[1]黎昌江,李秋慧,洪娟,等.黄芪甲苷对蓝光诱导损伤的视网膜色素上皮细胞的保护作用及其机制[J].眼科新进展,2021,41(11):1006-1011.[doi:10.13389/j.cnki.rao.2021.0212]
　LI Changjiang,LI Qiuhui,HONG Juan,et al.Protective effect of Astragaloside IV on the blue light-induced damage of retinal pigment epithelium and its mechanism[J].Recent Advances in Ophthalmology,2021,41(11):1006-1011.[doi:10.13389/j.cnki.rao.2021.0212]
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黄芪甲苷对蓝光诱导损伤的视网膜色素上皮细胞的保护作用及其机制/HTML
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《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
41卷
期数:
2021年11期
页码:
1006-1011
栏目:
实验研究
出版日期:
2021-11-05

文章信息/Info

Title:
Protective effect of Astragaloside IV on the blue light-induced damage of retinal pigment epithelium and its mechanism
作者:
黎昌江; 李秋慧; 洪娟; 陈方安; 郭翠玲
571400　海南省琼海市，琼海市人民医院眼科（黎昌江，李秋慧，洪娟，陈方安）；670102　海南省海口市，海南省中医院眼科（郭翠玲）
Author(s):
LI Changjiang1; LI Qiuhui1; HONG Juan1; CHEN Fangan1; GUO Cuiling2
1.Department of Ophthalmology,Qionghai People’s Hospital,Qionghai 571400,Hainan Province,China
2.Department of Ophthalmology,Hainan Provincial Hospital of Chinese Medicine,Haikou 670102,Hainan Province,China
关键词:
黄芪甲苷; 视网膜色素上皮细胞; 蓝光损伤; 线粒体; 活性氧
Keywords:
Astragaloside IV;  retinal pigment epithelium;  blue light-induced injury;  mitochondria;  reactive oxygen species
分类号:
R774.5
DOI:
10.13389/j.cnki.rao.2021.0212
文献标志码:
A
摘要:
目的　探讨黄芪甲苷（AS-IV）对蓝光诱导损伤的视网膜色素上皮细胞的保护作用及其机制。方法　将传代后的ARPE-19细胞分为5组。空白组：在暗环境中培养细胞，不采用蓝光照射；光照组：采用辐射强度为(4.0±0.5) mW·cm-2的蓝光照射细胞4 h；光照+AS-IV组：采用相同辐射强度的蓝光照射4 h +50 mg·L-1 AS-IV培养细胞24 h；光照+AS-IV+siNC组：将细胞转染对照siRNA后，采用相同辐射强度蓝光照射4 h +50 mg·L-1 AS-IV培养细胞24 h；光照+AS-IV+siPINK1组：将细胞转染siPINK1后，采用相同辐射强度蓝光照射4 h +50 mg·L-1 AS-IV培养细胞24 h。采用CCK-8法检测细胞增殖活性，流式细胞仪检测细胞凋亡情况，JC-1荧光探针检测细胞线粒体膜电位，DHE荧光探针法检测细胞内活性氧（ROS）含量，Western blot检测蛋白表达。结果　当连续蓝光照射4 h以上，ARPE-19细胞存活率均低于80%，故选择照射时间为4 h进行后续实验。光照+AS-IV组细胞存活率明显高于光照组［（93.85±1.79）%、（79.24±3.25）%，t=11.141、P<0.001］。光照+AS-IV组细胞凋亡率明显低于光照组［（11.03±1.65）%、（27.85±3.44）%，t=12.472、P<0.001］。光照组细胞线粒体膜电位高于空白组（绿/红色荧光强度比值分别为1.72±0.25、0.58±0.07，t=12.418、P<0.001），但是光照+AS-IV组细胞线粒体膜电位（绿/红色荧光强度比值为0.81±0.10)则低于光照组（t=9.559、P<0.001）。光照+AS-IV组细胞ROS含量少于光照组（t=6.341、P<0.001）。蓝光照射4 h后，光照+AS-IV组ARPE-19细胞PINK1蛋白（1.18±0.14，t=9.035、P<0.001）和Parkin蛋白（0.77±0.09，t=8.106、P<0.001）相对表达量则明显高于光照组。与光照组相比，光照+AS-IV组ARPE-19细胞的细胞质细胞色素C（Cyt C）蛋白相对表达量降低至0.67±0.08（t=17.059、P<0.001），线粒体Cyt C蛋白相对表达量升高至0.16±0.03（t=5.491、P<0.001），总蛋白中LC3B-II/I蛋白表达比值升高至3.44±0.27（t=34.047、P<0.001）。与光照+AS-IV组相比，光照+AS-IV+siNC组ARPE-19细胞的细胞质和线粒体Cyt C蛋白相对表达量（0.70±0.09、0.15±0.03）、总蛋白中LC3B-II/I蛋白表达比值（3.26±0.33）差异均无统计学意义（均为P>0.05），而光照+AS-IV+siPINK1组ARPE-19细胞的细胞质Cyt C蛋白相对表达量（1.20±0.15）较光照+AS-IV+siNC组升高（t=8.085、P<0.001），线粒体Cyt C蛋白相对表达量降低至0.02±0.01（t=11.628、P<0.001），总蛋白中LC3B-II/I蛋白表达比值则降低至0.85±0.13（t=19.224、P<0.001）。结论　AS-IV对蓝光诱导损伤的视网膜色素上皮细胞具有一定的保护作用，可抑制细胞产生大量ROS，缓解细胞线粒体损伤和细胞凋亡，维持细胞活力和自噬过程，其机制可能与AS-IV上调细胞PINK1/Parkin信号通路有关。
Abstract:
