microRNAs调控线粒体自噬减轻脊髓缺血/再灌注损伤的实验研究

Ischemic injury of spinal cords remains a devastating complication with a high incidence after surgery or endovascular repair of thoracoabdominal aneurysms. Mitochondria dysfunction plays a central role in the ischemia/reperfusion injury of myocardium and brain. Mitochondria dysfunction can disrupt energy metabolism, produce reactive oxygen species and activate proapoptotic and necrotic pathways, which induce cell death. Mitophagy is a special autophagy of selective clearance of excessive mitochondria or impaired mitochondria, which is important to maintain the quantity and quality of the mitochondria. It is indicated in the newly studies that mitophagy can induce protective effects against ischemia/reperfusion injury of heart and brain. As an important control mechanism after transcription, miRNAs contribute to the control of autophagy or mitophagy. However, it is still not known the function of mitophagy in the ischemia/reperfusion injury of spinal cords and whether miRNAs affect the activity of mitophagy in spinal cords. The current study is to investigate the protective effects of mitophagy in the ischemia/reperfusion injury of spinal cords. Further study is to attenuate the spinal cords injury by control of the activity of mitophagy with miRNAs targeted at the important proteins for inducing mitophagy. The final objective is to find a new protective strategy for ischemia/reperfusion injury of spinal cords.

缺血性脊髓损伤是胸腹主动脉瘤外科和介入治疗发生率较高的严重并发症。线粒体功能障碍在心、脑等组织缺血再灌注损伤中发挥重要作用。线粒体功能障碍可以导致细胞能量代谢障碍，损伤的线粒体可以产生过量的活性氧并启动凋亡和坏死信号途径，导致细胞死亡。线粒体自噬是一种选择性清除损伤或多余线粒体的自噬过程，对于维持细胞内线粒体的质量和数量至关重要。新近研究表明，线粒体自噬在心肌和脑组织缺血/再灌注损伤中发挥保护作用。miRNAs作为转录后调控的重要机制，也参与细胞自噬和线粒体自噬的调控。目前尚没有关于线粒体自噬在脊髓缺血/再灌注损伤中作用以及miRNAs对其调控机制的研究报告。本课题拟探讨线粒体自噬在脊髓缺血/再灌注损伤中作用及其miRNAs调控机制，并通过调控与介导线粒体自噬重要蛋白表达相关的一组miRNAs，靶向调控脊髓组织的线粒体自噬，减轻脊髓缺血/再灌注损伤，旨在探索一个全新的脊髓保护策略。

缺血性脊髓损伤所致的截瘫或轻瘫是外科或介入治疗主动脉夹层和胸腹主动脉瘤最为严重的并发症之一。本研究主要探讨一种以miRNA和自噬为主要靶点的脊髓保护策略。利用阻断锁骨下动脉远端降主动脉14min，建立大鼠脊髓缺血再灌注损伤模型。构建包含antagomiR-124、antagomiR-204、pro-miR-25的慢病毒载体。培养充间质干细胞 (MSC)以及转染pro-miR-25慢病毒的MSC并提取外泌体。鞘内注射转基因载体或外泌体。利用鞘内注射3-MA抑制线粒体自噬和细胞自噬过程。利用Motor Deficit Index (MDI) 评分，对大鼠后肢的运动功能进行评价。降主动脉阻断14分钟可以导致大鼠后肢严重的运动功能障碍，前角健存神经元数量明显减少。转染antagomiR-124可以显著降低脊髓组织中miR-124的表达，抑制p53的表达，上调iASPP、Beclin-1和LC3-II的表达；转染antagomiR-204可以降低脊髓组织中miR-204的表达，上调LC3-II/LC3-I的比值和Beclin-1、Bcl-2的表达，并降低caspase-3的表达。抑制miRNA-124和miRNA-204均能够显著降低再灌注后大鼠后肢MDI评分，改善运动功能。组织学检查结果表明，抑制miRNA-124和miRNA-204均显著减轻缺血再灌注后脊髓组织内的细胞凋亡和组织破坏。鞘内注射3-MA能够抑制antagomiR-124介导的线粒体自噬作用增强并能够部分取消其神经保护作用。鞘内注射3-MA也能够抑制antagomiR-204介导的细胞自噬增强，并取消其神经保护作用。鞘内注射MSC外泌体可以降低IL-1b及TNF-α的表达，减轻缺血再灌注后的脊髓损伤。转染pro-miR-25可显著提高间充质干细胞及其外泌体中的miR-25水平，富含miR-25的外泌体显著提高了脊髓中miR-25的表达水平，同时抑制了NOX 4的表达，并降低丙二醛的含量，提高了脊髓组织中的超氧化物歧化酶活性。同单纯外泌体组相比，富含miR-25的外泌体进一步提高了对脊髓缺血再灌注损伤的保护作用。结论：通过miRNAs调控线粒体自噬或细胞自噬，可以减轻脊髓缺血再灌注损伤；以MSC外泌体为载体，调控脊髓组织中特定miRNA的表达，可能成为减轻脊髓缺血再灌注损伤的新策略。