[1]杨倩,薛瑢,董怡辰,等.纳豆激酶对高糖缺氧诱导的视网膜色素上皮细胞损伤的保护作用[J].眼科新进展,2022,42(10):763-768.[doi:10.13389/j.cnki.rao.2022.0157]
　YANG Qian,XUE Rong,DONG Yichen,et al.Protective effect of nattokinase on retinal pigment epithelial cell injury induced by high glucose or hypoxia[J].Recent Advances in Ophthalmology,2022,42(10):763-768.[doi:10.13389/j.cnki.rao.2022.0157]
点击复制

纳豆激酶对高糖缺氧诱导的视网膜色素上皮细胞损伤的保护作用/HTML
分享到：

《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
42卷
期数:
2022年10期
页码:
763-768
栏目:
实验研究
出版日期:
2022-10-05

文章信息/Info

Title:
Protective effect of nattokinase on retinal pigment epithelial cell injury induced by high glucose or hypoxia
作者:
杨倩; 薛瑢; 董怡辰; 范夏莲; 余川; 万光明
450052　河南省郑州市，郑州大学第一附属医院眼科
Author(s):
YANG Qian; XUE Rong; DONG Yichen; FAN Xialian; YU Chuan; WAN Guangming
Department of Ophthalmology,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,Henan Province,China
关键词:
纳豆激酶; 人视网膜色素上皮细胞; 高糖; 缺氧; 血管内皮生长因子A
Keywords:
nattokinase;  human retinal pigment epithelial cells;  high glucose;  hypoxia;  vascular endothelial growth factor A
分类号:
R774.1
DOI:
10.13389/j.cnki.rao.2022.0157
文献标志码:
A
摘要:
目的　探讨纳豆激酶（NK）对高糖缺氧导致的人视网膜色素上皮细胞（ARPE-19）损伤的保护作用。方法　不同浓度的葡萄糖和氯化钴（CoCl2）诱导培养ARPE-19细胞24 h后，观察细胞形态变化，采用CCK-8法检测ARPE-19细胞活性。Western blot和RT-qPCR检测紧密连接蛋白-1（ZO-1）、缺氧诱导因子-1α（HIF-1α）、血管内皮生长因子A（VEGFA）、白细胞介素（IL）-1β、IL-18的表达情况，筛选最佳葡萄糖和CoCl2浓度构建体外高糖缺氧的细胞模型。CCK-8法筛选合适的NK浓度,将ARPE-19细胞分为正常糖组（5.5 mmol·L-1葡萄糖,NC组）、高糖+ CoCl2组（25.0 mmol·L-1葡萄糖+200 μmol·L-1 CoCl2，HG+CoCl2组）和NK处理组（1.0 μmol·L-1 NK+25.0 mmol·L-1葡萄糖+200 μmol·L-1 CoCl2，NK+HG+CoCl2组），采用Western blot和RT-qPCR检测各组ARPE-19细胞中ZO-1、HIF-1α及VEGFA的蛋白和mRNA表达水平。结果　CCK-8法检测结果显示，与NC组比较，CoCl2浓度高于200 μmol·L-1的干预组中ARPE-19细胞的活性均明显降低（均为P<0.01），且细胞形态发生明显改变。Western blot和RT-qPCR检测结果显示，与NC组比较，200 μmol·L-1CoCl2的干预组ARPE-19细胞中HIF-1α和VEGFA的蛋白和mRNA与IL-1β和IL-18的mRNA的表达水平均明显增加（均为P<0.01），而ZO-1蛋白的表达水平均明显降低（均为P<0.05）。CCK-8法检测结果显示，当NK 浓度高于1.0 μmol·L-1 时， ARPE-19细胞的活性较NC组均明显下降（均为P<0.01）。与HG+CoCl2组比较，NK+HG+CoCl2组ARPE-19细胞中HIF-1α、VEGFA蛋白和mRNA的表达水平均显著降低（均为P<0.001），而ZO-1蛋白的表达水平明显上升（P<0.05）。结论　NK可以降低高糖缺氧损伤的ARPE-19细胞中HIF-1α及VEGFA的蛋白和mRNA表达水平，对高糖缺氧引起的细胞损伤具有保护作用。
Abstract:
