【作者】 王娜；

【导师】 郭曲练；

【作者基本信息】 中南大学 ， 麻醉学， 2011， 博士

【摘要】 脑缺血一定时间恢复血液供应后,其功能不但未能恢复,却出现了更加严重的脑机能障碍,称之为脑缺血/再灌注(Ischemic-reperfusion,I/R)损伤。临床麻醉中颈动脉瘤手术、颅内动脉瘤切除术、心肺复苏过程中均不可避免地发生脑缺血再灌注损伤,影响了患者术后预后,这引起了麻醉工作者的重视,如何防治脑缺血再灌注损伤已成为科研的重要内容之一。环氧化酶(Cyclcoxygenase, COX),又称前列腺素内过氧化物合成酶,是前列腺素(Prostaglandin, PG)合成过程中一个重要限速酶,催化花生四烯酸经由PGG2转化成PGH2。 COX分为结构型(COX-1)和诱导型(COX-2)。COX-1被认为是“看家基因”,在大多数正常组织中的表达稳定；而COX-2被认为是“快速反应基因”,静息时不表达,仅在细胞受到刺激时迅速合成,细菌脂多糖、生长因子、促炎细胞因子、促肿瘤剂可促进COX-2表达增加。基于此人们推测COX-2是在炎症和其他病理情况中的主要存在形式。大量研究表明COX-2在缺血性脑损伤中至关重要。在离体实验以及不同脑缺血、脑出血、脑外伤实验模型中,抑制COX-2可以起到一定的神经保护保护作用。尽管2004年9月Merck公司自愿召回因长期慢性服用导致严重心血管事件的罗非考昔(商品名：万络),但是抑制缺血性神经元表达COX-2、 COX-2下游损伤因子仍然是令人感兴趣的研究方向。诸多动物实验都证实短期给予COX-2抑制剂并不加重脑损伤,但是真正应用于临床时,临床医师还是有所顾虑的。相对于其它COX-2抑制剂,临床上更易接受帕瑞昔布。帕瑞昔布(商品名：特耐)是第二代COX-2抑制剂,是唯一可以经静脉注射或肌内注射的高选择性COX-2抑制剂,现用于术后疼痛的短期治疗,不良反应少。目前研究发现帕瑞昔布能减轻心肌缺血性损伤,抑制心肌细胞死亡,改善心脏功能。但帕瑞昔布介导的脑保护机制目前仍不清楚,值得我们深入研究。越来越多的研究证据表明神经炎症在脑缺血损伤进展中发挥着举足轻重的作用。由于磷脂酶的协调作用,缺血后的膜磷脂释放大量游离的花生四烯酸,经由COX的花生四烯酸代谢途径在脑血管闭塞后的神经炎症中发挥着积极作用,是各种形式脑损伤包括脑缺血中触发和促进炎症进展的重要成分。人们已经在心肌缺血再灌注模型中发现抑制高迁移率族蛋白1(High mobility group box1protein, HMGB1)表达可以有效发挥心肌保护作用,我们推测抑制炎症介质HMGB1表达有可能介导帕瑞昔布的神经保护机制。蛋白激酶A(Protein kinase A, PKA)/cAMP反应元件结合蛋白(cAMP-response element-binding protein,CREB)信号转导途径是重要的神经保护机制之一,在神经发育、神经再生、记忆功能、突触可塑性及细胞修复中发挥着中枢性作用。自噬性程序性细胞死亡即Ⅱ型程序性细胞死亡,不同于细胞凋亡,吸引了越来越多研究人员的关注。在缺血、炎症等应激状态下,自噬可以被激活且与细胞死亡密切相关。研究发现Beclin1在心肌缺血再灌注阶段的自噬现象中发挥着重要的作用。因此,本课题选用远端大脑中动脉永久性阻断后再阻断双侧颈总动脉60min的缺血再灌注模型,于缺血前15min、缺血后12h经阴茎背静脉注射帕瑞昔布,探讨炎症反应、PKA/CREB、自噬是否参与了其神经保护作用。本课题首先通过评估再灌注后大鼠神经功能缺陷评分、脑梗死体积百分比,观察不同剂量的帕瑞昔布是否对Sprague-Dawley大鼠脑缺血再灌注损伤具有保护作用；其次,第二部分通过评估再灌注后大鼠神经功能缺陷评分、脑梗死体积百分比、缺血侧大脑皮层神经元HE染色及Niss1染色形态学变化、缺血半影区炎症介质TNF-α、HMGB1表达,提示帕瑞昔布可能通过抑制炎症反应及炎症介质表达而发挥有效的神经保护作用；随后,第三部分给予特异性PKA抑制剂,观察再灌注后大鼠神经功能缺陷评分、脑梗死体积百分比、缺血半影区p-CREB及脑源性神经营养因子(Brain-derived neurotrophic factor, BDNF)的表达,探讨帕瑞昔布介导的脑保护效应可能是激活PKA/CREB途径,增加p-CREB、BDNF的表达所致；最后,第四部分给予自噬抑制剂,观察再灌注后大鼠神经功能缺陷评分、脑梗死体积百分比、缺血侧大脑皮层神经元Nissl染色、缺血半影区Beclin1表达,推测帕瑞昔布可能通过抑制自噬Beclin1表达而减少神经细胞死亡,减轻脑缺血再灌注损伤。本课题研究结果证实：(1)静脉注射不同剂量的帕瑞昔布均能明显促进脑缺血后神经功能恢复,减小脑梗死体积,产生显著的神经保护作用。但未证实帕瑞昔布在本模型中的神经保护作用呈剂量依赖性；(2)帕瑞昔布可能通过抑制炎症反应及炎症介质表达,减少缺血再灌注所致的神经元死亡,发挥显著的神经保护作用；(3)帕瑞昔布可能通过激活PKA/CREB途径,增加p-CREB的表达,进而上调BDNF表达,减轻脑缺血再灌注损伤,发挥神经保护作用；(4)帕瑞昔布可能通过抑制自噬Beclin1表达,促进神经功能恢复,减少脑缺血再灌注造成的神经元死亡,发挥一定的神经保护作用。总之,脑缺血及再灌注所致的神经元损伤是一系列极为复杂的病理生理过程,而帕瑞昔布介导的神经保护机制也是一个多分子参与的多信号通路交织的复杂级联反应。第一章不同剂量帕瑞昔布对大鼠脑缺血再灌注损伤的保护作用目的：观察静脉注射帕瑞昔布4mg/kg或20mg/kg是否能发挥脑保护效应。方法：健康雄性SD大鼠,体重300-350g,随机分为四组：假手术组(Sham)、缺血再灌注组(I/R)、帕瑞昔布1组(Parecoxib1)、帕瑞昔布2组(Parecoxib2)。电凝左侧大脑中动脉永久性阻断(Permanent middle cerebral artery occlusion, pMCAO)后再阻断双侧颈总动脉60min,缺血前15min、缺血后12h经阴茎背静脉注射帕瑞昔布4mg/kg或20mg/kg。Sham组与I/R组注射等容积的生理盐水。各组于麻醉5min、大脑中动脉永久性阻断15min及再灌注15min时分别记录平均动脉压、心率、体温,并抽动脉血行血气分析。