Orexin-A/B对缺血性脑中风的神经保护作用及其机制研究

Ischemic stroke is the most common type of stroke and accounted for 80% of all stroke patients. Ischemic stroke has recurrent characteristics and is difficult to cure, so there is no effective treatment. In our previous studies, we find that Orexins has obvious neuroprotection. In addition, the HiSeq sequencing shows that miRNA expression is a significant change among ischemia, reperfusion and normal control, hinting that miRNA is involved in cerebral ischemia and the neuroprotection of Orexins. Therefore, we put forward the hypothesis: firstly, Orexins interact with its receptor on member, then regulate the different genes through different signaling pathways, eventually influencing cell biology function. miRNA plays a crucial role in this process, enhancing or inhibitting the expression of target genes, so as to realize the the neuroprotection of Orexins. In present study, using advanced experimental techniques, such as miRNA sequencing (HiSeq), digital gene expression profiling (RNA-Seq) and real time cellular analysis (RTCA), and conventional experimental techniques, including ELISA, qRT-PCR, western blotting, immunohistochemistry and flow cytometry, we study the treatment of Orexins on ischemic stroke. Meanwhie, miRNA and gene expression profiling are detected after Orexins reperfusion in ischem icstroke rat model, revealing the important factors involved in Orexins neuroprotective role, and the upstream regulatory mechanisms of miRNA. Therefore, the results will help us to understand the molecular mechanism of Orexins reperfusion from RNA and gene levels, and provide theoretical and experimental basis for clinical treatment and prevention of ischemic stroke. Finally, Orexins/miRNA is expected to become a new way of treatment of cerebral ischemia.

缺血性脑中风是最常见的中风类型，目前还没有行之有效的治疗方法。我们前期研究发现Orexins具有明显的神经保护作用；HiSeq测序表明miRNA表达谱在缺血、再灌注及对照标本间存在显著变化，提示miRNA参与了脑缺血及Orexins的神经保护作用。我们认为：Orexins与细胞膜上的受体作用后，影响胞质内不同的信号通路，从而调控核内不同基因的表达，最终导致细胞生物学功能的改变；在此过程中miRNA通过增强或抑制靶基因的表达，实现Orexins的神经保护作用。为此，本研究采用先进的实验技术及常规实验技术，研究缺血性脑中风模型再灌注Orexins后miRNA及基因表达谱的变化，从miRNA和基因不同层次揭示Orexins神经保护作用的分子机制，为临床治疗和预防缺血性脑中风提供理论与实验基础，使Orexins/miRNA有望成为缺血性脑中风治疗的新途径。

脑中风是严重危害人类健康和影响人类生活质量的疾病，目前还没有行之有效的治疗方法，因此研究脑缺血-再灌注损伤的机制和治疗具有重要的理论和临床意义。本项目主要研究Orexin对大鼠脑缺血-再灌注损伤模型的神经保护作用及其机制。目前本项目所取得的主要成果如下：（1）TTC实验表明侧脑室内注射Orexin-A/-B能明显减少大鼠缺血-再灌注损伤模型（MCAO）的脑梗死体积，流式细胞术检测表明Orexin-A/-B可以明显抑制SH-SY5Y细胞的凋亡，CCK-8实验表明Orexin-A显著改善由氧-糖剥夺诱导的神经元形态及突起的数量，这些结果表明Orexin-A/-B具有神经保护作用。（2）Orexin-A通过改变ERK蛋白的磷酸化水平参与缺血-再灌注损伤，且抑制剂LY294002表明Orexin-A引起的p-ERK的水平是由PI3K介导的；Orexin-B通过增强 p-AKT 活性，使磷酸化 GSK-3β增多，发挥促进神经元生存，抑制神经元凋亡。（3）HiSeq测序表明大鼠脑缺血-再灌注不同时间点引起miRNA的差异表达，其中miR-1298，miR-34C差异明显。体外实验表明miR-1298可明显抑制胶质瘤细胞C6的增值，并诱导C6的凋亡，且miR-1298可能是通过作用靶基因SETD 7而实验其功能的。（4）RNA-sequencing检测大鼠脑缺血-再灌注不同时间点差异基因表达情况。检测到≥2的上调的基因有156个，下调的基因有26个，而且RT-PCR结果表明差异基因的表达呈动态变化，在不同的缺血再灌注时间，其表达不尽相同，反应了缺血-再灌注损伤机制的复杂性。KEGG分析表明缺血-再灌注损伤激活了粘附信号通路、细胞骨架信号通路、MAPK信号通路、Jak-STAT信号通路等。本课题利用缺血-再灌注Orexin的大鼠模型，研究缺血-再灌注后miRNA及基因表达变化，从不同层次探讨缺血-再灌注的分子调控机制，为研究缺血性脑中风的发病机制和治疗方法奠定基础，结果将有助于揭示相关疾病的发病机理，并有助于探寻相关疾病治疗的药物靶点，具有潜在的应用和推广价值。