Objective　To investigate the protective effect of nattokinase (NK) on human retinal pigment epithelial cell (ARPE-19) injury induced by high glucose or hypoxia. Methods　ARPE-19 cells were cultured with different concentrations of glucose and cobalt chloride (CoCl2) for 24 hours, then cell morphological changes were observed and cell viability was detected by CCK-8. The expression levels of zonula occludens-1 (ZO-1), hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor A (VEGFA), interleukin (IL)-1β, and IL-18 were measured by Western blot and real-time quantitative polymerase chain reaction (RT-qPCR), respectively, and the optimal glucose and CoCl2 concentrations were screened to construct the high glucose or hypoxia induced cell models in vitro. The appropriate NK concentration was determined by CCK-8. ARPE-19 cells were divided into the normal control group (5.5 mmol·L-1 glucose, NC group), high glucose and CoCl2 group (25.0 mmol·L-1 glucose + 200 μmol·L-1 CoCl2, HG+CoCl2 group), and NK treatment group (1.0 μmol·L-1 NK + 25.0 mmol·L-1 glucose + 200 μmol·L-1 CoCl2, NK+HG+CoCl2 group). The protein and mRNA expression levels of ZO-1, HIF-1α and VEGFA in these groups were detected by Western blot and RT-qPCR. Results　CCK-8 assay results showed that compared with the NC group, the viability of ARPE-19 cells in the intervention groups with CoCl2 concentration higher than 200 μmol·L-1 was significantly decreased (both P<0.01), and the cell morphology was significantly changed. Western blot and RT-qPCR results showed that compared with the NC group, the protein and mRNA expression levels of HIF-1α and VEGFA, as well as the mRNA expression levels of IL-1β and IL-18 in the intervention groups (200 μmol·L-1 CoCl2) were significantly increased (all P<0.01), while the protein expression level of ZO-1 was significantly decreased (all P<0.05). CCK-8 assay results showed that when NK concentration was higher than 1.0 μmol·L-1, the viability of ARPE-19 cells was significantly decreased compared with the NC group (all P<0.01). Compared with the HG+CoCl2 group, the protein and mRNA expression levels of HIF-1α and VEGFA in the NK+HG+CoCl2 group were significantly decreased (all P<0.001), while the protein expression level of ZO-1 was significantly increased (P<0.05). Conclusion　NK can reduce the protein and mRNA expression levels of HIF-1α and VEGFA in ARPE-19 cells damaged by high glucose or hypoxia, thus protecting these cells.

