SIRT3在模拟失重大鼠脑动脉血管重塑中的作用和机制研究

Regional differential vascular remodeling results in postflight cardiovascular deconditioning in astronauts, which poses severe threat to human health and spaceflight safety. However, the mechanism is still unclear. Our previous work found that simulated microgravity modeled by hindlimb unweighting rat resulted in cerebrovascular mitochondrial oxidative injury and endoplasmic reticulum stress of rat cerebrovascular smooth muscle cells, but the underlying mechanism remains uncertain. The present work was designed to investigate the regulatory roles and mechanisms of SIRT3 in cerebrovascular remodeling of simulated microgravity rat using specific drug intervention, transgenic technology and intermittent artificial gravity. Firstly, we investigate the protein and mRNA levels of SIRT3/AMPK and PI3K/AKT pathways in hindlimb unweighting rat cerebrovascular and mesenteric arterioles to analyze the characteristics of their expression abundance. Then we clarify the regulatory roles and mechanisms of SIRT3/AMPK and PI3K/AKT pathways in mitochondrial function, mitochondrial fusion/fission, mitochondrial autophagy, endoplasmic reticulum stress and calcium homeostasis regulation in simulated microgravity rat vascular smooth muscle cells. And finally, we observe whether SIRT3/AMPK downregulation and PI3K/AKT upregulation participate in cerebrovascular remodeling of simulated microgravity rat. To answer the above key scientific issues benefits in clarifying the molecular mechanism of regional differential arterial remodeling during microgravity exposure and defining new molecular target for intervention, as well as developing preventive and countermeasures.

区域差异性动脉血管重塑是航天员航天飞行后心血管功能失调的重要原因，严重威胁航天员身体健康和飞行安全，其发生机理尚不明确。申请人前期研究发现模拟失重导致大鼠脑动脉血管平滑肌细胞线粒体氧化损伤和内质网应激，但机制不明。本课题拟针对SIRT3在模拟失重大鼠脑动脉血管重塑中的作用和分子机制展开研究，采用特异性药物干预、转基因和间断人工重力等技术手段，阐明SIRT3/AMPK和PI3K/AKT分子通路在模拟失重大鼠脑动脉（同步观察肠系膜小动脉）血管平滑肌细胞表达特征，及其对线粒体功能、线粒体融合/分裂、线粒体自噬、内质网应激和Ca2+稳态调控的影响，进而回答SIRT3/AMPK和PI3K/AKT分子通路是否参与模拟失重大鼠脑动脉血管重塑及其分子机制这一关键科学问题。本研究有助于阐明失重状态下区域差异性动脉血管重塑的分子机制和寻找新的分子干预靶点，进而发展有效的预防和对抗措施。

航天员医学保障是我国载人航天工程的核心任务，失重所致心血管损伤与防护已经成为空间生命科学的关键科学问题。本项目旨在阐明失重状态下血管重塑的调控机制，以细胞二维回转和尾部悬吊大鼠模拟失重，系统研究了模拟失重状态下血管平滑肌细胞SIRT3/雌激素受体α（ERα）信号通路表达特征、调控机制及其在血管重塑中的重要作用和分子机制。本项目的主要研究发现：（1）模拟失重导致血管平滑肌细胞SIRT3总蛋白以及线粒体、细胞核和细胞浆SIRT3蛋白表达水平下降而mRNA表达水平无明显变化，线粒体靶向抗氧化剂MitoTEMPO可以抑制SIRT3的泛素化降解，提示SIRT3蛋白泛素化水平升高与线粒体氧化应激损伤有关；（2）模拟失重导致血管平滑肌细胞线粒体未折叠蛋白反应标志物FOXO3a、SOD2、Catalase蛋白表达水平下降，ATP5A/MTCO1比值升高，内质网自噬标志物FAM134B、CCPG1以及FIP200、Atg9、LC3B 蛋白表达及mRNA水平升高，MitoTEMPO可部分逆转上述变化，提示模拟失重通过线粒体氧化应激损伤机制促进SIRT3泛素化降解进而调控血管平滑肌细胞线粒体未折叠蛋白反应和内质网自噬；（3）模拟失重抑制血管平滑肌细胞SIRT3/ERα-NRF1-OMI信号通路导致线粒体mtDNA拷贝数减少，Ppargc1α 和编码三羧酸循环及电子传递链蛋白的基因（Ppargc1β、NRF1、NRF2、Tfam、Plog1、Plog2 和 Polrmt）表达降低，线粒体融合蛋白MFN1/2表达下调，线粒体分裂蛋白Fis1和p-DRP S616表达上调，Pink1/Parkin 蛋白转换抑制，血管平滑肌细胞收缩型标记物SM-MHC、α-SMA、calponin、caldesmon表达水平降低，而合成及增殖型标记物OPN和PCNA表达水平升高，激活ERα-NRF1-OMI信号通路促进线粒体自噬，恢复线粒体稳态并促进血管平滑肌细胞由合成表型向收缩表型转换。这些证据揭示了模拟失重状态下血管平滑肌细胞SIRT3/ERα的表达特征和调控机制，并证实了SIRT3/ERα-NRF1-OMI信号通路在模拟失重大鼠动脉血管重塑中的重要作用和分子机制，为航天失重环境下心血管损伤与防护提供了理论依据。发表SCI收录论文4篇，核心/源期刊1篇，国际学术会议报告5人次，培养博士/硕士研究生4人。