【作者】 余国良；

【导师】 魏尔清；

【作者基本信息】 浙江大学 ， 药理学， 2005， 博士

【摘要】 半胱氨酰白三烯（cysteinyl leukotrienes,CysLTs,包括LTC₄,LTD₄,LTE₄）是花生四烯酸5-脂氧酶的代谢产物。CysLTs是很强的炎症介质,通过激活其受体（CysLT₁和CysLT₂受体）,参与多种炎症病理过程,介导平滑肌痉挛、微血管渗漏等反应。在中枢神经系统内,半胱氨酰白三烯作为一类炎症介质,参与许多疾病的发生发展,如:脑缺血、脑外伤、脑出血、脑肿瘤、脑脊髓炎、多发性硬化、癫痫以及衰老。脑缺血后,脑内半胱氨酰白三烯产生增加,与血脑屏障破坏、脑水肿发生相关。5脂氧酶抑制剂,如AA861,MK-886,和愈创木酸（NDGA）,对脑缺血有保护作用,本实验室也发现CysLT₁受体拮抗剂普鲁司特对脑缺血的保护作用。因此,干预脑内半胱氨酰白三烯通路可能是一条治疗脑缺血的有效途径。 半胱氨酰白三烯受体-1（CysLT₁）拮抗剂主要有普鲁司特（pranlukast,ONO-1078）、孟鲁司特（montelukast）、扎鲁司特（zafirlukast）等,由于其抗炎效应已在临床用于治疗支气管哮喘。我们发现,普鲁司特对大鼠和小鼠局灶性或全脑缺血有保护作用,除了保护神经元免受损伤外,还对血脑屏障破坏引起的脑微血管渗漏、脑水肿等缺血后炎症效应有明显抑制作用。然而,普鲁司特对脑缺血的保护是其特有的作用,还是CysLT₁受体拮抗剂共同的作用?对脑缺血后的炎症变化是否有抗炎浙江大学博士学位论文作用?都需进一步探讨。另外,目前仅证实CysLTI受体拮抗剂在缺血后短期（24一72h）内的抗脑缺血作用,在缺血后长时期（如4一10w）内是否依然有效?仍不清楚。CysLT,受体拮抗剂可能直接拮抗脑内CysLTI受体而起作用,但是,目前对CysLT,受体在脑内（特别是脑缺血后）的表达分布还不清楚,CysLT,受体拮抗剂究竟通过拮抗脑内哪些部位的CysLT,受体而起效?需进一步探讨。 因此,为了深入研究CysLTI受体拮抗剂对脑缺血的治疗作用及其可能机制。本研究在小鼠及大鼠持续性或短暂性脑缺血损伤模型上,探讨了cysLT,受体拮抗剂孟鲁司特、普鲁司特对局灶性脑缺血急性损伤、脑缺血后继发性的炎性损伤、脑缺血慢性损伤的神经保护作用,并探讨了脑缺血后CysLT;受体在脑内的表达改变,以明确CysLTI受体拮抗剂在脑内作用的分子生物学基础。第一部分半肤氨酸白三烯受体一1拮抗剂孟鲁司特对小鼠局灶性脑缺血时间及剂量依赖的保护作用 我们己报告CysLT,受体拮抗剂普鲁司特对脑缺血的保护作用,本实验进一步评价另一种CysLTI受体拮抗剂孟鲁司特的作用,以确定该保护作用是否是该类拮抗剂共同的作用;并且,观察了孟鲁司特作用的时间及剂量依赖性。以大脑中动脉堵塞（MCAo）诱导小鼠持续性局灶性脑缺血。孟鲁司特（0 .01¹ .Om眺g）在术前3天开始每天1次腹腔给药,术前30 min再注射1次;或在术前30 min、术后30 min或lh各给予单次注射;普鲁司特（0 .lm眺g）、依达拉奉（45m姚g）作为阳性对照药。脑缺血后24h,作神经症状评分,测定脑梗死体积、脑水肿、神经元密度和脑内伊文思蓝（EB）渗出。结果显示,孟鲁司特（0 .1、1.om沙g）术前多次或单次注射,能显著减轻各种脑损伤指标;术后30 min单次给药,也能显著减轻脑梗体积和脑水肿,但对减轻神经症状无效;而术后lh单次给药,不能减轻各种脑损伤指标。普鲁司特的作用和孟鲁司特相似,而依达拉奉仅术前多次给药有效。本实验证实孟鲁司特剂量及时间依赖性保护小鼠局灶性脑缺血急性损伤,有效剂量为0.1一1 .om眺g,治疗时浙江大学博士学位论文间窗为术后的作用。30 min;提示cysLTI受体拮抗剂抗脑缺血作用是CysLTI受体拮抗剂共同第二部分半肤氨酞白三烯受体一1拮抗剂普鲁司特对小鼠脑缺血后继发性炎性损伤的保护作用 鉴于CysLTI受体拮抗剂具有抗炎症作用,但对其抗脑缺血损伤中的抗炎作用未作系统研究。本实验评价CysLTI受体拮抗剂普鲁司特对小鼠脑缺血后继发性的炎性损伤及脑内炎症细胞的抑制作用。以大脑中动脉堵塞（MCAO）诱导小鼠持续性局灶性脑缺血,术前及术后30 min各腹腔注射1次普鲁司特（0.0卜o.lm吵g）,观察脑缺血后3d内的神经症状评分、脑梗死体积、神经元变性（Fluoro一JadeB荧光染色）、血脑屏障通透性增高（脑内IgG渗出）、NIPo+中性粒细胞及cD 1 lb+,J‘胶质/巨噬细胞数量。结果发现,普鲁司特0.1 mg/kg改善小鼠脑缺血3d后的神经症状;0.01、o.lm留kg能显著减小梗死体积、皮层缺血周边区变性神经元数目、缺血侧皮层纹状体梗死区lgG渗出,并抑制皮层缺血区中性粒细胞浸润,但对小胶质/巨噬细胞的活化无影响。这些结果证明,CysLT,受体抗剂普鲁司特对小鼠脑缺血后继发性炎症损伤有保护作用,可能部分通过抑制中性粒细胞浸润而发挥抗炎及脑保护作用。第三部分半肤氨酞白三烯受体一1拮抗剂普鲁司特对小鼠慢性缺血性脑损伤的保护作用及抑制胶质疤痕形成 我们已证实cysLT,受体拮抗剂普鲁司特等对脑缺血急性损伤的保护作用,但不清楚是否对慢性损伤也有保护作用。本实验探讨普鲁司特对小鼠脑缺血慢性损伤的持续性保护作用,以及对慢性损伤中胶质疤痕形成的抑制作用。以大脑中动脉堵塞浙江大学博士学位论文（MCAO）巧min诱导小鼠短暂性局灶性脑缺血。腹腔注射普鲁司特（0 .1 mg/kg）,MCAO术后30 min更多还原

