【作者】 赵敬；

【导师】 赵涌；

【作者基本信息】 重庆医科大学 ， 生物医学工程， 2010， 博士

【摘要】 研究背景:脑血管疾病是导致全球人口死亡的三大原因之一。缺血性脑血管疾病是脑血管病中最常见的类型。脑缺血再灌注损伤是一个复杂的病理过程,其具体机制目前尚不完全清楚。近年来的研究发现,脑血流中断和再灌注使脑细胞产生损伤的过程是一个快速的级联反应,这个级联反应包括能量障碍、氧自由基的大量释放、细胞酸中毒、兴奋性氨基酸释放增加、细胞内钙失稳态、自由基生成、凋亡基因激活等。这些环节互为因果,彼此重叠,并相互联系,形成恶性循环,最终导致细胞凋亡或坏死。寻找理想的能够中断缺血级联反应的神经保护剂是目前脑血管疾病治疗研究的热点之一。姜黄素是从植物姜黄根茎中提取的一种酚类化合物,具有抗肿瘤、抗炎、抗HIV、抗菌、抗氧化等多种药理作用,并且毒副作用低.具有良好的临床应用潜力,受到国内外的广泛关注。近年来发现姜黄素依靠其良好的抗氧化和抗炎等作用,在脑保护及治疗脑血管病方面具有良好的运用前景。目的:研究姜黄素对大鼠局灶性脑缺血再灌注损伤的神经保护作用并探讨其相关的信号转导机制。方法:第一部分:通过改良线栓法建立大鼠大脑中动脉缺血再灌注模型,随机分为假手术组、缺血再灌注损伤组及姜黄素处理组,对各组进行神经功能评分、TTC染色评价脑梗死体积、TUNEL法检测细胞凋亡、HE及Nissl染色进行组织学观察。同时检测脑组织中丙二醛(MDA)、超氧岐化物(SOD)的变化情况从而评价姜黄素对局灶性缺血再灌注脑损伤大鼠的神经保护作用。第二部分:采用TTC染色法、免疫组织化学法、Western blot法检测在有或无LY294002(一种特异性PI3K通路抑制剂)存在的情况下,姜黄素对局灶性缺血再灌注脑损伤大鼠的神经保护作用,及其对PI3K/AKT通路的Akt/pAkt蛋白,凋亡相关蛋白Bcl2/Bax、Caspase3以及转录因子Nrf2及二相解毒酶NQO1的表达的影响。结果:第一部分,姜黄素可以明显改善脑缺血再灌注损伤后大鼠的神经功能缺损,降低脑梗死体积和细胞凋亡率及神经元的死亡率。同时降低MDA水平及升高SOD水平。第二部分,姜黄素能够增强pAkt、凋亡相关蛋白Bcl-2/Bax及转录因子Nrf2、二相解毒酶NQO1的表达水平,降低Caspase3蛋白的表达;而当LY294002同时存在的情况下,姜黄素的神经保护作用受到抑制,动物神经功能缺损改善情况、脑梗塞面积的缩小均不如单纯给予姜黄素组; pAkt蛋白、Bcl-2/Bax以及Nrf2、NQO1表达水平低于单纯给予姜黄素组,而Caspase3的表达则高于单纯给予姜黄素组。结论:1.姜黄素对局灶性缺血再灌注脑损伤大鼠有确切的神经保护作用,可抑制脑缺血再灌注损伤后的神经元凋亡及死亡,减小脑梗死体积,改善神经功能2.姜黄素的神经保护主要通过抗氧化及抗凋亡的作用实现。而通过Pl3K/Akt途径调节凋亡相关蛋白及抗氧化酶的表达可能是其实现神经保护作用的重要机制之一。更多还原

【Abstract】 Background: Cerebrovascular disease is the third leading cause of death in the world. Ischemic cerebrovascular disease is the most common type in the cerebrovascular disease. Cerebral ischemia/reperfusion injury is a complex pathophysiologic process which is not been clarified now. Recent studies found that the injuries caused by cerebral blood flow cessation and reperfusion are a rapid cascade reaction which includes disfunction of ATP production, overproduction of reactive oxygen species, intracellular acidosis , increased release of excitatory amion acids, destabilization of intracellular calcium and apoptosis-related genes activation and so on. These pathophysiologic processes overlap and intercommunicate then form a vicious cycle which results in cell apoptosis or necrosis. It is a focus in neuroscience that looking for the ideal neuroprotective agents that can block ischemic cascade reaction. Curcumin is a phenol derived from tumeric which has anticancer, antiinflammation, anti-HIV and antioxidation properties and low toxic and side-effect. It has good potential in clinic. From recent years, curcumin was been found that it has understanding application prospect in neuroprotction and therapeutics of cerebrovascular disease because of its antioxidation and antiinflammation properties.Objecitve: To study neuroprotective effects of curcumin on focal cerebral ischemia/reperfusion injury in rats and explore its possible mechanisms and related signal transduction pathways.Methods: Part one : Transient cerebral ischemia/reperfusion model in rats was established by middle cerebral artery occlusion using modified suture occlusion technique.The rats were randomly divided into sham group, MCAO group, curcumin-treated group. Neuroscores of all groups were evaluated. Infarct volume was measured by TTC staining and morphologic changes were observed by H.E. and Nissl staining. TUNEL was used for detecting cell apoptosis. Meanwhile, we measured Malondialdehyde (MDA) and superoxide dismutase(SOD) of cerebral tissue to evaluate antioxidation activitis of curcumin . Part two: TTC staining, immunohistochemisty , Western blot technique were used to explore neuroprotection of curcumin on focal cerebral ischemia/reperfusion injury in rats ,with or without the existence of LY294002(a inhibitor of phosphatidylinositol-3- kinase ,PI3K).And also, the effects of curcumin on the expression of phospha-Akt, apoptosis-associated protein Bcl2/Bax、Caspase3 ,transcript factor Nrf2 and NQO1. Results: Part one: Curcumin could significantly improve the neurologic impairment, reduce cerebral infarct volume and decrease neuronal apoptosis and necrosis. And also, curcumin could decrease the level of MDA whereas increase the level of SOD. Part two: Curcumin could increase the expression of pAkt, antiapoptosis protein Bcl-2, transcript factor Nrf2 and II detoxifying enzymes NQO1 whereas decrease the expression of proapoptosis protein Bax and caspase-3. However, with the existence of LY294002, the neuroprotection of curcumin was inhibited. Improvement of neurologic impairment and reduction of cerebral infarct volume in Cur+LY group were less than that in CUR group. Moreover, the expression of pAkt, Bcl-2/Bax , Nrf2 and NQO1 in CUR+LY group were lower than that in CUR group whereas the expression of caspase-3 protein in CUR+LY group was higher than that in CUR group.Conclusions: 1.Curcumin has exact neuroprotective effects on focal cerebral ischemia/reperfusion injuries on rats which could inhibit the neuronal apoptosis and necrosis, reduce cerebral infarct volume and improve neurologic impairment. 2. The neuroprotection of curcumin was accomplished by antioxidation and antiapoptosis. It was one of important neuroprotective mechanisms of curcumin that regulating the expression of apoptosis-associated protein and antioxidative enzyme by PI3K/Akt pathway.更多还原