SIRT3介导的线粒体功能调节在白藜芦醇延缓血小板衰老中的机制研究

Mitochondria-derived reactive oxygen species (ROS) play an important role in nucleate cellular senescence. Currently, it is unknown that whether mitochondrial ROS plays a role in anucleate platelet senescence. Our latest study found that levels of mitochondrial ROS were time-dependly increased in platelet stored at RT, meanwhile the quantity of senescent platelet was increased. Mitochondria-targeted antioxidant and resveratrol decreased the levels of mitochodnrial ROS, and delayed platelet senescence. Contrast, antimycin A, which has been reported to promote mitochodnrial ROS production, accelerated platelet senescence. Resveratrol is an activator of SIRT3, which is a member of sirtuins. We found that SIRT3 was highly expressed in platelet, and mainly located in mitochondria. Focus on these findings, the aim of this study was to (1) confirm the role of mitochondrial ROS in platelet senescence and (2) demonstrate the reason that mitochondrial ROS augment in senescent platelet and (3) explore the mechanism of resveratrol delaying platelet senescence based on SIRT3/MnSOD/mitochondrial ROS signaling pathway. We hope our effort can help to further understand the mechanism of platelet storage lesion, provide theoretical basis for extending the time of platelet storage, and provide a new perspective for resveratrol clinically treating thrombocytopenia.

线粒体ROS在有核细胞衰老过程中起重要作用，血小板没有细胞核，线粒体ROS是否参与血小板衰老目前未明。申请者最新研究发现，体外保存的血小板，随着保存时间的延长，线粒体ROS逐渐增多、发生衰老的血小板也逐渐增多；线粒体靶向的ROS拮抗剂和白藜芦醇降低线粒体ROS浓度，延缓血小板衰老；相反，促进线粒体ROS产生的试剂则加速血小板衰老，提示线粒体ROS可能在血小板衰老过程中起重要作用。白藜芦醇是SIRT3活化剂，我们研究发现SIRT3在血小板高表达，主要位于线粒体。围绕这些发现，本课题拟进行以下研究：1、证实线粒体ROS在血小板衰老中的作用；2、探明随着血小板保存时间的延长，线粒体ROS增多的原因；3、基于SIRT3-MnSOD-线粒体ROS通路探讨白藜芦醇延缓血小板衰老的机制。预期结果有助于认识血小板储存损伤的机制，为延长血小板保存时间提供理论基础；同时为白藜芦醇治疗血小板减少症提供新的视角。

目的：探讨氧化应激对储存中血小板衰老的作用及白藜芦醇改善血小板储存损伤的研究。.方法：单采血小板置于22±2°C震荡保存7天，在特定时间点无菌取样后进行衰老标志、血小板活化、血小板凋亡、线粒体膜电位、细胞内氧自由基、线粒体氧自由基等水平的检测。接着分别采用细胞内及线粒体ROS拮抗剂NAC和Mito-TEMPO对储存血小板进行干预，观察氧自由基对血小板体外储存损伤（PSL）的影响。最后，将天然抗氧化物白藜芦醇添加至血小板储存袋中，标准保存条件下储存7天，观察其对延缓血小板衰老及改善血小板储存损伤的作用。.结果：1. 随着储存时间延长，衰老血小板比例逐渐增多；血小板活化及凋亡水平也同时升高，线粒体膜电位下降；细胞内及线粒体ROS均升高，提示血小板在储存过程中发生氧化应激。2. NAC和Mito-TEMPO作用后，血小板衰老比例下降，活化和凋亡水平降低；提示ROS拮抗剂可改善血小板储存损伤的发生。3. 白藜芦醇一定程度降低血小板衰老比例，改善血小板储存损伤；与对照组相比，有效延长了血小板体外储存时间；小鼠实验提示，白藜芦醇作用可延长血小板输注后体内存活时间。.结论：本文首次报道了血小板在体外储存时发生衰老改变。在此过程中，细胞及线粒体ROS水平均升高，提示储存血小板发生氧化应激。同时，ROS拮抗剂可以有效降低储存血小板ROS水平，并延缓血小板衰老。最后，白藜芦醇作为天然添加剂，可有效降低储存过程中血小板衰老水平，改善PSL的发生，并一定程度延长血小板体外储存时间和体内输注后存活时间，为今后延长血小板保存提供了有意义的指示。