miR-26a-5p通过靶向KCNJ2在β-淀粉样蛋白诱导的小胶质细胞炎症及氧化应激损伤  

miR-26a-5p alleviatesβ-amyloid induced glial cell inflammation and oxidatiue stress injury by targeting KCNJ2

作　　者：徐婉莹 赵洁 周游[2] XU Wanying;ZHAO Jie;ZHOU You(The Ninth People’s Hospital of Suzhou,Suzhou 215299,China;The Second Affiliated Hospital of Nanjing Medical University,Nanjing 210003,China)

机构地区：[1]苏州市第九人民医院,江苏苏州215299 [2]南京医科大学第二附属医院,江苏南京210003

出　　处：《中国实用神经疾病杂志》2023年第2期226-234,共9页Chinese Journal of Practical Nervous Diseases

基　　金：江苏省老年健康科研项目(编号:LD2021030)。

摘　　要：目的揭示miR-26a-5p对β-淀粉样蛋白诱导的炎症及氧化应激损伤的作用机制。方法选取2018-01—2020-01江苏省人民医院老年科实验室70例阿尔茨海默病(AD)患者外周血样本,同时采集80例健康体检者外周血作对照,通过qRT-PCR检测miR-26a-5p在AD患者体内的表达情况,并采用ELISA分析AD患者和健康体检者中炎性因子和活性氧(ROS)水平。通过Aβ_(1-42)(10μmol/L)刺激BV2小胶质细胞构建AD细胞模型,以等体积的DMSO刺激BV2细胞为对照组。采用ELISA检测正常BV2细胞和AD细胞模型的炎性和ROS水平,并通过qRT-PCR检测AD细胞模型中miR-26a-5p的表达趋势。转染miR-26a-5p调控质粒,采用CCK-8和ELISA分析检测miR-26a-5p对BV2细胞增殖、炎性因子表达和氧化应激的影响,生物信息分析和双荧光素酶报告基因预测miR-26a-5p的作用靶点并检测其对靶分子的影响,qRT-PCR、CCK-8和ELISA分析检测KCNJ2对BV2细胞增殖、炎性因子和氧化应激的影响,通过共同调控miR-26a-5ps和KCNJ2的表达,检测PI3K/AKT通路活性、细胞活力和ROS水平变化。结果miR-26a-5p在AD患者血清中的表达量低于健康患者,AD患者血清内炎性因子TNF-α和IL-18水平高于健康体检者,ROS水平也高于健康体检者。通过Aβ_(1-42)刺激构建的AD细胞模型中,TNF-α、IL-18和ROS水平高于正常BV2细胞,miR-26a-5p的表达量在AD细胞模型中低于正常BV2细胞。过表达miR-26a-5p促进AD细胞增殖,降低TNF-α、IL-18和ROS水平;干扰miR-26a-5p表达抑制AD细胞增殖,TNF-α、IL-18和ROS水平升高。生物信息学分析显示miR-26a-5p可靶向KCNJ2;KCNJ2在AD模型中高表达,过表达KCNJ2可抑制AD细胞增殖,提高TNF-α、IL-18和ROS的水平,干扰KCNJ2的表达可提高AD细胞增殖能力并降低TNF-α、IL-18和ROS水平;激活miR-26a-5p的表达可通过靶向降低KCNJ2的表达进而刺激PI3K/AKT通路活性来提高BV2细胞增殖减缓ROS释放水平。结论miR-26a-5p可通过靶向KCNJ2调节PI3K/AKT信Objective To reveal the impact of miR-26a-5p on the mechanism ofβ-amyloid induced inflam⁃mation and oxidative stress injury.Methods In this study,peripheral blood samples were collected from 70 AD patients in the Geriatric Laboratory of Jiangsu People’s Hospital from January 2018 to January 2020,and peripheral blood samples from 80 healthy people were collected as controls.The expression of miR-26a-5p in AD patients was detected by qRT-PCR,and the differences in inflammatory factors and reactive oxygen species(ROS)levels between AD patients and healthy people were analyzed by ELISA.BV2 microglia cells were constructed to AD cell model byAβ1-42(10μmol/L)stimulated,and BV2 cells stimulated by the same volume of DMSO were considered as the control group.The differences of inflammation and ROS between normal BV2 cells and AD cells models were detected by ELISA,and the expression trend of miR-26a-5p in AD model cells were detected by qRT-PCR.The miR-26a-5p regulatory plasmid was transfected,and the effects of miR-26a-5p on BV2 cell proliferation,inflammatory factor expression and oxidative stress were detected by CCK-8 and ELISA analysis.Bioinfor⁃matics analysis and double luciferase reporter gene were used to predict the target of miR-26a-5p and detect its in⁃fluence on the target molecule.qRT-PCR,CCK-8 and ELISA were used to detect the effects of KCNJ2 on the pro⁃liferation,inflammatory factors and oxidative stress of BV2 cells.By co-regulating the expression of miR-26a-5ps and KCNJ2,the activity of PI3K/AKT pathway,cell viability and ROS level were detected.Results The expres⁃sion of miR-26a-5p in the serum of clinical AD patients was lower than that of healthy patients.The levels of the inflammatory factor TNF-α,IL-18 and ROS in the serum of AD patients were higher than those of healthy people.In the AD cell model constructed by Aβ1-42 stimulation,TNF-α,IL-18 and ROS levels were higher than those of normal BV2 cells,and the expression of miR-26a-5p was lower than that of normal BV2 cells in AD ce

关 键 词：阿尔茨海默病 miR-26a-5p KCNJ2 PI3K/AKT信号通路 Β-淀粉样蛋白 小胶质细胞 细胞增殖 炎性反应 氧化应激 

分 类 号：R749.1[医药卫生—神经病学与精神病学]