【作者】 李姝雅；

【导师】 张祥建；

【作者基本信息】 河北医科大学 ， 神经病学， 2010， 硕士

【摘要】 目的:缺血性脑血管病(cerebral ischemia)是临床常见病和多发病,具有发病率、致残率和病死率高的特点。缺血性脑血管病的损伤机制十分复杂,主要包括能量代谢障碍,兴奋性毒性氨基酸作用,氧自由基损伤,钙超载和炎症反应。至今,对急性缺血性脑血管病继发脑损伤的病理生理机制认识尚未完善,在缺血区和坏死区的过度炎性反应导致的炎性损伤是其复杂损伤机制之一。Notch是一个广泛分布,进化十分保守的跨膜受体蛋白家族。Notch信号通路由受体,配体和DNA结合蛋白3部分组成,它可以通过局部细胞间相互作用,释放胞内段活性成分入核,通过与DNA结合调控下游基因的转录,具有多种生物学功能,并与多种神经系统疾病密切相关。已有报道Notch通过与NF-κB(nuclear factor-κB)共同参与多种癌细胞的调控,并成为癌症的治疗靶点。由此,Notch与NF-κB在疾病的病理生理学过程中存在联系。作为促炎反应的核心因子,NF-κB在脑缺血后脑损伤中表达活跃,起重要作用,是缺血后炎症反应所致脑损伤的核心环节。Notch与NF-κB的相关性提示Notch可能参与脑缺血后炎症反应导致脑损伤。姜黄素具有抗炎、抗氧化、抗癌、抗血管粥样硬化及保护肝肾等多种生物学作用,在缺血性脑血管病中有一定作用,其具体药理机制尚未明确。本实验通过观察大鼠局灶性脑缺血损伤后缺血灶周围Notch1的动态表达及其与NF-κB表达的联系,探讨在脑缺血损伤后脑组织炎性反应中Notch1所起的作用,并进一步明确炎症反应在缺血性脑组织中的损伤机制。用姜黄素干预,观察其对Notch1及NF-κB表达的影响及脑含水量和梗死体积的变化,探讨姜黄素对抗炎症反应的脑保护机制。方法:实验分为两个部分:实验一,Notch1在实验性大鼠脑梗死后脑组织中的动态表达。实验二,姜黄素通过Notch信号通路发挥脑保护作用。实验一采用成年健康雄性Sprague-Dawley大鼠,随机分为正常对照组,假手术1组(sham operated)和大脑中动脉闭塞模型组(middle cerebral artery occlusion, MCAO),用改良线栓法制作MCAO模型,假手术组不插线栓。将MCAO组和sham1组分为6 h、12 h、24 h、48 h、72 h五个亚组,分别在相应时间点用Longa评分法和Berderson评分法对大鼠进行行为学评分,2或3分者纳入实验组。归组后将动物断头处死,取病变脑组织,利用免疫组织化学和Westen blot方法测定Notch1和NF-κB的动态表达。实验二采用成年健康雄性Sprague-Dawley大鼠,随机分为假手术2组(sham operated),溶剂对照组(vehicle-control),姜黄素组(CUR)。MCAO术后立即腹腔注射姜黄素溶液(80mg/kg),溶剂对照组及假手术2组给予同体积含0.5MNaOH的0.01PBS。根据不同时间点每组分为6 h、12 h、24 h、48 h、72 h五个亚组,分别在相应时间点进行神经功能学评分,2或3分者纳入实验组。归组后将动物断头处死,留取病变侧脑组织利用免疫组织化学测定Notch1和NF-κB的阳性细胞表达情况。各组仅取24 h一个时间点进行脑含水量测定及2%2,3,5-三苯基四唑氮红(triphenyltetrazolium chloride,TTC)染色观测梗死体积。结果:1 MCAO组术后各时间点Notch1表达高于sham组(P < 0.05),各点相比,Notch1在梗死后6 h表达已经升高,48 h达高峰,72 h有所回落,但较sham组相比仍较高。2 MCAO组术后各时间点NF-κB表达高于sham组(P < 0.05),各点相比,NF-κB在梗死后6 h表达已经升高,48 h达高峰,72 h有所回落,但较sham组相比仍较高。表达规律与Notch1一致。3脑组织含水量测定:用干湿重法测得的脑组织含水量结果显示:sham组脑含水量为78.16%±8.00%,vehicle-control组为83.71%±7.00%,CUR组的脑组织含水量较vehicle-control组有所下降,为80.42%±9.00%,差异有统计学意义(P < 0.05)。4脑梗死体积测定:TTC染色显示,sham组脑组织均匀红染无梗死灶,MCAO组可见大面积白色梗死灶,多位于右侧基底节、顶叶和颞叶皮质。脑梗死体积测定显示,CUR组脑梗死体积低于MCAO组,差异有统计学意义(P < 0.05)。5 CUR对Notch1和NF-κB表达的影响:免疫组织化学法显示,与vehicle-control组相比,CUR组中Notch1和NF-κB表达下调,差异有统计学意义(P < 0.05)。结论:Notch1在大鼠大脑中动脉闭塞后脑组织中早期既有表达增高,48小时达高峰,随后有所下降,其动态表达规律和NF-κB有相似性。姜黄素干预后,MCAO模型病变脑组织含水量降低,梗死体积减小,Notch1和NF-κB表达水平同步下调,推测姜黄素有脑保护作用,Notch1通过与NF-κB相互作用参与缺血后炎症反应所致的脑损伤,姜黄素可能通过抑制Notch1和NF-κB的表达对缺血性脑组织起到脑保护作用。更多还原