再灌注1d、3d、7d时评估神经功能缺陷评分,再灌注3d时2,3,5一三苯基氯化四氮唑(2,3,5-triphenyltetrazolium chloride,TTC)法测定大鼠脑梗死体积百分比。结果：各组pH值、动脉血二氧化碳分压(PaCO2)、动脉血氧分压(Pa02)、平均动脉压(MAP)及体温(T)差异无统计学意义(P>0.05)；与I/R组相比,再灌注1d、3d、7d时神经功能缺陷评分显示Parecoxib1组和Parecoxib2组均能明显改善神经功能(P<0.05),但两组之间比较无统计学差异(P>0.05)；与I/R组相比,再灌注3d时TTC染色显示Parecoxib1组和Parecoxib2组均能明显减少脑梗死体积百分比(P<0.05),但两组之间比较无统计学差异(P>0.05)。结论：静脉注射不同剂量的帕瑞昔布均能明显促进脑缺血后神经功能恢复,减小脑梗死体积,产生显著的神经保护作用。但未证实帕瑞昔布在本模型中的神经保护作用呈剂量依赖性。第二章帕瑞昔布对脑缺血再灌注损伤炎症反应的影响目的：观察帕瑞昔布对大鼠脑缺血再灌注损伤后缺血半影区炎症介质TNF-α、HMGB-1表达的影响。方法：健康雄性SD大鼠,体重300-350g,随机分为三组：假手术组(Sham)、缺血再灌注组(I/R)、帕瑞昔布组(Parecoxib)。缺血再灌注模型为pMCAO并阻断双侧颈总动脉60min,缺血前15min、缺血后12h经阴茎背静脉注射帕瑞昔布4mg/kg。Sham组与I/R组注射等容积的生理盐水。再灌注1d、3d、7d时评估神经功能缺陷评分、缺血侧大脑皮层神经元HE染色及Niss1染色形态学变化、缺血半影区炎症介质TNF-α、 HMGB1表达；再灌注3d时TTC法测定大鼠脑梗死体积百分比。结果：与I/R组相比,再灌注1d、3d、7d时,帕瑞昔布能明显改善神经功能、减少TNF-a阳性细胞及HMGB1的表达(P<0.05)；再灌注3d时,与I/R组相比,Parecoxib组能明显减少脑梗死体积百分比(P<0.05)。结论：帕瑞昔布可能通过抑制炎症反应及炎症介质表达,减少缺血再灌注所致的神经元死亡,发挥显著的神经保护作用。第三章PKA/CREB信号途径在帕瑞昔布脑保护效应中的作用目的：探讨PKA/CREB信号途径在帕瑞昔布脑保护效应中的作用。方法：健康雄性SD大鼠,体重300-350g,随机分为五组：假手术组(Sham)、缺血再灌注组(I/R)、帕瑞昔布组(Parecoxib)、帕瑞昔布+H-89组(Parecoxib+H-89)、H-89组(H-89)。缺血再灌注模型为pMCAO并阻断双侧颈总动脉60min,缺血前30min腹腔注射PKA抑制剂H-89,缺血前15min、缺血后12h经阴茎背静脉注射帕瑞昔布4mg/kg。再灌注1d、3d、7d时观察神经功能缺陷评分、缺血半影区p-CREB及BDNF的表达；再灌注3d时TTC法测定大鼠脑梗死体积百分比。结果：与I/R组相比,再灌注1d、3d、7d时,帕瑞昔布能明显改善神经功能、增加p-CREB及BDNF的表达(P<0.05)；再灌注3d时,与I/R组相比,Parecoxib组能明显减少脑梗死体积百分比(P<0.05)；PKA抑制剂H-89能拮抗帕瑞昔布的脑保护效应(P<0.05)。结论：帕瑞昔布可能通过激活PKA/CREB途径,增加p-CREB的表达,进而上调BDNF表达,减轻脑缺血再灌注损伤,发挥神经保护作用。第四章帕瑞昔布对脑缺血再灌注损伤自噬的影响目的：观察帕瑞昔布对大鼠脑缺血再灌注损伤后白噬的影响。方法：健康雄性SD大鼠,体重300～350g,随机分为四组：假手术组(Sham)、缺血再灌注组(I/R)、帕瑞昔布组(Parecoxib)、3-甲基腺嘌呤组(3-MA)。缺血再灌注模型为pMCAO并阻断双侧颈总动脉60min,缺血前15min、缺血后12h经阴茎背静脉注射帕瑞昔布4mg/kg,3-MA组于缺血前15min侧脑室给予自噬抑制剂3-MA600nmol。再灌注1d、3d、7d时观察大鼠神经功能缺陷评分、缺血侧大脑皮层神经元Nissl染色、缺血半影区Beclinl表达；再灌注3d时TTC法测定大鼠脑梗死体积百分比。结果：与I/R组相比,再灌注1d、3d、7d时,帕瑞昔布、3-MA均能明显改善神经功能、增加存活神经元、减少Beclinl表达(P<0.05)；再灌注3d时,与I/R组相比,Parecoxib组、3-MA组均能明显减少脑梗死体积百分比(P<0.05)。结论：帕瑞昔布可能通过抑制自噬Beclinl表达,促进神经功能恢复,减少脑缺血再灌注造成的神经元死亡,发挥一定的神经保护作用。更多还原

【Abstract】 Part I:The neuroprotection induced by parecoxib treatment against cerebral ischemia/reperfusion injuryObjective:To investigate the neuroprotective effect of4mg/kg or20mg/kg parecoxib treatment against cerebral ischemia/reperfusion injury in rats. Methods:Adult male Sprague-Dawley rats (300～350g) were randomly assigned into four groups:the sham group (Sham), ischemia/reperfusion group (I/R), ischemia/reperfusion+4mg/kg parecoxib group (Parecoxibl), ischemia/reperfusion+20mg/kg parecoxib group (Parecoxib2). The left middle cerebral artery was permanently occluded by bipolar electrical coagulation and both commom carotid arteries were occluded with miniature clips for60min. The Parecoxib groups received