益气活血化痰方作用于巨噬细胞外泌体miRNA-let-7-5p/TAB2信号通路抗动脉粥样硬化的实验研究  

Effect of Yiqi Huoxue Huatan Decoction on Antiatherosclerosis through Macrophage Exosome miRNA-let-7-5p/TAB2 Signaling Pathway

作　　者：黄宇 赵汉君 苏立杰[1] 陈婕[1] 阮小芬[1] 姚轶立 王肖龙[1] 薛金贵[1] HUANG Yu;ZHAO Hanjun;SU Lijie;CHEN Jie;RUAN Xiaofen;YAO Yili;WANG Xiaolong;XUE Jingui(Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China)

机构地区：[1]上海中医药大学附属曙光医院,上海201203 [2]上海健康医学院附属周浦医院

出　　处：《中西医结合心脑血管病杂志》2022年第5期835-840,共6页Chinese Journal of Integrative Medicine on Cardio-Cerebrovascular Disease

基　　金：上海市卫计委临床专项(No.201840309);上海市医学重点专科建设基金项目(No.zk2019B25)。

摘　　要：目的通过验证巨噬细胞外泌体miRNA-let-7-5p/TAB2的信号通路,探寻益气活血化痰方抗动脉粥样硬化(AS)的分子机制,为益气活血化痰方抗AS提供依据。方法小鼠巨噬细胞RAW 264.7培养与构建AS模型细胞并分组;巨噬细胞外泌体的提取;免疫印迹法(Western Blot)测定外泌体标记表达,评价外泌体的质量;提取总RNA,反转录,实时荧光定量聚合酶链式反应(RT-qPCR)检测miRNA表达;双荧光素酶报告基因检测实验分析miRNA-let-7-5p与预测的靶向基因之间的结合关系;益气活血化痰方干预过的小鼠大动脉组织芯片进行比对,寻找差异性基因。结果①外泌体的鉴定:对照组(RAW264.7+磷酸缓冲盐溶液)与AS模型组细胞上清液中CD_(61)与CD_(81)几乎没有表达,而对照组(RAW264.7+磷酸缓冲盐溶液)与AS模型组提取的外泌体中CD_(61)与CD_(81)均高表达,表明外泌体提取成功。②外泌体微小RNA(miRNA)qPCR Array与生物信息学分析:检索3个与AS相关的数据库,有12个miRNA共存在于这3个数据库。③通过芯片分析这12个miRNA在外泌体中的表达,与对照组相比,外泌体组有4个miRNA(miRNA-885、miRNA-21、miRNA-451与miRNA-let-7-5p)表达异常。④外泌体与主动脉内皮细胞(HAEC)共培育后,RT-qPCR检测miRNA表达,与HAEC组相比,只有miRNA-let-7-5p在外泌体与HAEC共培养组中显著表达。⑤生物信息学分析差异miRNA-let-7-5p调控的靶向基因;应用双荧光素酶报告基因实验分析验证miRNA-let-7-5p与预测的靶向基因之间的结合关系,发现TAB2为miRNA-let-7-5p的靶基因。在HAEC中,过表达miRNA-let-7-5p抑制了TAB2的表达。通过查找益气活血化痰方干预过的小鼠大动脉组织芯片,发现miRNA-let-7-5p和TAB2均为差异性基因。结论益气活血化痰方可能通过作用于巨噬细胞外泌体miRNA-let-7-5p/TAB2的信号通路发挥抗AS作用。Objective To explore the molecular mechanism of Yiqi Huoxue Huatan Decoction on antiatherosclerosis(AS)by the signaling pathway of miRNA-let-7-5p/TAB2 of macrophage exosomes.Methods Mouse macrophages RAW 264.7 were cultured,AS model cells were constructed and grouped.Exosomes were extracted from macrophages.The quality of exosomes was evaluated by Western Blot.Total RNA was extracted.miRNA expression was detected by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR).The binding relationship between miRNA-let-7-5p and the predicted target gene was analyzed by double luciferase reporter assay.The microchips of the aorta tissues of the mice after the intervention of Yiqi Huoxue Huatan Decoction were compared to search for the different genes.Results Identification of exosomes:CD_(61) and CD_(81) in the supernatant of the control group(RAW264.7+PBS)and the AS model group were almost not expressed,while CD_(61) and CD_(81) in the exosome extracted from the control group(RAW264.7+PBS)and the AS model group were both highly expressed.The results showed that exosomes were extracted successfully.Exosome miRNA qPCR Array and bioinformatics analysis:3 databases related to AS were retrieved,in which 12 miRNAs were co-existed.The expression of these 12 miRNAs in exosomes was analyzed by microarray.Compared with the control group,4 miRNAs(miRNA-885,miRNA-21,miRNA-451,and miRNA-let-7-5p)were abnormally expressed in exosome.After co-culture of exosome and aortic endothelial cells,miRNA expression was detected by RT-qPCR.Compared with aortic endothelial cell group,only miRNA-let-7-5p cous significantly expressed in co-culture group with exosomes and aortic endothelial cells.Double luciferase reporter assay was used to verify the binding relationship between differential miRNAs and the predicted target genes.TAB2 was found to be the target gene of miRNA-let-7-5p.In aortic endothelial cells,the over-expression of miRNA-let-7-5p inhibited the expression of TAB2.By searching the chips in aorta tissue of mice those

关 键 词：动脉粥样硬化 益气活血化痰方 外泌体 miRNA-let-7-5p TAB2 实验研究 

分 类 号：R73[医药卫生—肿瘤]