参考文献/References:

［1］　DONG L,ZHANG Z,LIU X,WANG Q,HONG Y,LI X,et al.RNA sequencing reveals BMP4 as a basis for the dual-target treatment of diabetic retinopathy［J］.J Mol Med (Berl),2021,99(2):225-240.
［2］　WU M Y,YIANG G T,LAI T T,LI C J.The oxidative stress and mitochondrial dysfunction during the pathogenesis of diabetic retinopathy［J］.Oxid Med Cell Longev,2018,2018:3420187.
［3］　KIM J,KIM C S,SOHN E,KIM J S.Cytoplasmic translocation of high-mobility group box-1 protein is induced by diabetes and high glucose in retinal pericytes［J］.Mol Med Rep,2016,14(4):3655-3661.
［4］　CHEN J F,LUO Q H,HUANG C,LIU W T,ZENG W,GAO Q,et al.Expression of VEGF and PEDF in early-stage retinopathy in diabetic Macaca mulatta［J］.J Southern Med Univ,2017,37(9):1217-1221.
［5］　AL-SHABRAWEY M,MUSSELL R,KAHOOK K,TAWFIK A,ELADL M,SARTHY V,et al.Increased expression and activity of 12-lipoxygenase in oxygen-induced ischemic retinopathy and proliferative diabetic retinopathy:implications in retinal neovascularization［J］.Diabetes,2011,60(2):614-624.
［6］　FOREMAN J,DEMIDCHIK V,BOTHWELL J H,MYLONA P,MIEDEMA H,TORRES M A,et al.Reactive oxygen species produced by NADPH oxidase regulate plant cell growth［J］.Nature,2003,422(6930):442-446.
［7］　XIAO Q,ZENG S,LING S,LV M.Up-regulation of HIF-1alpha and VEGF expression by elevated glucose concentration and hypoxia in cultured human retinal pigment epithelial cells［J］.J Huazhong Univ Sci Tech Med Sci,2006,26(4):463-465.
［8］　URANO T,IHARA H,UMEMURA K,SUZUKI Y,OIKE M,AKITA S,et al.The profibrinolytic enzyme subtilisin NAT purified fromBacillus subtilis cleaves and inactivates plasminogen activator inhibitor type 1［J］.J Biol Chem,2001,276(27):24690-24696.
［9］　WU H,WANG Y,ZHANG Y,XU F,CHEN J,DUAN L,et al.Breaking the vicious loop between inflammation,oxidative stress and coagulation,a novel anti-thrombus insight of nattokinase by inhibiting LPS-induced inflammation and oxidative stress［J］.Redox Biol,2020,32:101500.
［10］　HUANG Z,NG T K,CHEN W,SUN X,HUANG D,ZHENG D,et al.Nattokinase attenuates retinal neovascularization via modulation of Nrf2/HO-1 and glial activation［J］.Invest Ophthalmol Vis Sci,2021,62(6):25.
［11］　VUJOSEVIC S,ALDINGTON S J,SILVA P,HERNNDEZ C,SCANLON P,PETO T,et al.Screening for diabetic retinopathy:new perspectives and challenges［J］.Lancet Diabetes Endocrinol,2020,8(4):337-347.
［12］　CHEN Q,TANG L,XIN G,LI S,MA L,XU Y,et al.Oxidative stress mediated by lipid metabolism contributes to high glucose-induced senescence in retinal pigment epithelium［J］.Free Radic Biol Med,2019,130:48-58.
［13］　SPARROW J R,HICKS D,HAMEL C P.The retinal pigment epithelium in health and disease［J］.Curr Mol Med,2010,10(9):802-823.
［14］　LI J,ZHANG R,WANG C,WANG X,XU M,MA J,et al.Activation of the small GTPase RAP1 inhibits choroidal neovascularization by regulating cell junctions and ROS generation in rats［J］.Curr Eye Res,2018,43(7):934-940.
［15］　ZHANG C,XIE H,YANG Q,YANG Y,LI W,TIAN H,et al.Erythropoietin protects outer blood-retinal barrier in experimental diabetic retinopathy by up-regulating ZO-1 and occludin［J］.Clin Exp Ophthalmol,2019,47(9):1182-1197.
［16］　WANG Y,TANG Z,XUE R,SINGH G K,LIU W,LV Y,et al.Differential response to CoCl2-stimulated hypoxia on HIF-1α,VEGF,and MMP-2 expression in ligament cells［J］.Mol Cell Biochem,2012,360(1/2):235-242.
［17］　BAHRAMI B,SHEN W,ZHU L,ZHANG T,CHANG A,GILLIES M C.Effects of VEGF inhibitors on human retinal pigment epithelium under high glucose and hypoxia［J］.Clin Exp Ophthalmol,2019,47(8):1074-1081.
［18］　REN N N,CHEN H J,LI Y,MCGOWAN G W,LIN Y G.A clinical study on the effect of nattokinase on carotid artery atherosclerosis and hyperlipidaemia［J］.Natl Med J Chin,2017,97(26):2038-2042.
［19］　FUJITA M,OHNISHI K,TAKAOKA S,OGASAWARA K,FUKUYAMA R,NAKAMUTA H.Antihypertensive effects of continuous oral administration of nattokinase and its fragments in spontaneously hypertensive rats［J］.Biol Pharm Bull,2011,34(11):1696-1701.
［20］　JY J,KIM T S,CAI J,KIM J,KIM Y,SHIN K,et al.Nattokinase improves blood flow by inhibiting platelet aggregation and thrombus formation［J］.Lab Anim Res,2013,29(4):221-225.
［21］　FADL N N,AHMED H H,BOOLES H F,SAYED A H.Serrapeptase and nattokinase intervention for relieving Alzheimer’s disease pathophysiology in rat model［J］.Hum Exp Toxicol,2013,32(7):721-735.
［22］　HSU R L,LEE K T,WANG J H,LEE L Y,CHEN R P.Amyloid-degrading ability of nattokinase from Bacillus subtilis natto［J］.J Agric Food Chem,2009,57(2):503-508.

相似文献/References:

