油莎草干预缺血性脑中风功能成分靶向分离鉴定与分子机制研究

There has been an increasing attention to the intervention of active ingredient from plant on ischemic stroke in recent years. Cyperus esculentus L.var.sativus Boeck. has many regional features，including drought resistance and alkali resistance. Our preliminary test results show that its alcohol extracts has significant intervention effect on ischemic/reperfusion injury of the middle cerebral artery occlusion (MCAO) model of rats. Furthermore, we have confirmed the structure identification of active monomer compounds by means of morden chromatographic separation techniques and NMR spectrum analysis technology and selected out of four kind of monomer compounds from target components with orientin as the main composition. However, it remains unclear that whether the protective effect of C. esculentus L. on neurons in ischemic stroke is associated with resistance to oxidative stress and inflammatory reaction, therefore, in our study, we plan to make use of the methods of biochemistry, molecular biology and histology and reveal at molecular, cellular and integral level (i) target component and monomer compounds of C. esculentus L. possess antioxidant activity; (ii) target component and monomer compounds of C. esculentus L. have the ability of resist inflammation; (iii) target component and monomer compounds of C. esculentus L. play the role of antioxidant and anti-inflammatory effects through the inhibition of JNK/c-Jun and Nrf2/ARE signal transduction pathways; (ⅴ) to reveal the active portion of C. esculentus L. through high-throughput microarray to investigate the differences in gene expression and its effect target of active monomer compounds on focal cerebral ischemia-reperfusion injury. The multiple targets for protection and the molecular mechanism of functional constituent from C. esculentus L. intervened ischemic stroke was finally demonstrated.

近年植物活性成分干预缺血性脑中风的研究备受关注。油莎草具有抗旱、耐盐碱等地域特色，前期大鼠脑缺血再灌注（MCAO）损伤的预试验结果表明油莎草醇提物显著干预缺血性脑中风，并且我们已经采用现代色谱分离和NMR波谱分析技术筛选出了以荭草苷（化合物4）为主的4种单体化合物。但油莎草对缺血性中风神经元的保护作用机制是否与抗氧化应激、炎症反应相关，目前仍不清楚。本课题拟采用生物化学、分子生物学、组织学等方法，在分子、细胞、整体水平揭示：（1）油莎草目标组分和单体化合物具有抗氧化活性；（2）油莎草目标组分和单体化合物具有抗炎症反应能力；（3）油莎草目标组分和单体化合物通过抑制JNK/c-Jun与Nrf2/ARE信号转导通路发挥其抗氧化及抗炎作用；（4）结合基因芯片高通量技术研究单体化合物干预缺血脑组织损伤的基因表达差异及作用靶点，揭示活性物质基础。最终阐明油莎草干预缺血性脑中风多靶点保护作用及分子机制。

脑中风引起的氧化应激和炎症反应对脑缺血损伤起着重要的作用。油莎草具有天然药物的一些功能，开展油莎草干预缺血性脑中风功能成分及分子机制研究有现实意义。.项目对油莎草干预缺血性脑中风功能成分进行了靶向分离、筛选及活性单体化合物结构，确定了目标组分，得到4个活性单体化合物；通过大脑中动脉闭塞(MCAO)大鼠动物模型验证目标组分的干预作用；体外实验基于HT22细胞与BV2细胞的CoCl2诱导的模拟动物缺血再灌注（I/R）模型、缺氧缺糖再灌注（OGD/R）损伤模型，研究目标组分及单体荭草苷对模型细胞损伤的保护作用及机制、对模型细胞氧化应激及炎症反应的抑制作用及分子机制。结果表明，目标组分及荭草苷对I/R损伤的HT22、BV2细胞均具有不同程度的改善作用；荭草苷通过降低MDA含量、下调caspase-3水平及减少蛋白质氧化损伤而对CoCl2诱导的HT22细胞I/R损伤产生保护作用。荭草苷通过Keap1-Nrf2-ARE 信号通路来提高细胞的抗氧化能力，它能促进BV2细胞中Nrf2蛋白向胞核内转移，从而抑制IR损伤的BV2细胞氧化应激的作用；荭草苷对OGD/R损伤的BV2细胞有一定炎症抑制作用，通过抑制P65蛋白向核内的转移，抑制IĸB蛋白的降解，减少下游炎症因子的表达，减轻了炎症反应带来的损伤。.采用BV2/PC12缺糖缺氧（OGD/R）模型，研究油莎草醇提物及其30%、50%乙醇洗脱部位对OGD/R损伤的BV2、PC12细胞的保护作用，并基于PI3K/Akt、NF-κB以及JNK/p38信号通路研究油莎草对BV2、PC12细胞损伤发挥保护作用的分子机制。油莎草醇提物均能通过激活PI3K/Akt通路而有效地抑制OGD/R损伤导致的BV2、PC12细胞的凋亡，减轻细胞氧化损伤；油莎草醇提物通过降低JNK/p38信号通路中JNK、p38蛋白磷酸化水平而抑制OGD/R诱导的PC12细胞损伤；油莎草醇提物可能通过蛋白的磷酸化来抑制NF-κB信号通路的激活，而减少炎症因子的表达，对OGD/R损伤的BV2细胞发挥保护作用。.项目揭示了油莎草干预缺血性脑中风多通路、多靶点作用机制，具有很好的抗炎抗氧化作用。研究结果不仅对丰富潜在防治脑中风医药的研究具重要学术价值，而且将大力促进油莎草高产药用植物资源的开发利用。