基于外泌体microRNAs调控EPCs 功能研究经方侯氏黑散促进脑缺血后血管新生的分子机制

To promote angiogenesis is the key to the treatment of ischemic stroke. Recent studies have found that exosomes miRNAs plays an important role in regulation of EPCs function and repair of injury vessels. Our previous research has found that houshiheisan can stimulate angiogenesis while exosomes modified by houshiheisan can protect endothelial cells. The improved micro environment of angiogenesis has a big role to play, though it is still unclear about the molecular mechanism of houshiheisan to promote angiogenesis. Based on these researches, a scientific hypothesis is put forward that houshiheisan can affect exosomes miRNAs derived from endothelial cells and regulate EPCs function so as to promote angiogenesis. The focus of our research is to be on the micro environment of endothelial cells. We will probe into the major mechanism of houshiheisan to regulate signaling of angiogenesis through exosomes miR-126 and miR-21 and their target gene Spred-1、P13KR2、SDF-1、Hmga2、WWP1 using techniques such as 3D TOF -MRA, pCASL-PWI, NTA, miRNA interference based on pMCAO rats model combined with oxygen glucose deprivation cell model. Objectives of the research are to provide new ideas for promoting angiogenesis after ischemic stroke and further develop the classical formula and academic thoughts of apoplexy in the tradition of Zhongjing.

促进血管新生是缺血性脑卒中治疗关键。内皮微环境中的外泌体miRNAs对EPCs功能及损伤血管的修复起重要调控作用。前期发现经方侯氏黑散可上调促血管新生因子刺激脑缺血后血管新生，其修饰的外泌体可促进内皮细胞增殖，在这一过程中，侯氏黑散对外泌体miRNAs产生了怎样的关键调控作用尚不清楚。课题提出侯氏黑散可通过影响内皮细胞外泌体miRNAs，调节EPCs功能，促进脑缺血后血管新生的科学假说。拟通过pMCAO大鼠模型结合OGD细胞模型，以内皮微环境为重点，采用3D TOF-MRA、pCASL-PWI、NTA、miRNA干扰等技术，深入研究侯氏黑散对外泌体miR-126和miR-21及靶基因Spred-1、P13KR2、SDF-1、Hmga2、WWP1在调控血管新生相关信号中的作用，分析其促进脑缺血后血管新生的关键作用机制，为脑缺血后促血管新生研究提供新的思路，促进仲景中风学术思想传承及经方发展。

促进血管新生是缺血性脑卒中治疗关键。内皮微环境中外泌体miRNAs对内皮细胞功能重塑和EPCs修复血管损伤的功能起重要调控作用。侯氏黑散如何通过影响外泌体miRNAs，调节内皮细胞和EPCs功能，促进脑缺血后血管新生，是本研究的主要内容。采用pMCAO大鼠模型结合OGD细胞模型，从miR-126和miR-21及靶基因Spred-1、P13KR2、SDF-1α、Hmga2和WWP1探究侯氏黑散促进脑缺血后血管新生信号的分子机制。网络药理学表明侯氏黑散抗脑缺血的靶点显著富集于MAPK、PI3K/AKT和HIF1-α/VEGF等信号通路，涉及到炎症和血管新生。神经功能评分、横木行走实验、旷场实验等结果表明侯氏黑散可以改善脑缺血后神经功能。病理学检结果表明侯氏黑散可以抑制脑缺血后神经元的脱失，减轻血管肿胀，增加缺血脑组织血管管腔数。磁共振成像结果表明侯氏黑散缩小脑缺血后梗死体积，减轻脑组织损伤，增加血管信号强度，改善脑灌注。侯氏黑散可明显上调miR-126的表达，抑制其靶基因Spred1、PIK3R2和SDF-1α的表达，激活SDF-1α/CXCR4、Ras/ERK1/2和PI3K/AKT通路，促进内皮细胞增殖、迁移和成管，改善缺血后血管新生。侯氏黑散也可明显抑制miR-21的表达，上调其靶基因Hmga2和WWP1的表达，抑制TGF-β/SMAD7、p16/p19通路的激活，促进内皮细胞增殖和迁移，改善缺血后血管新生。以上结果表明侯氏黑散通过外泌体miR-126和miR-21调控血管新生关键信号，改善内皮细胞增殖、迁移和成管功能，促进脑缺血后血管新生。