Objective　To investigate the protective effect of Astragaloside IV (AS-IV) on the blue light-induced damage of retinal pigment epithelium (RPE) and its mechanism. Methods　The passaged ARPE-19 cells were divided into five groups: blank group (cells were cultured in the dark environment without irradiation of blue light), light group ［cells were irradiated with blue light of (4.0±0.5) mW·cm-2 for 4 h］, light+AS-IV group (cells were irradiated with blue light of the said intensity for 4 h and cultured with 50 mg·L-1 AS-IV for 24 h), light+AS-IV+siNC group (cells which were transfected with siRNA were irradiated with blue light of the said intensity for 4 h and cultured with 50 mg·L-1 AS-IV for 24 h), and light+AS-IV+siPINK1 group (cells which were transfected with siPINK1 were irradiated with blue light of the said intensity for 4 h and cultured with 50 mg·L-1 AS-IV for 24 h). Cell counting kit-8 and flow cytometry were used to detect cell proliferation and apoptosis. The JC-1 probe was used to detect the mitochondrial membrane potential, and the DHE probe was used to detect the level of reactive oxygen species (ROS). The Western blot was used to detect the expression of proteins. Results　The survival rate of ARPE-19 cells was lower than 80% after continuous blue right irradiation for more than 4 h; therefore, 4-hour irradiation was selected for the follow-up experiments. The cell viability of the light+AS-IV group was significantly higher than that of the light group ［(93.85±1.79)%, (79.24±3.25)%, t=11.141, P<0.001］. The apoptosis rate of the light+AS-IV group was significantly lower than that of the light group ［(11.03±1.65)%, (27.85±3.44)%, t=12.472, P<0.001］. The mitochondrial membrane potential of the light group (1.72±0.25) was higher than that of the blank group (0.58±0.07)(t=12.418, P<0.001), and was also higher than that of the light+AS-IV group (0.81±0.10)(t=9.559, P<0.001). The ROS content of cells in the light+AS-IV group was lower than that in the light group (t=6.341, P<0.001). After 4-hour irradiation by blue light, the relative expression levels of PINK1 protein (1.18±0.14, t=9.035, P<0.001) and Parkin protein (0.77±0.09, t=8.106, P<0.001) in ARPE-19 cells in the light+AS-IV group were significantly higher than those in the light group. Compared with the light group, the relative expression of cytoplasmic Cyt C protein in ARPE-19 cells decreased to 0.67±0.08 (t=17.059, P<0.001), the relative expression of mitochondrial Cyt C protein increased to 0.16±0.03 (t=5.491, P<0.001), and the LC3B-II/I expression in the total protein increased to 3.44±0.27 (t=34.047, P<0.001) in the light+AS-IV group. In the light+AS-IV+siNC group, the relative expression levels of Cyt C protein in ARPE-19 cytoplasm and mitochondria were 0.70±0.09 and 0.15±0.03, respectively, and the expression level of LC3B-II/I protein in the total protein was 3.26±0.33, without statistically significant differences compared with the light+AS-IV group (all P>0.05). Compared with the light+AS-IV+siNC group, the relative expression of cytoplasmic Cyt C protein in ARPE-19 cells in the light+AS-IV+siPINK1 group increased to 1.20±0.15 (t=8.085, P<0.001), while the relative expression of mitochondrial Cyt C protein decreased to 0.02±0.01 (t=11.628, P<0.001), and the expression of LC3B-II/I protein in the total protein decreased to 0.85±0.13 (t=19.224, P<0.001). Conclusion　AS-IV can protect RPE from blue light-induced injury by inhibiting the ROS production, alleviating mitochondrial injury and apoptosis, and maintaining cell vitality and autophagy. The mechanism may be related to the up-regulated PINK1/parkin signaling pathway by AS-IV.

