Das et al., 2017). Fisetin 뼇諛곗텛, 뼇뙆, 궗怨, 벑뿉 룷븿릺뼱 엳怨, 빆븫, 빆 삁愿 떊깮 諛 떊寃 蹂댄샇 슚怨쇨 엳뒗 寃껋쑝濡 엯利앸릺뿀떎(Chirumbolo, 2014). Fisetin씠 H2O2뿉 쓽빐 쑀룄맂 궛솕 뒪듃젅뒪 꽭룷 넀긽쑝濡 遺꽣 떖洹 꽭룷瑜 蹂댄샇븯뒗 寃껋쑝濡 蹂닿퀬릺뿀떎(Bai et al., 2016). 뵲씪꽌 떖옣洹쇱쑁 젅젣 썑 옱깮쓣 븯뒗 zebrafish (Danio rerio)-Tg (cmlc2: egfp)濡 H2O2 쑀諛 궛솕 넀긽뿉 븳 媛뺣젰븳 떖옣諛뺣룞쓣 넻븯뿬 떖옣 蹂댄샇 슚怨쇰 諛쒗쐶븳떎뒗 寃껋쓣 諛쒓껄븯떎.

寃쎈턿븰援 젣釉뚮씪뵾돩 뻾쑝濡쒕꽣 젣釉뚮씪뵾돩(Danio rerio) 뻾벑濡앸쾲샇 ZOMB 0006 씪諛섎챸 Tg [cmlc2: egfp], 냼옱遺꾨쪟룞臾쇱 泥숈텛룞臾, 냼옱醫낅쪟뒗 媛쒖껜, 냽紐, Danio, 醫낅챸 rerio, 솗蹂댁씪 1996-03-01, Line Type transgenic, Phenotype, 떖옣(myocardium), Target Gene cardiac myosin light chain쓣 궛 48떆媛 맂 寃껋쓣 怨듦툒諛쏆븯떎.

궛 48떆媛 맂 zebrafish (Danio rerio)-Tg (cmlc2: egfp) embryo瑜 6 well plate뿉 媛 well뿉 10~20媛쒕줈 꽔怨, fisetin (0, 1, 3, 5 μM)瑜 遺꾩< 썑 28.5°C꽌 24떆媛 씤걧踰좎씠뀡 븯뿬 궛 72떆媛 썑 쁽誘멸꼍쑝濡 愿李고븯떎(100X, 400X (Fig. 1). 30珥 룞븞 떖옣諛뺣룞닔媛 0 μM뒗 28±2.4, 1 μM뒗 25±1.5, 3 μM뒗 28±0.3, 5 μM뒗 29±0.3쑝濡 fisetin뿉 븳 룆꽦씠 뾾뒗 寃껋쑝濡 굹궗떎(Fig. 1).

Zebrafish (Danio rerio)-Tg (cmlc2: egfp) 諛곗 E3 embryonic stuck solution 60X濡 씗꽍븯뿬 궛 썑 48떆媛 吏궃 zebrafish (Danio rerio)-Tg [cmlc2: egfp]瑜 28.5°C뿉꽌 24떆媛 씤걧踰좎씠뀡 썑 媛쒖껜濡 遺솕떆耳 10씪媛 湲몃떎.

Fig. 1.

Fisetin is not toxic to zebrafish (Danio rerio)-Tg (cmlc2: egfp). After scattering for 48 hours, 10~20 zebrafish (Danio rerio)- Tg (cmlc2: egfp) embryos were placed in each well of a six well plate, Different concentrations of fisetin (0, 1, 3, 5 μM) were dispensed into each well and the embryos were incubated at 28.5°C. for 24 hours. After 72 hours of scattering, the cells were observed under a microscope (100-×, 400-×). The heart rate/30 sec was 28 ± 2.4 for 0 μM of fisetin, 25 ± 1.5 for 1 μM of fisetin, 28 ± 0.3 for 3 μM of fisetin, and 29 ± 0.3 for 5 μM of fisetin, showing that fisetin was toxic to the embryos.


