依达拉奉联合灯盏花素对脑缺血大鼠神经元、胶质细胞及微血管影响的体内、体外实验研究

Cerebral ischemic disease with its high risk in causing debilitation is one the major cerebral vascular diseases affecting many especially the aged population. Indeed, the incidence of cerebral stroke is rising exponentially in recent years thus threatening the health of many globally. The neurovascular unit (NVU) in the brain is made up of neurons, astrocytes, microglia and cerebral microvessels. Collectively, they maintain the normal function of the brain. Thus, any perturbations to the unit such as an ischemic insult would invariably result in neuronal damage and hence the normal function of the brain. Hence, NVU and its subcomponents in cerebral ischemia would be the key therapeutic target for restoring the normal functions of the brain. This study is aimed to investigate the combined therapy effects of edaravone and breviscapine on NVU in the ischemic cortex in rats subjected to MCAO. To better understand the molecular mechanisms of both drugs, the study will be extended to in vitro investigation using BV-2 cells, a cell line of microglial cells which are known to play a pivotal role in neuroinflammation. Different investigative tools including immunofluorescence, RT-PCR, Western blot (also ELISA), cell culture etc will be adopted to assess the expression levels of various inflammatory mediators such as TNF-α, IL-1β, reactive oxygen species (ROS), nitric oxide (NO) before and after the drug treatments. Along with this, signaling pathway(s) e.g. MAKPs and NF-κB that may be involved in edaravone and breviscapine actions will be elucidated. The information obtained based on the present experimental studies both in vivo and in vitro would be useful for design of a more effective therapeutic strategy on the combined use of edaravone and breviscapine for treatment of cerebral vascular disease patients. The study will also provide a morphological, biochemical and molecular basis for clinical investigation and use of both drugs unique to Yunnan.

缺血性脑血管疾病是当前严重威胁人类健康的一类重要疾病。目前脑缺血治疗的新策略强调对NVU（即神经血管单元：由神经元、神经胶质细胞、微血管等组成的结构复合体）的整体保护。依达拉奉和云南地方特色药物灯盏花素分别有一定的神经保护作用，但两药联合对脑缺血NVU的整体作用及分子机制仍缺乏研究。本项目应用免疫组织化学、RT-PCR、Western（或ELISA）及细胞培养等技术，对两种药物联合治疗大鼠脑缺血后NVU各组成部分的病理变化及相互关系进行系统研究。体外实验拟采用在神经炎症起重要作用的小胶质细胞系（BV-2细胞），对两种药物干预后各种炎症介质如：TNF-α、IL-1β、ROS、NO表达的影响及其可能涉及的MAPKs和NF-κB信号通路进行探讨。预期将在阐明依达拉奉联合灯盏花素治疗脑缺血对NVU的整体保护及分子机制方面有新的发现，为临床两药联用提高脑缺血的治疗效果提供可靠的体内、体外实验依据。

脑缺血是中枢神经系统最常见的疾病，严重威胁人类健康。目前脑缺血治疗强调对NVU（即神经血管单元：由神经元、胶质细胞、微血管等组成）的整体保护。本项目主要研究依达拉奉联合云南地方特色药灯盏花素对大鼠脑缺血NVU各部分的影响及可能涉及的MAPKs、NF-κB、Notch信号通路。结果显示：①依达拉奉联合灯盏花素能明显减小脑梗塞体积，改善大鼠缺血后的神经功能，大脑皮层凋亡细胞和变性固缩神经元数量减少，凋亡相关蛋白Bcl-2表达增强，Bax表达减弱，与单独用药相比差异有显著性。②脑缺血后小胶质细胞激活，体积变大，突起变短，联合用药后小胶质细胞多恢复为分枝状。星形胶质细胞增生、肥大，免疫阳性增强，缺血后1w，胞体逐渐变小，在梗死灶边缘形成胶质纤维网。③脑缺血大鼠大脑皮质炎性介质iNOS、TNF-α、IL-1β，趋化因子MCP-1及毒性介质ROS、NO等表达显著增高，依达拉奉、灯盏花素单独或联合治疗后，这些炎性介质表达明显降低，联合用药TNF-α、MCP-1和NO表达更低，与单药组相比差异有显著性。④两种药物干预后可通过MAPKs (ERKS、JNK、p38)、NF-κB和Notch (NICD，RBP-Jk，Hes-1) 信号通路抑制小胶质细胞激活，减轻小胶质细胞介导的神经炎症反应和星形细胞胶质化，联合用药比单独用药的作用更显著。⑤联合用药与单独用药相比，微血管内皮及基膜的变化不明显。⑥灯盏花乙素可抑制小胶质细胞迁移，增加细胞黏附和细胞内微丝、微管，使小胶质细胞形态和功能发生变化；还可通过小胶质细胞介导，影响星形细胞胶质化。结果表明：依达拉奉联合灯盏花素治疗大鼠脑缺血在减小脑梗死体积和凋亡细胞数量，改善大鼠神经功能，抑制小胶质细胞激活和减少部分炎症介质的表达，减轻神经炎症反应和神经损害等方面具有一定的协同作用，为临床两药联用增强神经保护，提供脑缺血的治疗效果提供可靠的理论和实验依据。