参考文献/References:

［1］　GHEORGHE A,MAHDI L,MUSAT O.Age-related macular degeneration［J］.Rom J Ophthalmol,2015,59(2):74-77.
［2］　LIM L S,MITCHELL P,SEDDON J M,HOLZ F G,WONG T Y.Age-related macular degeneration［J］.Lancet,2012,379(9827):1728-1738.
［3］　赵芳芳,杨冬萍,俞洋.枸杞多糖对兔视网膜色素上皮细胞光损伤凋亡线粒体信号传导途径的影响［J］.湖北中医杂志,2018,40(10):3-8.
ZHAO F F,YANG D P,YU Y.Effect of lycium barbarum polysaccharide on rabbit retinal pigment epithelial cells photodamage apoptosis mitochondrial signal transduction pathway［J］.Hubei J Tradit Chin Med,2018,40(10):3-8.
［4］　ROBERTS R F,TANG M Y,FON E A,DURCAN T M.Defending the mitochondria:the pathways of mitophagy and mitochondrial-derived vesicles［J］.Int J Biochem Cell Biol,2016,79(7):427-436.
［5］　古贤君,林栩,凌霄雁,郑心彤,黄海庭,梁钊,等.miR-155调控线粒体自噬对足细胞损伤的机制研究［J］.右江医学,2020,48(5):326-333.
GU X J,LIN X,LING X Y,ZHENG X T,HUANG H T,LIANG Z,et al.Study on the mechanism of miR-155 regulating mitophagy on podocyte injury［J］.Youjiang Med J,2020,48(5):326-333.
［6］　赵夏,许传文,徐艳梅.黄芪皂苷调节TGF-β1/Smad通路对尿毒症腹膜透析大鼠钙磷代谢的影响［J］.中药药理与临床,2019,35(4):24-28.
ZHAO X,XU C W,XU Y M.Effects of astragaloside on calcium-phosphorus metabolism in rats with uremic peritoneal dialysis by regulating TGF-β1/Smad pathway［J］.Pharmacol Clin Chin Mater Medic,2019,35(4):24-28.
［7］　ZHANG Y,XI X,MEI Y,ZHAO X,ZHOU L,MA M,et al.High-glucose induces retinal pigment epithelium mitochondrial pathways of apoptosis and inhibits mitophagy by regulating ROS/PINK1/Parkin signal pathway［J］.Biomed Pharmacother,2019,111(5):1315-1325.
［8］　AMMAR M J,HSU J,CHIANG A,HO A C,REGILLO C D.Age-related macular degeneration therapy:a review［J］.Curr Opin Ophthalmol,2020,31(3):215-221.
［9］　MEHRZADI S,HEMATI K,REITER R J,HOSSEINZADEH A.Mitochondrial dysfunction in age-related macular degeneration:melatonin as a potential treatment［J］.Expert Opin Ther Targets,2020,24(4):359-378.
［10］　MARIE M,BIGOT K,ANGEBAULT C,BARRAU C,GONDOUIN P,PAGAN D,et al.Light action spectrum on oxidative stress and mitochondrial damage in A2E-loaded retinal pigment epithelium cells［J］.Cell Death Dis,2018,9(3):287.