10씪 옄 zebrafish (Danio rerio)-Tg (cmlc2: egfp) 媛쒖껜瑜 6 well plate뿉 媛곴컖쓽 well뿉 3~5留덈━ 젙룄 遺꾪븷븯뿬 꽔怨, well留덈떎 2.5 mL瑜 湲곗쑝濡 fisetin (0, 1, 3, 5 μM)瑜 泥④ 썑 28.5°C 씤걧踰좎씠꽣뿉꽌 24떆媛 썑 利, 궛 11씪吏 쁽誘멸꼍 A12.0706 Nikon E200 100X, 400X濡 떖옣諛뺣룞쓣 痢≪젙쓣 븯떎. 옄猷뚯쓽 넻怨꾨텇꽍 Windows SPSS Program (ver. 20, Chicago, IL, USA)쓣 씠슜븯뿬 쑀쓽 李⑤ compare means ones-sample test濡 寃利앺븳 썑 P < 0.05 닔以뿉꽌 95% 떊猶곌뎄媛꾩쑝濡 몴떆븯떎.

궛 48떆媛 zebrafish (Danio rerio)-Tg (cmlc2: egfp)쓽 embryo瑜 6 well plate뿉 媛곴컖쓽 well留덈떎 10~20媛쒖뵫 꽔怨, 媛곴컖쓽 well뿉 2.5 mL瑜 湲곗쑝濡 600 μM H2O2瑜 遺꾩<瑜 븯怨 1떆媛 썑 fisetin (0, 1, 3, 5 μM)瑜 遺꾩<븯떎. 諛곗븘瑜 28.5°C뿉꽌 24떆媛 씤걧踰좎씠꽣뿉꽌 諛곗뼇 썑 利 궛 썑 72떆媛 吏궃 寃껋쓣 쁽誘멸꼍쑝濡 30珥덇컙 떖옣諛뺣룞쓣 痢≪젙쓣 븳 寃곌낵 fisetin쓣 泥섎━븯吏 븡 0 μM 紐⑤몢 궗硫명븯뿬 諛곗븘媛 源⑥졇 엳뿀떎. 1 μM뒗 8.0±5.0 (P < 0.005) 떖옣씠 媛꾪뿉쟻쑝濡 쎇뿀怨, 쟾泥댁쟻쑝濡 삁瑜섎굹 泥댁븸쓽 쓲由꾨룄 嫄곗쓽 蹂댁씠吏 븡븯쑝硫, 諛곗븘 궡뿉꽌 紐몄껜媛 떖엳 뮘由щʼn 슂룞쓣 移섎뒗 寃껋쓣 솗씤븯떎. 3 μM 25±7.6% (P < 0.001), 5 μM 29±5.0% (P < 0.000)濡 fisetin쓽 냽룄 쓽議댁쟻쑝濡 떖옣諛뺣룞닔媛 뒛뼱 굹뒗 寃껋쓣 솗씤븯떎(Fig. 2).

Fig. 2.

Fisetin protects the heart against damage induced by H2O2 in zebrafish (Danio rerio)-Tg (cmlc2: egfp) embryo. 10- zebrafish (Danio rerio)-Tg (cmlc2: egfp) embryos were placed in each well of a six-well plate for 48 hrs. 600 μM H2O2 was then dispensed into each well and after one hour, the heart rate/30 sec was measured with a microscope at 100× and 400× after incubation in an incubator at 28.5°C for 72 hours. All the measurements were completed, and the embryos not treated with fisetin (0 μM) were found to be killed and broken. The heart rate/30 sec was 8.0 ± 5.0 (P < 0.005) for 1 μM of fisetin: the heart was found to beat intermittently, Overall, there was almost no flow of blood or body fluid, and the body was severely twisted in the embryo. The heart rate/30 sec was 25 ± 7.6% (P < 0.001) for 3 μM of fisetin and 29 ± 5.0% (P < 0.000) for 5 μM of fisetin, indicating that fisetin increased the heart rate in a dose- dependent manner.


