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  

Protective effect of nattokinase on retinal pigment epithelial cell injury induced by high glucose or hypoxia

作　　者：杨倩[1] 薛瑢 董怡辰 范夏莲 余川 万光明[1] 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)

机构地区：[1]郑州大学第一附属医院眼科,河南省郑州市450052

出　　处：《眼科新进展》2022年第10期763-768,共6页Recent Advances in Ophthalmology

基　　金：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)。

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(CoCl_(2))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^(-1)8的表达情况,筛选最佳葡萄糖和CoCl_(2)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+CoCl_(2)组(25.0 mmol•L^(-1)葡萄糖+200μmol•L^(-1) CoCl_(2),HG+CoCl_(2)组)和NK处理组(1.0μmol•L^(-1) NK+25.0 mmol•L^(-1)葡萄糖+200μmol•L^(-1) CoCl_(2),NK+HG+CoCl_(2)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,CoCl_(2)浓度高于200μmol•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,200μmol•L^(-1) CoCl_(2)的干预组ARPE-19细胞中HIF-1α和VEGFA的蛋白和mRNA与IL^(-1)β和IL^(-1)8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1.0μmol•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+CoCl_(2)组比较,NK+HG+CoCl_(2)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。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(CoCl_(2))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^(-1)8 were measured by Western blot and real-time quantitative polymerase chain reaction(RT-qPCR),respectively,and the optimal glucose and CoCl_(2) 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 CoCl_(2) group(25.0 mmol•L^(-1) glucose+200μmol•L^(-1) CoCl_(2),HG+CoCl_(2) group),and NK treatment group(1.0μmol•L^(-1) NK+25.0 mmol•L^(-1) glucose+200μmol•L^(-1) CoCl_(2),NK+HG+CoCl_(2) group).The protein and mRNA expression levels of ZO-1,HIF-1αand 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 CoCl_(2) concentration higher than 200μmol•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-1αand VEGFA,as well as the mRNA expression levels of IL^(-1)βand IL^(-1)8 in the intervention groups(200μmol•L^(-1) CoCl_(2))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 1.0μmol•L^(-1),the viability of ARPE-19 cells was significantly

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 

分 类 号：R774.1[医药卫生—眼科]