［11］　陈健明,黄健英,秦璐,黄绮云.黄芪皂苷对不同证型C57BL/6小鼠血清IL-2、IL-4、IgG1、IgG2水平的影响［J］.儿科药学杂志,2018,24(8):1-4.
CHEN J M,HUANG J Y,QIN L,HUANG Q Y.Astragaloside on serum IL-2,IL-4,IgG1 and IgG2 levels in different C57BL/6 mice［J］.J Pediatr Pharm,2018,24(8):1-4.
［12］　兰天野,陈婷,王彦红,刘平,张丽娟.黄芪甲苷对高糖条件下人晶状体上皮细胞氧化应激的保护作用［J］.哈尔滨医科大学学报,2018,49(1):6-10.
LAN T Y,CHEN T,WANG Y H,LIU P,ZHANG L J.The protective effect of astragaloside Ⅳ against oxidative stress induced by high glucose in human lens epithelium cells［J］.J Harbin Med Univ,2018,49(1):6-10.
［13］　周云丰,李琳,葛争艳,周立东,郭宇洁,金龙,等.黄芪甲苷对甲基乙二醛诱导的人视网膜色素上皮细胞损伤的保护作用研究［J］.中国药理学通报,2017,33(7):915-921.
ZHOU Y F,LI L,GE Z Y,ZHOU L D,GUO Y J,JIN L,et al.Protective effect of astragalosides IV on retinal pigmentepithelium injury induced by methylglyoxal［J］.Chin Pharmacol Bull,2017,33(7):915-921.
［14］　洪萌,洪道先,石荣先,魏聪,赵晓丽,李权达.木犀草素调控Nrf2/HO-1通路保护视网膜色素上皮细胞氧化损伤［J］.国际眼科杂志,2021,21(1):21-26.
HONG M,HONG D X,SHI R X,WEI C,ZHAO X L,LI Q D.Protective effect of Luteolin on oxidative damage of retinal pigment epithelium cells by regulating the Nrf2/HO-1 pathway［J］.Int Eye Sci,2021,21(1):21-26.
［15］　NEWSHOLME P,CRUZAT V F,KEANE K N,CARLESSI R,DE BITTENOURT P I.Molecular mechanisms of ROS production and oxidative stress in diabetes［J］.Biochem J,2016,473(24):4527-4550.
［16］　LIU L,LIAO X,WU H,LI Y,ZHU Y,CHEN Q.Mitophagy and its contribution to metabolic and aging-associated disorders［J］.Antioxid Redox Signal,2020,32(12):906-927.
［17］　SUN R,ZHANG A,GE Y,GOU J,YIN T,HE H,et al.Ultra-small-size astragaloside-IV loaded lipid nanocapsules eye drops for the effective management of dry age-related macular degeneration［J］.Expert Opin Drug Deliv,2020,17(9):1305-1320.
［18］　CHEN X,JIANG C,SUN R,YANG D,LIU Q.Circular noncoding RNA NR3C1 acts as a miR-382-5p sponge to protect RPE functions via regulating PTEN/AKT/mTOR signaling pathway［J］.Mol Ther,2020,28(3):929-945.
［19］　靳晓飞,周晓红,武密山,张颖,赵艳萌,李媛,等.黄芪甲苷通过激活自噬抑制缺氧缺糖/复氧复糖诱导的PC12细胞凋亡［J］.北京中医药大学学报,2017,24(5):405-412.
JIN X F,ZHOU X H,WU M S,ZHANG Y,ZHAO Y M,LI Y,et al.Inhibitory effect of astragaloside IV on PC12 apoptosis induced by oxygenglucose deprivation/oxygen-glucose rehabilitation through activating autophagy［J］.J Beijing Univ Tradit Chin Med,2017,24(5):405-412.
［20］　EIYAMA A,OKAMOTO K.PINK1/parkin-mediated mitophagy in mammalian cells［J］.Curr Opin Cell Biol,2015,33(4):95-101.
［21］　TANAKA K.The PINK1-Parkin axis:an overview［J］.Neurosci Res,2020,159(10):9-15.

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备注/Memo

备注/Memo:
海南省卫生健康行业科研项目（编号：20A200367）
更新日期/Last Update: 2021-11-05