遺솕릺뼱 10씪 옄 zebrafish (Danio rerio)-Tg (cmlc2: egfp) 媛쒖껜瑜 6 well plate쓽 媛곴컖 well留덈떎 3~5留덈━ 젙룄 遺꾪븷븯뿬 꽔怨, 媛곴컖쓽 well뿉 2.5 mL瑜 湲곗쑝濡 600 μM H2O2瑜 遺꾩<瑜 븳 썑 1떆媛 썑뿉 fisetin (0, 1, 3, 5 μM)瑜 泥④븯뿬 遺꾩<븯떎. 洹몃━怨 28.5°C 씤걧踰좎씠꽣뿉꽌 24떆媛 썑 利 궛 11씪吏 쁽誘멸꼍쑝濡 30珥덇컙 떖옣諛뺣룞쓣 痢≪젙쓣 븳 寃곌낵 control 27±5.0% (P < 0.000), 0 μM 8.0±5.0 (P < 0.005)濡 zebrafish (Danio rerio)-Tg (cmlc2: egfp) 媛쒖껜쓽 떖옣씠 遺덇퇋移숉븯怨 븘媛誘몃 떖븯寃 踰뚮━硫 옒뱾寃 샇씉쓣 뻽쑝硫, 1 μM뒗 14.3±7.6 (P < 0.004)濡 떖옣諛뺣룞씠 媛꾪뿉쟻쑝濡 遺덇퇋移숉븯寃 쎇뿀떎. 3 μM뒗 27±5.0% (P < 0.000), 5 μM 29±4.0% (P < 0.000)쑝濡 fisetin쓽 냽룄 쓽議댁쟻쑝濡 떖옣諛뺣룞 닔媛 뒛뼱 궃 寃껋쓣 솗씤븯떎(Fig. 3).

Fig. 3.

Fisetin protects the heart against damage induced by H2O2 in zebrafish (Danio rerio)-Tg (cmlc2: egfp) that was maintained for 10 days. 3~5 zebrafish (Danio rerio)-Tg (cmlc2: egfp) embryos grown for 10 days were placed in each well of a six-well plate, 600 μM H2O2 was than dispensed into each well, and after one hour, different concentrations of fisetin (0, 1, 3, 5 μM) were added to each well. After incubation for 24 hours at 28.5°C in an incubator, that is, on day 11 of scattering, the heart rate/30 sec was measured for 30 seconds by a microscope at 100× and 400×. The results showed that the heart of the zebrafish (Danio rerio)-Tg (cmlc2: egfp) embryos was irregular, and the gills were severely dilated; heavy breathing was also observed. The heart rate/30 sec was 27 ± 5.0% (P < 0.000), in case of the control and 8.0 ± 5.0 for 0 μM of fisetin, and 14.3 ± 7.6 (P < 0.004) for 1 μM of fisetin; andintermittent jerking of the heart beat was observed. The heart rate/30 sec was 27 ± 5.0% (P < 0.000) for 3 μM of fisetin and 29 ± 4.0% for 5 μM of fisetin, confirming that the heart rate was increased by fisetin in a concentration dependent manner (P < 0.000).


醫낇빀쟻쑝濡 蹂 븣 fisetin (3, 7, 3’, 4’-tetrahydroxyflavone) (Alireza et al., 2017) 벑쓽 뿰援ъ뿉 빆궛솕 슚怨쇨 엳쓬씠 蹂닿퀬媛 릺뼱議뚭퀬(Sona et al., 2016) 벑뿉 뵲瑜대㈃ 븣젅瑜닿린꽦 湲곌吏솗옣젣濡쒕룄 슚怨쇨 엳쓬씠 蹂닿퀬릺뿀떎. 씠윭븳 寃곌낵뒗 fisetin쓽 옞옱쟻씤 떖옣 蹂댄샇 슚怨쇨 zebrafish (Danio rerio)-Tg (cmlc2: egfp)쓽 諛곗븘 媛쒖껜 紐⑤몢 떖옣諛뺣룞닔瑜 냽룄 쓽議댁쟻쑝濡 利앷瑜 떆궓 寃껋쓣 蹂 븣 뿀삁/옱愿瑜섏뿉 쓽븳 떖옣 넀긽怨 궛솕쟻 뒪듃젅뒪瑜 넻빐 쑀룄맂 떖옣삁愿吏덊솚쓽 삁諛 諛 移섎즺뿉 媛뺥븳 슚怨쇨 엳쓬쓣 떆궗븳떎.

ACKNOWLEDGEMENTS

None.

CONFLICT OF INTEREST

The authors have no conflicts of interest to disclose.

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