【Abstract】 Cysteinyl leukotrienes (CysLTs, including LTC4, LTD4 and LTE4) are the 5-lipoxygenase (5-LOX) metabolites of arachidonic acid. CysLTs are potent inflammatory mediators and involved in various diseases via activating their receptors (CysLT1 and CysLT2). They can induce smooth muscle spasm, microvascular leakage and other pathophysiological processes. In the central nervous system, CysLTs are also involved in pathogenesis of various diseases, such as cerebral ischemia, trauma, hemorrhage, tumor, encephalomyelitis, multiple sclerosis, epileptic seizures and aging. Production of CysLTs is increased in the ischemic brain, and the increased CysLTs are correlated to blood-brain barrier (BBB) dysfunction and brain edema. It has been reported that 5-LOX inhibitors, such as AA861, MK-886 and nordihydroguaiaretic acid, have protective effects on cerebral ischemia, and our previous studies also show the protective effect of CysLTi receptor antagonist, pranlukast (ONO-1078). Therefore, these findings suggest that intervening pathway of CysLTs may be a possible therapeutic strategy of cerebral ischemia.A lot of selective CysLTi receptor antagonists have been developed, for example pranlulukast (ONO-1078), montelukast and zafirlukast. Some of them are used for treatment of bronchial asthma clinically because of their potent anti-inflammatory effects.Recently, we found that pranlukast, a CysLT1 receptor antagonist, protects mice and rats against focal and global cerebral ischemia. In addition to protection of neurons against ischemic injury, it can also inhibit BBB dysfunction, microvascular leakage and brain edema after cerebral ischemia. However, whether the effect of pranlukast is its special effect or a common effect of CysLTi receptor antagonists is unknown. The anti-inflammatory effects of CysLTi receptor antagonists needs to be further investigated. In addition, only short-term neuroprotective effects of CysLTi antagonists have been evaluated within 24-72 h in stroke models. Whether these effects are long lasting (such as 4-10 w after ischemia) is still unknown. Since the effects of CysLTi receptor antagonists may be mediated by antagonizing CysLTi receptor in the brain, the expression and distribution of CysLTi receptor in the brain, especially after ischemia, should be clarified to explain the effects of CysLTi receptor antagonists.Therefore, the purpose this study was to further determine the neuroprortective effects of CysLTi receptor antagonists and its possible mechanisms. We evaluated the neuroprotective effect of montelukast on acute cerebral ischemia, observed the anti-inflammatory effect of pranlukast on inflammatory reactions following cerebral ischemia, and its long-term neuroprotective effect on chronic cerebral ischemia in mice, and investigated the expression of CysLTi receptor in the brain after ischemia in rats.PartiMontelukast, a cysteinyl leukotriene receptor-1 antagonist, dose- andtime-dependently protects against focal cerebral ischemia in miceWe have reported that pranlukast, a CysLTi receptor antagonist, protects rats and mice against cerebral ischemia. We, in this part, further determined the neuroprotective effect of montelukast, another CysLTi receptor antagonist, on focal cerebral ischemia in mice to confirm whether this protective effect is a common effect of CysLTi receptor antagonists. Moreover, the dose- and time-dependencies of the effect of montelukast were also assessed. Permanent focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) in mice. Montelukast was injected intraperitoneally either withmultiple doses (once a day for 3 days and 30 min before MCAO) or single doses (at 30 min before, 30 min after, or 1 h after MCAO) respectively, and pranlukast and edaravone were used as controls. The neurological deficits, infarct volumes, brain edema, neuron density, and Evans blue extravasation in the brain were determined 24 h after MCAO. We found that pretreatment with multiple doses or single dose of montelukast (0.1 and 1.0 mg/kg) before MCAO significantly attenuated all更多还原