天麻活性成分调控VEGF/VEGF-R-Ang/Tie2动态平衡促进脑缺血后血管新生的作用及机制研究

There is close association between the collateral circulations of the brain and the occurrence, development and prognosis of the ischemic cerebrovascular disease. As an important part of the collateral circulations of the brain, the accelerated neovascularization in the ischemic penumbra may promote the repair of nerves in the ischemic area. The balance between the VEGF/VEGF-R and the Ang/Tie2 signaling pathways plays a pivotal role in the formation and maturation of the nascent vasculatures. In our preliminary experiments three extractives from Gastrodia elata were found to enter the brain and show the protective effects on the nerves. Meanwhile, these extractives reduced the cerebral ischemia and reperfusion injury, protected the blood brain barrier and increased the expression of VEGF in the cerebral cortex from MCAO/R rats. In vitro studies also showed that these extractives significantly promoted the migration, proliferation and the tube formation of the cerebrovascular endothelial cells. These findings indicate that these three extractives from Gastrodia elata may promote the formation of the nascent vasculatures. However, it is largely unknown whether or not these extractives could promote the development of the nascent vasculatures into the functional mature vessels. Based on the data from our preliminary experiments and other documented studies, the present project is designed to further investigate the dynamic regulations between the VEGF/VEGF-R and the Ang/Tie2 signaling pathways. In addition, in vivo studies will be performed to demonstrate the effects of these three extractives from Gastrodia elata on the formation and maturation of the nascent vasculatures in different areas of the brain, including the central and the penumbra of the ischemic areas, at different courses of the disease, including the acute phase, transition phase and the subacute phase. Moreover, in vitro studies will also be performed to evaluate the mechanisms involved by applying the small interfering RNA (siRNA) to knock down the expressions of the according genes in the cerebrovascular endothelial cells and the neurocytes.

“血管新生”是脑侧支循环的重要组成部分，促进脑缺血半暗带的“血管新生”可加速机体对缺血区神经的修复。VEGF/VEGF-R-Ang/Tie2系统平衡是“血管新生”过程中新生血管网是否能够形成并最终发展为功能性血管的关键因素。课题组前期研究发现天麻3个入脑成分具有抗大鼠脑缺血再灌注损伤，保护血脑屏障，升高大鼠MCAO/R模型大脑皮层VEGF表达的作用，在体外可促进血管内皮细胞迁移、增殖及血管成环，提示它们可能具有促进缺血后新生血管网形成的作用，但是否能够促进新生血管发育为功能性血管尚不清楚。本课题拟从脑缺血后VEGF/VEGF-R-Ang/Tie2系统的动态调控入手，于不同的空间（缺血中心区、缺血半暗带）、时间（缺血后急性期、过渡期、亚急性期），从整体水平对天麻3个入脑成分促进新生血管网形成并发育为功能性血管的作用进行研究，运用基基因沉默技术，从细胞及分子水平探究它们的作用机制。

本课题从脑缺血后调节VEGF/VEGF-R-Ang/Tie2的动态平衡的角度对天麻三个入脑成分促进脑缺血再灌注损伤后血管新生的作用机制进行了探讨。通过复制大鼠脑缺血再灌注损伤（Middle cerebral artery cclusion/reperfusion, MCAO/R）模型，观察连续给予天麻酚性成分7d、14d、28d后天麻酚性成分3、4、8#在不同时间点都有不同程度升高脑血流量，增加脑皮层及海马齿状回微血管密度，通过调节VEGF/VEGF-R-Ang/Tie2蛋白表达，具有促进脑缺血后血管新生，通过调控脑内皮细胞介导的TGF-β1/Smad信号通路发挥促进新生血管成熟的作用，从而改善神经功能的作用。同时研究表明天麻酚性成分3#具有促进CIRI大鼠早期血管新生和保护BBB的作用，作用机制与上调促血管新生因子VEGF-A、Ang-1和紧密连接Occludin、Claudin-5 mRNA的表达相关。为进一步明确天麻酚性成分促血管新生的作用及机制，本研究还通过小鼠下肢缺血模型研究天麻酚性成分3#具有促进下肢缺血小鼠血管新生及成熟的作用，作用机制与上调VEGF-A及其受体VEGFR-2、Ang-1/Ang-2及其受体Tie-2、Smad-3、ɑ-SMA mRNA表达相关；天麻酚性成分4#、8#具有促进下肢缺血小鼠血管新生的作用，作用机制与上调VEGF-A及其受体VEGFR-2 mRNA的表达相关。3#、4#、8#还有促进脑缺血后NeuN表达，能明显改善MCAO/R模型大鼠的神经功能缺失症状，提示3#、4#、8#在促进血管新生的同时还具有促脑缺血后神经功能恢复的作用。. 天麻三个入脑成分可通过调节脑缺血再灌注损伤后VEGF/VEGF-R-Ang/Tie2的动态平衡促进血管新生及成熟，并具有保护BBB及促进神经修复的作用。其中3#的作用更为明显。