PPARα在脑缺血中改善脑血流的机制研究

Stroke is the first leading cause of death and the primary cause for severe functional disability in China. The poor prognosis of stroke patients is largely due to a lack of effective therapies. The investigators had reported that the agonists of PPARα, fenofibrate, Wy14643 and Gemfibrizol, have been proved to attenuate brain tissue injury after focal ischemia on mice through activating PPARα, promoting SODs expression, inhibiting MMP9 expression and inflammatory response and improving cerebral blood flow. However, how to improve cerebral blood flow and the link among PPARalpha, SODs and cerebral blood flow is not clear. Following the previous research, we further investigate the related mechanism among PPARalpha, SODs and cerebral blood flow with SOD1,3 deficiency mice on MCAO model. To investigate the effects of PPARalpha and SODs on tube formation with culturing endothelial cells in vitro; the effects of SOD1,3 on the function of brain microvessel isolated from SOD1,3 deficiency mice; the regulation of PPARalpha on SOD1,2,3 and MMP9 expression with luciferase report plasmids containing the promoter sequence of gene SOD1,2,3,or MMP9. Using RT-PCR, western blotting for molecular mechanism, cerebral blood flow with Laser Speckle image system and endothelial cell staining for investigating how to attenuate cerebral blood flow under MCAo, and infarct size analysis to investigate the neuroprotection of PPARalpha.. This project will make the neuroprotective effects of PPARalpha clearly through improving the cerebral blood flow with PPARalpha through promoting SODs expression, and the mechanism of PPARalpha regulated SOD1,2,3 and MMP9 expression clearly. And using luciferase report plasmids containing the promoter sequence of gene SOD1,2,3,or MMP9 research the mechanisms how PPARalpha regulates the SOD1,2,3 or MMP9 expression.

缺血性脑卒中是我国第一位致残和致死原因，目前尚缺少较有效的治疗手段。申请者前期研究工作表明，在小鼠脑缺血/再灌流模型中，SODs表达及活性降低，MMP9活性升高，PPARα可以促进SODs表达及酶活性升高，抑制MMP9的活性，PPARα的神经保护机制是改善脑血流和抑制炎症反应。本研究用SOD1,3基因敲除小鼠在脑缺血/再灌流模型中，研究PPARα-SODs-脑血流三者之间的关系；在体外实验中，用培养的内皮细胞研究PPARα-SODs影响内皮细胞增殖和内皮细胞微小管形成的机制，从SOD1,3基因敲除小鼠分离微血管并进行培养，以研究SOD1,3在血管功能方面的作用；用含基因SOD1,2,3和MMP9的启动子序列的荧光素酶报告质粒研究PPARα调控基因SOD1,2,3和MMP9表达的机制；本项目从PPARα改善脑血流和调控SOD1,2,3及MMP9的机制入手，阐述PPARα的神经保护机制。

缺血性脑卒中是我国第一位致残和致死原因，目前尚缺少较有效的治疗手段。本研究中首先研究非诺贝特的保护作用依赖于激活PPARα，然后调节SOD1,2,3的表达以保护I/R损伤。小鼠经非诺贝特处理后，其PPARα和SOD1,2,3的表达增强，脑血流侧支循环改善，从而起神经保护作用；利用PPARα基因敲除小鼠进行I/R模型时，非诺贝特的神经保护作用消失，说明非诺贝特的神经保护作用依赖于PPARα的激活；利用SOD2转基因小鼠，在缺血/再灌注损伤中坏死面积减小及脑血流部分恢复；利用SOD3基因敲除小鼠经Feno处理后，Feno的保护作用部分消失；进一步说明激活的PPARα起作用依赖于调控SODs的表达；在此基础上，利用荧光素酶报告系统研究非诺贝特调控SOD1,2,3表达的机制，结果表明非诺贝特调控SOD1,2,3表达依赖于PPARα的活化，而与非诺贝特是否存在无关；利用PPARα的不同结构域构建质粒，转染细胞后，研究含SOD1,2,3基因启动子的荧光素酶活性，研究结果发现PPARα调控SOD1,2,3表达依赖于DBD结构域，而与LBD结构域无关。另外，利用法舒地尔处理小鼠后，进行I/R模型，结果发现法舒地尔通过抑制ROCK表达，以调控PPARα的表达，PPARα表达改变后，一方面通过影响SODs的表达以调节脑血流侧支循环以改善脑血流；另外一方面通过影响MMP9的表达及活性，维持血脑屏障完整性，从而保护缺血/再灌注损伤，即法舒地尔通过ROCK-PPARα-NOX或者SODs轴以影响缺血/再灌注模型小鼠的脑血流和血脑屏障完整性，从而保护缺血/再灌注损伤。本项目通过非诺贝特和法舒地尔两种药物阐述了PPARα改善脑血流和调控SOD1,2,3及NOX和MMP9的表达，并在细胞水平上研究PPARα调控SOD1,2,3表达的分子机制，以阐明PPARα的神经保护机制。