人参皂苷Rg1诱导组蛋白H3K27去甲基化调控缺血性脑卒中后血管新生的作用与机制研究

Cerebral angiogenesis is the most crucial factor determining neurological function recovery and prognosis of ischemic stroke.However,previous studies have been limited to the induction of certain growth factor or receptors related to angiogenesis after drug intervention,whereas the deep and detailed mechanisms are rarely explored.It was found in the preliminary experiments by the research group that ginsenoside Rg1 could significantly induce two histone demethylases specific to H3K27 demethylation,JMJD3 and UTX,and promote migration and tube formation ability of microvascular endothelial cell.Recent studies have shown that histone methylation modification regulate activation of VEGF and Notch signaling,the key signals of angiogenesis.Therefore,we hypothesize that ginsenoside Rg1 firstly induce the expression of H3K27 demethylase JMJD3 and UTX,reducing H3K27 methylation,which leads to the transcriptional activation of key molecules such as VEGF and Jagged1,thus finally promotes angiogenesis.In this project we intend to construct ginsenoside Rg1 nano vesicles,combined with in vitro microvascular endothelial cells (BMECs) system,in vivo rat middle cerebral artery occlusion (MACO) model,the application of WB,ChIP and RNA-seq technology,aiming to explore the function of ginsenoside Rg1-induced epigenetic modifications in angiogenesis.By elucidating the pathways,targets and mechanism of ginsenoside Rg1 function,we provide new ideas for the treatment of ischemic stroke.

血管新生是决定缺血性脑卒中后神经功能恢复及预后的重要因素。以往的研究多限于药物干预对血管新生相关生长因子或受体的诱导表达，更深层次的机制鲜有涉及。项目组前期实验发现：人参皂苷Rg1可显著诱导H3K27特异性去甲基化酶JMJD3和UTX表达，并促进微血管内皮细胞的迁移和成管能力。新近研究显示，血管新生关键信号VEGF与Notch通路的激活受组蛋白甲基化修饰调控。因此我们推测：人参皂苷Rg1可能通过诱导JMJD3和UTX的表达，下调H3K27甲基化水平，进而导致VEGF、Jagged1等关键基因的转录激活，促进血管新生。本项目拟构建人参皂苷Rg1纳米囊泡，应用体外微血管内皮细胞系统与体内大鼠大脑中动脉闭塞模型，通过WB、ChIP与RNA-seq等技术探索人参皂苷Rg1诱导的表观遗传修饰改变在缺血性脑卒中后血管新生中的作用，解析其发挥功能的途径、靶点及作用机理，为缺血性脑卒中的治疗提供新的思路。

缺血性脑卒中是当今严重危害人类健康的多发性疾病。脑部血管新生是决定缺血性脑卒中治疗效果的重要因素。VEGF和Notch是参与血管新生的重要信号通路。人参皂苷Rg1是传统中药人参的主要活性成分，其能促进血管新生的功能已得到广泛证实，但其在表观遗传学层面的调控机制和具体靶分子还需进一步研究。纳米材料药物相对于传统药物具有提高难溶性药物的溶解度、提高生物利用度、提高吸收速率等优势。本研究中采用复乳-溶剂挥发法制备人参皂苷Rg1复合纳米颗粒，分别通过体内大鼠缺血性脑卒中模型和体外分离培养的脑皮质微血管内皮细胞干预处理，利用MTT、定量PCR、transwell、Western blot、co-IP、ChIP、ChIP-seq等技术观察 Rg1-CNV对缺血性脑卒后脑组织血管新生的影响、细胞内Jagged1、VEGF表达以及表观遗传学修饰变化。研究发现Rg1-CNV复合纳米颗粒在体内外缺血性脑卒中模型中对细胞增殖和血管新生的促进作用均优于人参皂苷Rg1。进一步研究发现Rg1-CNV促进去甲基化酶UTX、JMJD3的表达，抑制Jagged1、VEGF启动子区H3K27me3甲基化修饰，从而促进基因的转录和表达，通过VEGF、Notch信号通路激活促进脑卒中后的血管新生。本项目研究主要阐明表观遗传调控在VEGF和Notch通路上的具体作用方式，并为缺血性脑卒中的治疗提供新的分子调控研究基础和备选药物。