miR-155靶向Nrf2/HO-1通路对高糖作用下视网膜血管内皮细胞氧化应激损伤的影响-东南大学学报医学版

miR-155靶向Nrf2/HO-1通路对高糖作用下视网膜血管内皮细胞氧化应激损伤的影响

单位：琼海市人民医院眼科, 海南琼海 571400

关键词：miR-155 氧化应激高糖 Nrf2蛋白 HO-1蛋白视网膜血管内皮细胞

分类号：R774.1

出版年·卷·期（页码）：2021·40·第五期（666-674）

摘要：

目的：探讨miR-155对高糖作用下视网膜血管内皮细胞（HRECs）氧化应激的调控作用及作用机制。方法：体外培养HRECs，使用不同浓度D-葡萄糖作用于HRECs 48 h或72 h，CCK-8法检测细胞存活率。将HRECs分为正常组（5.5 mmol·L^-1 D-葡萄糖培养）、高渗组（50 mmol·L^-1甘露醇培养）、高糖组（30 mmol·L^-1 D-葡萄糖培养）、高糖+miR-NC组（转染miRNA scramble+30 mmol·L^-1 D-葡萄糖培养）、高糖+miR-155-i组（转染miR-155 inhibitor+30 mmol·L^-1 D-葡萄糖培养）、高糖+miR-155-i+siNC组（转染miR-155 inhibitor和siRNA空质粒+30 mmol·L^-1 D-葡萄糖培养）、高糖+miR-155-i+siNrf2组（转染miR-155 inhibitor和Nrf2 siRNA+30 mmol·L^-1 D-葡萄糖培养）。RT-qPCR法检测miR-155和Nrf2 mRNA相对表达量。流式细胞术分别用于检测细胞凋亡和细胞内活性氧簇（ROS）水平。ELISA检测各组细胞培养上清中丙二醛（MDA）、超氧化物歧化酶（SOD）和谷胱甘肽过氧化物酶（GSH-Px）含量。Western blotting法检测胞质和胞核Nrf2蛋白及细胞总蛋白中HO-1蛋白表达。双荧光素酶报告基因用于验证miR-155和Nrf2 mRNA之间的靶向关系。结果：与D-葡萄糖0 mmol·L^-1组相比，30 mmol·L^-1D-葡萄糖作用48 h时HRECs中miR-155相对表达量显著上调（P<0.05），且细胞存活率<70%，可用于后续实验。与正常组相比，高糖组细胞凋亡率增加，细胞培养上清中SOD、GSH-Px含量降低，同时MDA和ROS含量（平均荧光强度，MFI）升高，Nrf2和HO-1蛋白表达下调（均P<0.05）；而与高糖组相比，高糖+miR-155-i组细胞凋亡率降低，细胞培养上清中SOD、GSH-Px含量升高，同时MDA和ROS含量降低，Nrf2和HO-1蛋白表达上调（均P<0.05）；与高糖+miR-155-i组相比，高糖+miR-155-i+siNrf2组上述指标则被逆转。经双荧光素酶报告基因分析，miR-155 mimics和Nrf2-3'UTR-WT共转染可降低荧光素酶活性（P<0.05）。结论：高糖可诱导HRECs miR-155表达上调，敲低miR-155表达后能够保护高糖作用下HRECs凋亡和氧化应激损伤，其机制与激活Nrf2/HO-1通路有关。

Objective: To study the effect of miR-155 on the oxidative stress injury of retinal vascular endothelial cells (HRECs), and to further explore its mechanism. Methods: HRECs were cultured in vitro. The survival rate of HRECs was determined by CCK-8 assay after 48 h or 72 h treatment of different concentrations of D-glucose. HRECs were divided into normal group (cultured in a medium containing 5.5 mmol·L^-1 D-glucose), hypertonic group (cultured in medium containing 50 mmol·L^-1 mannitol), high glucose (HG) group (cultured in medium containing 30 mmol·L^-1 D-glucose), HG+miNC group (transfected with miR-155 mimics inhibitor and cultured in medium containing 30 mmol·L^-1 D-glucose), HG+miR-155-i group (transfected with miR-155 inhibitor and cultured in medium containing 30 mmol·L^-1 D-glucose), HG+miR-155-i+siNC group (transfected with miR-155 inhibitor+siRNA vector and culture in medium containing 30 mmol·L^-1 D-glucose) and HG+miR-155-i+siNrf2 group (transfected with miR-155 inhibitor+Nrf2 siRNA and culture in medium containing 30 mmol·L^-1 D-glucose). RT-qPCR was used to detect the relative expression of miR-155 and Nrf2 mRNA. FCM methods were used to detect cell apoptosis and the level of reactive oxygen species (ROS). The levels of malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were detected by enzyme-linked immunosorbent assay (ELISA). The protein expressions of Nrf2 in nuclear or cytoplasm protein and HO-1 were detected by Western blotting. Moreover, the relationship between miR-155 and Nrf2 mRNA was verified by the dual-luciferase reporter gene assay. Results: Compared with D-gluocse (0 mmol·L^-1) group, the relative expression of miR-155 in HRECs under the condition of 30 mmol·L^-1 D-gluocse for 48 h was significantly up-regulated (P<0.05) and the survival rate of HRECs was<70%, which could be used in the follow-up experiment. Compared with normal group, in HG group the apoptosis rate increased, SOD and GSH-Px levels in the supernatant of cell culture decreased, MDA and ROS (MFI) contents increased, Nrf2 and HO-1 protein expressions decreased (P<0.05). Compared with HG group, the apoptosis rate in the HG+miR-155-i group decreased, SOD and GSH-Px levels in the supernatant of cell culture increased, MDA and ROS contents decreased, and the expressions of Nrf2 and HO-1 protein increased (P<0.05). However, the above indexes were reversed in HG+miR-155-i+siNrf2 group (P<0.05). Co-transfection of miR-155 mimics and Nrf2-3'UTR-WT reduced luciferase activity (P<0.05) by dual-luciferase reporter gene assay. Conclusion: High glucose can induce up-regulation of miR-155 expression in HRECs, and down-regulation of miR-155 can protect HRECs from apoptosis and oxidative stress induced by high glucose, which may be related to the activation of Nrf2/HO-1 pathway.

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