MiR-199a修饰的间充质干细胞来源外泌体通过Akt/HIF-1α/DRP1轴促进缺糖缺氧/复糖复氧模型心肌细胞H9c2线粒体修复  

MiR-199a-modified mesenchymal stem cell-derived exosomes promote mitochondrial repair in H9c2 cardiomyocytes of hypoxia/reoxygenation model through Akt/HIF-1α/DRP1 axis

作　　者：郑君毅[1] 李晓凤[2] 郭绪昆[1] ZHENG Junyi;LI Xiaofeng;GUO Xukun(Department of Cardiology,Tianjin Chest Hospital,Tianjin Institute of Cardiovascular Disease,Tianjin 300222,China;Department of Cardiology,Second Affiliated Hospital of Tianjin University of Traditional Chinese Medicine,Tianjin 300150,China)

机构地区：[1]天津市胸科医院心内科,天津大学附属胸科医院心内科,天津市心血管病研究所,天津300222 [2]天津中医药大学第二附属医院心内科,天津300150

出　　处：《药物评价研究》2024年第10期2317-2325,共9页Drug Evaluation Research

基　　金：国家自然科学基金资助项目(82004329);天津市医学重点学科(专科)建设项目。

摘　　要：目的 探讨miR-199a修饰的间充质干细胞(MSCs)来源的外泌体修复缺糖缺氧/复糖复氧模型心肌细胞H9c2线粒体的作用机制。方法体外培养MSCs,转染miR-199a mimics或miR-NC, 48~72 h后收集外泌体,实时荧光定量PCR(qRT-PCR)法检测外泌体的miR-199a水平。将H9c2细胞分为对照组、模型组、miR-199a修饰外泌体(Exos^(mimic),终质量浓度50μg·mL^(-1))组、miRNA阴性对照修饰外泌体(Exos^(NC),终质量浓度50μg·mL^(-1))组和miR-199a修饰外泌体+蛋白激酶B(Akt)抑制剂(Exos^(mimic)+MK2206 10μg·mL^(-1))组,除对照组外,制备缺糖缺氧/复糖复氧模型。应用CCK-8法检测各组细胞存活率,酶标仪检测各组细胞三磷酸腺苷(ATP)、超氧化物歧化酶(SOD)、丙二醛(MDA)水平以及上清液8-羟基脱氧尿苷(8-OHdG)、乳酸脱氢酶(LDH)水平;共聚焦显微镜检测各组线粒体膜电位(ΔΨm)和线粒体动力学变化;Western blotting法检测各组缺氧诱导因子1α(HIF-1α)和线粒体动力相关蛋白1(DRP1)蛋白表达变化。结果 与对照组比较,Exos^(mimic)中miR-199a表达水平明显升高(P<0.05),提取的外泌体直径平均为109.3 nm,浓度为1×10^(6)颗粒·mL^(-1)。与对照组比较,模型组细胞存活率和ATP、SOD、ΔΨm水平显著下降,LDH、MDA、8-OHdG水平和HIF-1α和DRP1蛋白表达水平显著升高(P<0.01、0.001),线粒体分裂水平增加;与模型组比较,Exos^(mimic)组细胞存活率和ATP、SOD和ΔΨm水平显著升高,LDH、MDA、8-OHdG水平和HIF-1α及DRP1蛋白表达水平显著下降(P<0.01、0.001),线粒体分裂水平减少;MK2206能明显逆转Exos^(mimic)效果(P<0.05、0.01)。结论 miR-199a修饰的MSCs外泌体通过Akt/HIF-1α/DRP1轴促进缺糖缺氧/复糖复氧心肌细胞线粒体修复。Objective To investigate the mechanism of miR-199a-modified mesenchymal stem cell(MSC)-derived exosomes in repairing mitochondria of H9c2 cardiomyocytes in a model of hypoglycemia/hypoxia/reoxygenation with hypoglycemia.Method Transfect miR-199a mimics or miR-NC into MSCs and collect exosomes 48-72 h later.Fluorescence quantitative PCR(qRT-PCR)method was used to detect miR-199a levels in different exosomes.H9c2 cells were divided into control group,model group,Exosmimic group(final concentration of 50μg·mL^(−1)),Exos^(NC) group(final concentration of 50μg·mL^(−1)),and Exos^(mimic)+MK2206(10μg·mL^(−1))group.Except for the control group,the model of hypoglycemia/hypoxia/reoxygenation with hypoglycemia was established.The survival rate of cells in each group was detected by CCK8 assay,and the levels of ATP,8-hydroxy-2-deoxyguanosine(8-OHdG),lactate dehydrogenase(LDH),superoxide dismutase(SOD),malondialdehyde(MDA)in each group were detected by enzyme-linked immunosorbent assay(ELISA).The changes of mitochondrial membrane potential(ΔΨm)and dynamics were detected by confocal microscopy.The expression changes of hypoxia-inducible factor 1α(HIF-1α)and mitochondrial dynamics-related protein 1(DRP1)were detected by Western blotting.Result Compared with control group,the expression level of miR-199a in Exosmimic was significantly increased(P<0.05),and the average diameter of extracted exosomes was 109.3 nm,with a concentration of 1×10^(6) particles·mL−1.Compared with control group,the model group showed a significant decrease in cell survival rate and ATP,SOD,andΔΨm levels,while LDH,MDA,8-OHdG levels,HIF-1α and DRP1 protein expression levels were significantly increased(P<0.01,0.001),and mitochondrial division levels were increased.Compared with model group,the Exosmimic group showed a significant increase in cell survival rate,ATP,SOD,andΔΨm levels,while LDH,MDA,8-OHdG levels,HIF-1α,and DRP1 protein expression levels were significantly decreased(P<0.01,0.001),and mitochondrial division levels were

关 键 词：miR-199a 外泌体 间充质干细胞 心肌细胞H9c2 缺糖缺氧/复糖复氧 线粒体 Akt/HIF-1α/DRP1轴 

分 类 号：R363[医药卫生—病理学]