山西医科大学学报2025，Vol.56Issue(7)：767-775,9.DOI:10.13753/j.issn.1007-6611.2025.07.006
miR-320介导氧化应激在糖尿病视网膜病变中的作用机制
Role and mechanism of miR-320-mediated oxidative stress in diabetic retinal microangiopathy
摘要
Abstract
Objective To explore the mechanism of miR-320-mediated oxidative stress response involving in diabetic retinopathy(DR).Methods The oxidative stress model of retinal endothelial cells was induced by H2O2,and the cells were transfected with miR-320 knockdown or overexpression plasmids and NKAP overexpression plasmids.Retinal endothelial cells(hRECs)were divided into PBS group,H2O2(50,100,200 μmol/L)groups,agomir-NC group,agomir-320 group,antagomir-NC group,antagomir-320 group,PBS+agomir-NC group,PBS+agomir-320 group,H2O2+agomir-NC group,H2O2+agomir-320 group,agomir-NC+Vector group,agomir-NC+NKAP oe group,agomir-320+Vector group,and agomir-320+NKAP oe group.Cell proliferation was detected by CCK-8 assay,cell apoptosis was detected by flow cytometry,the levels of oxidative stress-related indicators such as catalase(CAT),malondi-aldehyde(MDA),and superoxide dismutase(SOD)were detected by ELISA,and the mRNA levels of miR-320 and NKAP were de-tected by qRT-PCR.The protein levels of inflammatory factors IL-1β,IL-6,IκBα,p-NF-κB,NF-κB and NKAP were detected by Western blot.Luciferase reporter gene experiment(LUC)was used to verify the targeted binding of miR-320 to NF-κB activating pro-tein(NKAP).Results Compared with 0 μmol/L H2O2,different concentrations of H2O2 significantly reduced the viability of hRECs cells,and increased the apoptosis rate and intracellular ROS levels(all P＜0.05).Western blot results confirmed that H2O2 induced the expression of inflammation-related proteins IL-1β and IL-6 and the activation of NF-κB signaling pathway in a concentration-depen-dent manner(P＜0.01).To ensure the validity of the experiment,100 μmol/L H2O2 was finally selected for the subsequent experiments.The hRECs with low and high expression of miR-320 were successfully constructed.Compared with PBS+agomir-NC group or H2O2+agomir-NC group,the apoptosis rate of hRECs,the levels of CAT,MDA,SOD,and inflammatory factors and p-NF-κB in hRECs were significantly decreased in PBS+agomir-320 group or H2O2+agomir-320 group,and the expression of IκBα protein was signifi-cantly upregulated(P＜0.05).The LUC experiment verified that miR-320 could targetedly bind to NKAP and downregulate the expres-sion of NKAP.Compared with agomir-NC+Vector group or agomir-320+Vector group,the apoptosis rate of hRECs,the levels of CAT,MDA,SOD and inflammatory factors,and p-NF-κB in hRECs were significantly upregulated in agomir-NC+NKAP oe group or agomir-320+NKAP oe group,and the level of IκBα protein was significantly decreased(P＜0.05).Conclusion miR-320 can target and bind to NKAP,and inhibit the oxidative stress response of cells by downregulating the expression of NKAP,thereby alleviating the progres-sion of DR.关键词
糖尿病视网膜病变/视网膜内皮细胞/氧化应激/炎症/miR-320/NF-κB激活蛋白/细胞凋亡Key words
diabetic retinopathy/human retinal endothelial cells/oxidative stress/inflammation/miR-320/NF-κB acti-vating protein/apoptosis分类
医药卫生引用本文复制引用
高静,马存花,吴沈昊,热娜古丽·斯迪克..miR-320介导氧化应激在糖尿病视网膜病变中的作用机制[J].山西医科大学学报,2025,56(7):767-775,9.基金项目
新疆维吾尔自治区自然科学基金项目(2021D01C436) （2021D01C436）
新疆维吾尔自治区卫生健康委员会"天山英才"医药卫生高层次人才培养计划项目(TSYC202301B064) （TSYC202301B064）