【Abstract】 Objective: Cerebral ischemia, with high mortality and serious disability, is the most common type of cerebral vascular disease. The mechanism of impairment in acute ischemia is so complicated that it is not totally understood. It is the excessive inflammatory reaction residing in necrosis and ischemic region that participates in the complex pathogenesis. Notch is a protein family of conservative transmembrane receptors in a wide distribution. Notch signaling pathway is related with many nervous system diseases by modulating the intercellular reaction. It has been reported that Notch is associated with NF-κB in some way. As a radical factor of inflammation, NF-κB plays an important role in cerebral ischemic pathogenesis. It can be speculated that Notch signaling pathway takes part in the inflammatory reaction that causes the brain damage. Curcumin is extractive confirmed to possess a variety of biological activities including antiinflammatory, antioxidant and anticancer.This study tested the dynamic expression of Notch1 and the relationship between the dynamic expression of Notch1 and NF-κB in rat MCAO modelin order to detect the role of Notch1 in cerebral ischema. Also, this study evaluated the influence of curcumin on brain water content, infarction volume and the expresion of Notch1 and NF-κB to testify the neuroprotective effect of curcumin on cerebral ischemia and the underlying mechanism.Methods: This study included two parts: Part one evaluated the dynamic expression of Notch1 and NF-κB in rat MCAO model. Part two detected the influence of curcumin on brain water content, infarction volume and the expresion of Notch1 and NF-κB.Part one: Male Sprague-Dawley rats were randomly divided into normal control group, sham1 group and MCAO group. Each group was assigned to 5 subgroups randomly: 6 h, 12 h, 24 h, 48 h and 72 h after operation. MCAO animal model was established by modified Longa method. neurological deficit was evaluated at each time point before the rats was sacrificed, 2 or 3 scores were brought into this study. Immunohistochemistry and Westen blot were used to analyse the expression of Notch1 and NF-κB.Part two: Male Sprague-Dawley rats were randomly divided into sham2 group, vehicle control group and curcumin group (80 mg/kg). Curcumin solution was administrated by intraperitoneal injection immediately after MCAO, sham2 group and vehicle control accepted equal volumn 0.01PBS with 0.5M NaOH. The rats, which were evaluated neurological deficit, were sacrificed at 6 h, 12 h, 24 h, 48 h and 72 h after operation. the ischemic brain tissues were used for analyse the expression of Notch1 and NF-κB. At 24 h after operation, brain water content and infarct size was analyzed.Results:1 Notch1 expression in MCAO group is higher than sham group at each time point. The elevation was found at 6 h , peaking at 48 h, and decreased at 72 h, still higher than sham group.2 NF-κB expression in MCAO group is higher than sham group at each time point. the expressive regulation of NF-κB is consistent with the expression of Notch1.3 Brain water content measurement:brain water content in curcumin group was lower than vehicle control group, the difference was statistically significant (P < 0.05) .4 Infarct size analysis:Compared with vehicle control group, curcumin group significantly decreased the infarct size.5 Curcumin’s effect on the expression of Notch1 and NF-κB:Curcumin down regulated the expression of Notch1 and NF-κB,the difference was statistically significant(P < 0.05) .Conclusions:Notch1 were induced at the early stage after MCAO, the expressive regulation of Notch1 is consistent with NF-κB. Curcumin protected the brain damage caused by MCAO, decreased the brain water content and infarct size, down regulated the expression of Notch1 and NF-κB. Notch1 could take part in the inflammatory reaction by interacting with NF-κB, curcumin may protect the brain against ischemic injury through down regulating Notch signaling pathway.更多还原