parecoxib4mg/kg or20mg/kg intravenously15min before ischemia and again at12h after ischemia, and the Sham and I/R groups received normal saline in the same way. Physiological variables (mean arterial blood pressure, heart rate, temperature, and arterial blood gases) were measured at5min after induction of anesthesia,15min after ischemia, and15min after reperfusion. The neurologic deficit scores (NDSs) were evaluated at Id,3d and7d after reperfusion. Infarct volume was assessed with2,3,5-triphenyltetra-zolium chloride (TTC) staining at3d after reperfusion. Results:No difference was found in rectal temperature, mean arterial blood pressure, arterial pH, PaCO2and PaO2in the four groups (P>0.05). Compared with the I/R group, the Parecoxib1and Parecoxib2groups improved the neurological functions at1d,3d and7d after reperfusion (P<0.05). Animals in the Parecoxibl and Parecoxib2developed smaller brain infarct volumes than the I/R group at3d after reperfusion (P<0.05). But no difference was found in NDSs and infarct volumes between the Parecoxib1and Parecoxib2groups (P>0.05). Conclusions:Parecoxib treatment could induce the neuroprotection against cerebral ischemia/reperfusion injury, but the protective effect was not dose-dependent in this model. Part II:The neuroprotection induced by parecoxib treatment via anti-inflammation pathway against cerebral ischemia/reperfusion injuryObjective:To investigate the neuroprotective effect of parecoxib via anti-inflammation pathway against cerebral ischemia/reperfusion injury in rats. Methods:Adult male Sprague-Dawley rats (300～350g) were randomly assigned into three groups:the sham group (Sham), ischemia/reperfusion group (I/R), parecoxib group (Parecoxib). The left middle cerebral artery was permanently occluded by bipolar electrical coagulation and both commom carotid arteries were occluded with miniature clips for60min. The Parecoxib group received parecoxib4mg/kg intravenously15min before ischemia via vena dorsalis penis and again at12h after ischemia. The NDSs, Hematoxylin and eosin staining, Nissl staining, and expression of Tumor necrosis factor-a (TNF-a) and High mobility group box1protein (HMGB1) were evaluated at1d,3d and7d after reperfusion. Infarct volume was assessed with TTC staining at3d after reperfusion. Results:Compared with the I/R group, parecoxib administration significantly improved NDSs, reduced infarct volume, and decreased TNF-a and HMGB1expressin (P<0.05). Conclusions:Treatment with intravenous parecoxib was neuroprotective. Its effects may be associated with the attenuation of inflammatory reaction and the inhibition of inflammatory mediators. Part III:The neuroprotection