波生坦在大鼠脊髓缺血再灌注中调控VEGF表达的作用机制研究

Spinal cord ischemia-reperfusion injury (SIRI) is a common clinical condition affecting the nervous system. Because the mechanism is not clear, there is not a very effective therapy. In order to study the pathogenesis and explore possible therapeutic strategies, we have established a rat model of spinal cord ischemia reperfusion according to Zivin method and found that Bosentan can promote endothelin-1 (ET-1) expression through the feedback mechanism, which in turn upregulated the expression of vascular endothelial growth factor (VEGF). After comprehensive literature review and preliminary experiment results, we hypothesize that Bosentan can improve the microcirculation after SIRI by blocking the ET-1 receptors, while elevated ET-1 results in upregulated expression of VEGF through the endoplasmic reticulum stress signalling pathway involving XBP1. VEGF can not only protect neuron from apotosis, but also improve microcirculation of ischemia tissue through angiogenesis. In this project, we will explore the mechanism of SIRI by using the well-established rat model of SIRI in vivo and in glial cells in vitro. Experiments will be carried out using methods from histology, molecular biology and cell biology. We will to provide reliable evidence for the clinical use of Bosentan and contribute to the molecular biologic mechanism involved in SIRI.

脊髓缺血再灌注是临床常见的一种神经系统损伤，由于机制尚不明确，目前还没有一种十分有效的防治方法。为了深入研究其发病机理及探讨治疗措施，我们参照Zivin法建立了大鼠脊髓缺血再灌注的模型并发现波生坦可以通过反馈机制上调大鼠脊髓组织内皮素1（ET-1）和血管内皮细胞生长因子(VEGF)的表达。结合文献检索和预实验的结果，我们推断波生坦在阻断ET-1受体、缓解再灌注后微循环障碍的同时上调ET-1，而后者则通过内质网应激的信号传导通路激活VEGF表达。VEGF不仅具有神经元保护作用，而且可以通过血管再生改善组织缺血。为了进一步探索其中的分子细胞学机制，我们拟通过我们建立的大鼠脊髓缺血再灌注模型和体外胶质细胞培养从组织学、分子生物学和细胞生物学对其进行研究。希望通过这项研究为波生坦在脊髓缺血再灌注的治疗提供可靠的证据，并为缺血再灌注分子生物学机制提供更新的诠释。

脊髓缺血再灌注损伤（Spinal cord ischemia reperfusion injury，SIRI）的发生发展机制及其防治措施已成为近年来多个学科领域的研究重点。本课题参照ZIVIN法建立大鼠的脊髓缺血再灌注模型，给予波生坦（一种内皮素受体A/B非选择性拮抗剂）干预，从神经功能评分、形态学等方面探讨了波生坦对SCIR的积极作用，并深入研究了机制。我们的实验发现，建立大鼠SCRI模型，给予波生坦干预，可以提高大鼠行为学评分，TTC染色显示缺血梗死灶明显减少，HE染色显示细胞学损伤减轻，ELISA发现血清中ET-1浓度增高，免疫组化及RT-PCR证实组织中ETRB表达均上调，通过免疫组化检测细胞凋亡相关蛋白证实细胞凋亡明显减少，以上结果说明Bosentan可能是通过ET-1-ETRB信号通路减少脊髓缺血再灌注引起的细胞凋亡来发挥保护作用的，该结果已经发表在文章《Bosentan reduces neuronal apoptosis following spinal cord ischemic reperfusion injury》中；血管内皮生长因子（VEGF）在中枢神经系统中具有促进血管重建及神经营养作用的双重功能，本实验发现，建立大鼠SCIR模型，给予Bosentan干预，ELISA发现血清中VEGF浓度增高，免疫组化及RT-PCR证实组织中VEGF受体表达上调, 以上结果说明Bosentan上调VEGF及其受体的表达，促进血管重建和神经重建，从而促进神经功能恢复。根据上述内容已经撰写完成文章《Bosentan Enhances Recovery in Rats Through Up-regulation of Vascular Endothelial Growth Factor and its Receptors after Spinal Cord Ischemia Reperfusion Injury》，该文目前已投，正在送外审。本课题已发表论文2篇，其中SCI收录1篇，核心期刊收录1篇，目前的实验结果预计还可以发表两篇SCI文章，已投稿1篇。