Liu Liang, Jin Xian, Hu Cui-Fen, Li Rong, Zhou Zhong'e, Shen Cheng-Xing
Department of Cardiology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, China.
Department of Ultrasound, the First Affiliated Hospital of Soochow University, Suzhou, China.
Cell Physiol Biochem. 2017;43(1):52-68. doi: 10.1159/000480317. Epub 2017 Aug 25.
BACKGROUND/AIMS: Reperfusion after an ischaemic insult might cause infarct extension. Mesenchymal stem cell (MSC)-derived exosomes could attenuate myocardial remodelling in animal models of myocardial ischaemia reperfusion injury (MIRI), and the present study aimed to explore the related mechanisms.
In vitro, rat H9C2 cardiomyocytes (H9C2s) were exposed to H2O2. Cell viability was detected by the CCK-8 assay, apoptosis was detected by Annexin V-PE/7-AAD staining, ROS production was detected by fluorescence microscopy and flow cytometry, and apoptosis-related proteins and signalling pathway-related proteins were detected by western blot analysis. Autophagic flux was measured using the tandem fluorescent mRFG-GFP-LC3 assay. MSC-derived exosomes were extracted using the total exosome isolation reagent. Apoptosis, myocardial infarction size, heart function and myocardial LC3B expression were examined in an in vivo I/R model by the TUNEL assay, TTC/Evan blue staining, echocardiography and immunohistochemicalstaining, respectively.
In vitro, H2O2 dose-dependently increased ROS production and cell apoptosis in H9C2s and blocked autophagic flux after 3 h of exposure; autophagy gradually decreased thereafter, and the lowest level was detected at 12 h after exposure. MSC-derived exosomes reduced H2O2-induced ROS production and cell apoptosis and enhanced autophagy at 12 h after exposure. In H9C2 cells exposed to H2O2 for 12 h, treatment with exosomes enhanced autophagy via the AMPK/mTOR and Akt/mTOR pathways. Likewise, in vivo exosome injections in rats that underwent I/R injury significantly reduced apoptosis and the myocardial infarct size and upregulated myocardial LC3B expression as well as improved heart function.
Our results indicate that MSC-derived exosomes could reduce MIRI by inducing cardiomyocyte autophagy via AMPK/mTOR and Akt/mTOR pathways.
背景/目的：缺血损伤后的再灌注可能导致梗死面积扩大。间充质干细胞（MSC）衍生的外泌体可减轻心肌缺血再灌注损伤（MIRI）动物模型中的心肌重塑，本研究旨在探讨其相关机制。
在体外，将大鼠H9C2心肌细胞（H9C2s）暴露于过氧化氢。通过CCK-8法检测细胞活力，通过Annexin V-PE/7-AAD染色检测细胞凋亡，通过荧光显微镜和流式细胞术检测活性氧（ROS）生成，通过蛋白质免疫印迹分析检测凋亡相关蛋白和信号通路相关蛋白。使用串联荧光mRFG-GFP-LC3法测量自噬通量。使用总外泌体分离试剂提取MSC衍生的外泌体。在体内缺血/再灌注（I/R）模型中，分别通过TUNEL法、TTC/伊文思蓝染色、超声心动图和免疫组织化学染色检测细胞凋亡、心肌梗死面积、心脏功能和心肌LC3B表达。
在体外，过氧化氢剂量依赖性地增加H9C2s中的ROS生成和细胞凋亡，并在暴露3小时后阻断自噬通量；此后自噬逐渐减少，在暴露后12小时检测到最低水平。MSC衍生的外泌体减少了过氧化氢诱导的ROS生成和细胞凋亡，并在暴露后12小时增强了自噬。在暴露于过氧化氢12小时的H9C2细胞中，外泌体处理通过AMPK/mTOR和Akt/mTOR途径增强了自噬。同样，在经历I/R损伤的大鼠体内注射外泌体可显著减少细胞凋亡和心肌梗死面积，上调心肌LC3B表达，并改善心脏功能。
我们的结果表明，MSC衍生的外泌体可通过AMPK/mTOR和Akt/mTOR途径诱导心肌细胞自噬，从而减轻MIRI。