induced by parecoxib treatment via the PKA/CREB pathway against cerebral ischemia/reperfusion injuryObjective:To investigate the neuroprotective effect of parecoxib via PKA/CREB pathway against cerebral ischemia/reperfusion injury. Methods:Adult male Sprague-Dawley rats (300～350g) were randomly assigned into five groups:Adult male Sprague-Dawley rats (300～350g) were randomly assigned into three groups:the sham group (Sham), ischemia/reperfusion group (I/R), parecoxib group (Parecoxib), ischemia/reperfusion+H-89group (H-89), parecoxib+H-89group (Parecoxib+H-89). The left middle cerebral artery was permanently occluded by bipolar electrical coagulation and both commom carotid arteries were occluded with miniature clips for60min. The Parecoxib groups received parecoxib4mg/kg intravenously15min before ischemia via vena dorsalis penis and again at12h after ischemia. The NDSs, and expression of CREB and BDNF were evaluated at Id,3d and7d after reperfusion. Infarct volume was assessed with TTC staining at3d after reperfusion. Results:Compared with the I/R group, parecoxib administration significantly improved NDSs, reduced infarct volume, and increased CREB and BDNF expressin (P<0.05). The effect was abolished by H-89(P<0.05). Conclusions:Treatment with intravenous parecoxib was neuroprotective. The neuroprotection of parecoxib may be mediated by PKA/CREB pathway. Part IV:Effect of parecoxib treatment on autophagy against cerebral ischemia/reperfusion injuryObjective:To investigate the neuroprotective effect of parecoxib via autophagy pathway against cerebral ischemia/reperfusion injury in rats. Methods:Adult male Sprague-Dawley rats (300～350g) were randomly assigned into four groups:the sham group (Sham), ischemia/reperfusion group (I/R), parecoxib group (Parecoxib), ischemia/reperfusion+3-MA group (3-MA). The left middle cerebral artery was permanently occluded by bipolar electrical coagulation and both commom carotid arteries were occluded with miniature clips for60min. The Parecoxib group received parecoxib4mg/kg intravenously15min before ischemia via vena dorsalis penis and again at12h after ischemia. The NDSs, Nissl staining, and Beclinl expression were evaluated at Id,3d and7d after reperfusion. Infarct volume was assessed with TTC staining at3d after reperfusion. Results:Compared with the I/R group, parecoxib and3-MA administration significantly improved NDSs, reduced infarct volume, increased alive neurons and decreased Beclinl expression (P<0.05). Conclusions:Treatment with intravenous parecoxib was neuroprotective. Its effects may be associated with the attenuation of autophagy and the inhibition of Beclinl expression.更多还原