[1]司艳芳 王君 关娟 韩泉洪 惠延年.α-平滑肌肌动蛋白在PVR增生膜中的表达以及PDGF对其在人RPE细胞中表达的影响[J].眼科新进展,2012,32(11):000.
[2]李姣 王兴荣 崔彦 刘正峰 王影 李洋.雷珠单抗选择性体外抑制睫状体恶性黑色素瘤[J].眼科新进展,2013,33(11):000.
[3]俞永珍,徐哲,邹秀兰,等. 蓝光诱导氧化应激反应参与视网膜色素上皮细胞凋亡机制研究[J].眼科新进展,2015,35(6):520.[doi:10.13389/j.cnki.rao.2015.0141]
　YU Yong-Zhen,XU Zhe,ZOU Xiu-Lan,et al. Role of oxidative stress induced by blue light in human retinal pigment epithelium cells apoptosis[J].Recent Advances in Ophthalmology,2015,35(10):520.[doi:10.13389/j.cnki.rao.2015.0141]
[4]彭素芬,韩泉洪,李丽华,等.LED光源对人视网膜色素上皮细胞分泌单核细胞趋化因子-1和白细胞介素-8的影响[J].眼科新进展,2016,36(3):201.[doi:10.13389/j.cnki.rao.2016.0055]
　PENG Su-Fen,HAN Quan-Hong,LI Li-Hua,et al.Effects of light emitting diode on expression of MCP-1 and IL-8 in cultured human retinal pigment epithelial cells[J].Recent Advances in Ophthalmology,2016,36(10):201.[doi:10.13389/j.cnki.rao.2016.0055]
[5]张照华,杨玲玲,张盼盼,等.贝伐单抗对视网膜色素上皮细胞抗氧化功能的影响[J].眼科新进展,2017,37(4):305.[doi:10.13389/j.cnki.rao.2017.0077]
　ZHANG Zhao-Hua,YANG Ling-Ling,ZHANG Pan-Pan,et al.Effects of bevacizumab on antioxidative function in human retinal pigment epithelial cells[J].Recent Advances in Ophthalmology,2017,37(10):305.[doi:10.13389/j.cnki.rao.2017.0077]
[6]赵洁文,李明新.康柏西普联合PDGF受体抑制剂对缺氧条件下视网膜色素上皮细胞增殖、迁移及VEGF表达的影响[J].眼科新进展,2017,37(12):1127.[doi:10.13389/j.cnki.rao.2017.0284]
　ZHAO Jie-Wen,LI Ming-Xin.Effects of conbercept combined with PDGF receptor inhibitor on the proliferation，migration of RPE cells and VEGF expression in hypoxic conditions[J].Recent Advances in Ophthalmology,2017,37(10):1127.[doi:10.13389/j.cnki.rao.2017.0284]
[7]张盼盼,董琪,华英彬,等.贝伐单抗对人视网膜色素上皮细胞形态、凋亡率及凋亡相关因子表达的影响[J].眼科新进展,2018,38(4):314.[doi:10.13389/j.cnki.rao.2018.0074]
　ZHANG Pan-Pan,DONG Qi,HUA Ying-Bin,et al.Effects of bevacizumab on morphology and apoptosis of human retinal pigment epithelial cells as well as the expression of apoptosis-related factors[J].Recent Advances in Ophthalmology,2018,38(10):314.[doi:10.13389/j.cnki.rao.2018.0074]
[8]邱梅园,丁芝祥,廖妙云,等.表没食子儿茶素没食子酸酯对视网膜色素上皮细胞增殖及相关因子表达的影响[J].眼科新进展,2018,38(7):638.[doi:10.13389/j.cnki.rao.2018.0150]
　QIU Mei-Yuan,DING Zhi-Xiang,LIAO Miao-Yun,et al.Effects of epigallocatechin gallate on proliferation of retinal pigment epithelium cells and the expression of P21，P27，PDGFR，NF-κB and IκB[J].Recent Advances in Ophthalmology,2018,38(10):638.[doi:10.13389/j.cnki.rao.2018.0150]
[9]孙凯,刘丽娟.雷公藤甲素对人视网膜色素上皮细胞氧化损伤的抑制作用[J].眼科新进展,2018,38(12):1133.[doi:10.13389/j.cnki.rao.2018.0267]
　SUN Kai,LIU Li-Juan.Inhibition effects of triptolide on human retinal pigment epithelium cells against oxidative damage[J].Recent Advances in Ophthalmology,2018,38(10):1133.[doi:10.13389/j.cnki.rao.2018.0267]
[10]李静,李迪,李雪颖,等.上调microRNA-27b对血小板衍生因子诱导下的人视网膜色素上皮细胞增殖的抑制作用[J].眼科新进展,2020,40(9):822.[doi:10.13389/j.cnki.rao.2020.0187]
　LI Jing,LI Di,LI Xueying,et al.Up-regulation of microRNA-27b inhibits PDGF-induced cell proliferation in ARPE-19 cells[J].Recent Advances in Ophthalmology,2020,40(10):822.[doi:10.13389/j.cnki.rao.2020.0187]

备注/Memo

备注/Memo:
国家自然科学基金面上项目（编号：81970824）；国家自然科学基金青年基金（编号：82000865）
更新日期/Last Update: 2022-10-05