S1P/SPC-S1PR-MAPK介导无创远端肢体缺血预处理脊髓保护作用

Perioperative spinal cord ischemia-reperfusion injury (SCII) caused by spinal orthomophia or excision of thoraco-abdomianl aortic aneurysm which can lead to immediate or delayed paraplegia should be solved urgently. We have proved that noninvasive limb ischemic preconditioning (NLIPC) is an effective way for preventing perioperative SCII. However, the mechanism underlying this approach is still unclear. Accumulating evidence indicates that mitochondria serve a major role in the cellular bioenergetics, function and survival of SCII. Currently, sphingomyelin metabolites have become the focus of intense interest on SCII and the mechanism may be through the mitochondrial functional protection. This project is designed to investigate the possible involved mechanism of NLIPC on SCII through in vivo and in vitro experiments, morphology and molecular biology. According to the preliminary experimental results, we hypothesize that NLIPC is one protective method for SCII by increasing sphingomyelin metabolites in blood, stimulating spinal sphingosine-1-phosphate receptor, activating cellular mitogen-activated protein kinase (MAPK) pathway and improving mitochondria function, ultimately resulting in ischemic tolerance effect. By exploring the possible mechanism of NLIPC, we aim to provide more theoretical basis for this new promising approach for large-scale clinical application for preventing SCII in future.

围术期脊髓缺血损伤是临床亟待解决的难题。本团队率先研究发现无创远端肢体缺血预处理（NLIPC）对围术期脊髓缺血损伤具有保护作用，然而其作用机制不明确。新近文献显示，NLIPC脊髓保护与线粒体功能密切相关。最新研究表明，鞘磷脂代谢物（SPC/S1P）及其受体S1PR对脊髓缺血性损伤防护具有重要作用，其机制可能是通过对线粒体功能产生保护作用从而诱导脊髓缺血耐受。结合前期预实验结果，我们推测: NLIPC通过增加血液中SPC/S1P含量，局部作用于脊髓S1PR，激活MAPK通路，继而开放kATP通道、减少ROS释放和钙超载、阻止mPTP开放从而对线粒体功能产生保护作用，最终诱导脊髓缺血耐受。本项目拟在我们的研究基础上，通过在体及离体实验，应用功能、形态、分子生物学等综合方法，以SPC/S1P-S1PR-MAPK为切入点，深入探索NLIPC脊髓保护作用的新机制，为其将来应用于临床提供新的理论依据。

研究背景：课题负责人所在实验室在国内率先发现远程缺血预处理（Remote Ischemic Preconditioning，RIPC）可模拟脊髓缺血耐受，产生显著的脊髓保护作用（Chin J Clin Rehab, 2003）；并且在国际上率先将RIPC用于围术期脊髓保护，取得了一定的临床效果（J Neurosurg Anesthesiol,2010）。然而其确切机制仍不明确。最新研究表明，鞘磷脂代谢物（SPC/S1P）及其受体S1PR对脊髓缺血性损伤防护具有重要作用，其机制可能是通过对线粒体功能产生保护作用从而诱导脊髓缺血耐受。这些结果提示鞘磷脂代谢物（SPC/S1P）及其受体S1PR可能参与RIPC产生的脊髓保护作用。.课题假设:结合前期预实验结果，我们推测: NLIPC通过增加血液中SPC/S1P含量，局部作用于脊髓S1PR，激活MAPK通路，继而开放kATP通道、减少ROS释放和钙超载、阻止mPTP开放从而对线粒体功能产生保护作用，最终诱导脊髓缺血耐受。.研究内容及结果:首先我们对脊髓缺血再灌注损伤大鼠和正常大鼠脊髓差异蛋白进行了筛选，采用2-DE-MALDI-TOF-MS等差异蛋白组学技术，筛选出重合的差异点共19个。经初步鉴定，有6个蛋白点在NLIPC组和对照组中存在明显差异。其次，远程缺血预处理后大鼠脊髓缺血再灌注24h后血液和脊髓组织中SPC/S1P表达较对照组明显升高，给予S1PR受体阻断剂后表达降低并消除RIPC的脊髓保护作用，证实RIPC诱导脊髓缺血耐受与鞘磷脂代谢物（SPC/S1P）及其受体密切相关。最后，我们对NLIPC是否介导S1PR兴奋通过激活MAPK信号通路产生脊髓缺血耐受作用进行了证实。.项目结论：鞘磷脂代谢物（SPC/S1P）及其受体参与了RIPC诱导的脊髓缺血耐受作用；其机制与MAPK信号通路的激活有关。.以上研究结果为RIPC诱导脊髓缺血耐受的机制提供了新的学术见解，为其将来应用于临床提供新的理论依据。