基于NVU和miRNA基因网络研究三七总皂苷对脑缺血保护作用的分子机制

With the occurrence of ischemic brain damage, each component of the neurovascular unit (NVU) such as neurons, glial cells and microvascular were damaged at the same time,and it provided a new target for comprehensive study of ischemic brain injury.It was already know that miRNA involved in the gene network regulation of NVU changes after ischemic brain damage.Through the study of pharmacological intervention of specific miRNA can help to find new target point and new mechanism of drugs. Panax Notoginseng Saponins(PNS) was widely used in the clinical treatment of cerebral infarction. Studies have found that PNS has multiple targets in the protection of ischemic brain injury.PNS can be applied to multiple components of the NVU after cerebral ischemia, but the exact molecular mechanism is still unclear. This research will apply PNS in cerebral ischemia injury rat model, to study the difference of miRNA expression between brain tissue of rats after cerebral ischemic injury and after PNS intervention, to screen differentially expressed miRNA after drug,to forecast the target gene of the differentially expressed miRNA and the signal pathway of target gene involved in, to verify the differentially expressed miRNA, to analysis the relationship between the changes of neurovascular unit and the differentially expressed miRNA with it target gene and the signal pathway after the treatment, to show the molecular mechanism of that PNS has a multitargets in ischemic brain injury through the miRNA gene regulation network.

缺血性脑损伤发生时，神经血管单元（NVU）中的神经元、胶质细胞及微血管同时受到损伤，NVU为多方位研究缺血性脑损伤提供新靶点。miRNA参与缺血性脑损伤后NVU变化的基因网络调控，通过对药物干预后相关的miRNA进行研究，可为发现药物作用的分子机制提供新思路。三七总皂苷（PNS）临床上广泛用于脑梗死的治疗。研究发现PNS对脑缺血后的NVU具有保护作用，但是通过哪些信号通路、哪些分子靶点作用于NVU，目前不清楚。为此，本研究项目将给脑缺血损伤模型大鼠PNS干预，研究脑缺血后和PNS干预后大鼠脑组织miRNA表达谱的差异，筛选出用药后差异表达的miRNA；预测差异表达miRNA的靶基因和靶基因参与的信号通路；验证差异表达的miRNA，深入分析相关差异表达miRNA及其靶基因和信号通路与用药后NVU变化的关系；从miRNA基因调控网络层面阐明PNS在缺血性脑损伤中多作用靶点的分子机制。

三七总皂苷（PNS）在缺血性脑卒中治疗方面，有多作用靶点，可参与缺血损伤后神经血管单元（NVU）的修复。但是，PNS通过哪些作用靶点、哪些信号通路作用于脑缺血后的NVU，目前不明确。. 我们对脑缺血再灌注损伤模型大鼠给予PNS干预，观察PNS对大鼠脑缺血后NVU形态学变化、相关指标表达和神经功能的影响；采用高通量测序技术检测缺血性脑损伤后和PNS干预后大鼠脑组织 miRNA表达谱的差异，筛选出用药后差异表达的miRNA；应用生物信息学等方法和技术对差异表达 miRNA的靶基因和靶基因参与的信号通路进行预测和验证；验证差异表达的miRNA，分析相关差异表达miRNA及其靶基因和信号通路与用药后NVU的关系。. 我们研究发现，PNS能改善脑缺血再灌注损伤模型大鼠的神经功能和NVU的结构，促进NVU相关标志物的表达。转录组分析：共观察到28792个基因，48173个转录本的表达数据，并对其做了功能注释；采用blast-short搜索Rfam v11数据库和miRbase 21.0数据库，一共观察到961个成熟miRNA的表达，其中760个是拥有注释信息的已知miRNA，另外201个为新预测的未知miRNA，在有注释的miRNA中，一共观察到了27个成熟miRNA在组间表现出显著差异。mir-21，mir-1298，mir-204，mir-34c，mir-199a，mir-34b， mir-448，mir-375，mir-214，mir-155，mir-223，mir-1912和mir-3743等13个小分子表现为既积极参与I/R调控又对PNS的干预响应。对发现的miRNA做了靶基因预测，共发现40个miRNA与2294个转录本间有2665对靶向调控关系。其中10个差异miRNA获得了38个靶基因。对显著表达差异miRNA之一的miR-155通过细胞培养离体研究进行验证，结果发现PNS通过miR-155调节IL-1β、IL-6和TNF-α的表达，对OGD/R-诱导的SH-SY5Y细胞损伤有保护作用。. 本研究从miRNA基因调控网络层面阐明了PNS在缺血性脑损伤中具有多作用靶点，整体调节的分子机制，为临床使用PNS治疗脑梗死提供理论依据。