【作者】 邱丽颖；

【导师】 陈崇宏；

【作者基本信息】 福建医科大学 ， 内科学， 2004， 博士

【摘要】 阿司匹林对大鼠局灶性脑缺血再灌注损伤的保护作用及机制阿司匹林(acetylsalicylic acid / aspirin, ASA)作为非甾体抗炎药,不仅有较好的抗血小板聚集作用,许多实验还证实其有直接的神经保护作用。本研究旨在阐明其对脑缺血-再灌注损伤(cerebral ischemia-reperfusion injury, CIRI)的保护作用、作用机制、有效剂量及最佳用药时间,为临床治疗用药提供理论基础和实验依据。线栓法制作SD大鼠大脑中动脉缺血-再灌注损伤模型,分为假手术组、溶媒组、6 mg. kg-1 ASA治疗组及60mg. kg-1 ASA治疗组。不同组于缺血2 h再灌注同时分别灌胃给予溶媒或不同剂量ASA,观察ASA 再灌注24 h、72 h的脑保护作用及可能机制;缺血2 h、再灌注0 h、1 h、3 h分别给予ASA 60 mg.kg-1 后,观察再灌注24 h的脑保护作用;从形态学角度观察上述各组CIRI时远隔重要器官的损伤及ASA的保护作用。结果:1.不同剂量ASA对CIRI 24 h的保护作用及机制6 mg. kg-1 和60 mg. kg-1剂量ASA均可明显缩小CIRI时脑损伤范围,减轻脑水肿程度,降低大鼠死亡率,抑制脑锥体神经元的丢失,改善脑组织病理形态,以60 mg. kg-1 剂量为显著。60 mg. kg-1 剂量ASA可降低血清中CK和LDH。 6 mg. kg-1 和60 mg. kg-1剂量ASA均可明显提高血浆PGI2/TXA2,阻止炎性介质产生,且有剂量依赖关系;均可提高bcl-2/bax,有明显的抗凋亡作用。6 mg. kg-1 剂量可抑制血清NO水平,升高血浆ET。60 mg. kg-1 剂量可改善脑组织ATP水平;降低脑组织MDA含量。两个剂量ASA对脑组织MPO和CaN活性无明显影响。2.不同剂量ASA对CIRI 72 h的保护作用及机制再灌注72 h与24 h比较,脑损伤范围缩小,脑组织病理形态有所改善,脑水肿程度、大鼠死亡率及脑锥体神经元的密度无显著差异。6 mg. kg-1 和60 mg. kg-1剂量ASA均可明显缩小CIRI 72 h时脑损伤范围,减轻脑水肿程度,降低大鼠死亡率,抑制脑锥体神经元的丢失,改善脑组织病理形态,以60 mg. kg-1为显著。(2)再灌注72 h与24 h比较,脑组织ATP含量进一步下降;脑细胞凋亡数目减<WP=4>少;bcl-2/bax提高;钙调神经磷酸酶(CaN)活性升高。6 mg. kg-1 和60 mg. kg-1剂量ASA均可明显改善再灌注72 h脑组织ATP含量,以60 mg. kg-1 剂量为显著。60 mg. kg-1 剂量可抑制脑细胞凋亡,提高bcl-2/bax,抑制CaN活性升高。3.ASA不同治疗时间窗对CIRI 24 h的保护作用及机制再灌注0 h、1 h、3 h分别给予ASA 60 mg.kg-1 后,均可明显缩小CIRI时脑损伤范围,降低大鼠死亡率,三组间无显著差异;均降低脑含水量,但3 h组效应低于其它两组。抑制脑锥体神经元的丢失,改善脑组织病理形态,但0 h组效果明显好于其它两组。(2) 三个治疗时间窗ASA,只有再灌注0 h给药组可明显改善脑组织ATP含量,与1 h 和3 h组有显著差异。4.ASA对CIRI时远隔重要器官损伤的保护作用6 mg. kg-1 和60 mg. kg-1剂量ASA均可明显改善CIRI 24 h时心、肺、肝和肾的病理形态。再灌注72 h与24 h比较,心、肺、肝和肾的病理形态有所改善,6 mg. kg-1 和60 mg. kg-1剂量ASA均可发挥明显的保护效应。结果表明:(1)6 mg.kg-1 ASA剂量通过阻止炎性介质产生及抗凋亡作用,发挥脑保护效应;60 mg.kg-1剂量ASA通过改善能量代谢、抗氧化、阻止炎性介质产生及抗凋亡发挥脑保护效应。本实验新发现,6 mg. kg-1 ASA主要通过提高bcl-2表达,发挥抗凋亡效应;而60 mg. kg-1 ASA除提高bcl-2表达外,更主要的是抑制bax表达。(2)CIRI 24 h时脑损伤重于72 h; 6 mg. kg-1 和60 mg. kg-1剂量ASA均有明显的保护作用。还新发现CaN的活性在CIRI 72 h明显升高,ASA可抑制其异常升高。(3)再灌注0 h给药效果最佳。(4)6 mg. kg-1 和60 mg. kg-1剂量ASA均可改善CIRI引起的远隔重要器官损伤。更多还原

【Abstract】 Aspirin (acetylsalicylic acid, ASA) as a nonsteroidal anti-inflammatory agent not only has well-established efficacy in anti- thromboxane A2, but there also have several reports about neuroprotective effects. In this study, we design to investigate its mechanism, effective doses, and effective treatment time-window on focal cerebral ischemia-reperfusion injury (CIRI) in rats.Right middle cerebral artery was occluded by inserting a thread through internal carotid artery for 2 h, and then reperfused for 24 h or 72 h. 6 mg·kg-1 and 60 mg·kg-1 doses of aspirin were ig administrated at reperfusion 0 h. The neuroprotective effects and its mechanism were estimated. 60 mg·kg-1 dose of aspirin was ig administrated at reperfusion 0 h, 1 h or 3 h , respectively. The effects on reperfusion 24 h were measured. The protective effects of ASA on remote-organs injury were also studied.RESULTS:The protective effects and its mechanism of ASA on CIRI rats for 24 h (1) With the use of 6 mg. kg-1 and 60 mg·kg-1 doses of ASA, the brain injured area, cerebral edema of occluded side, and mortality were dramatically reduced, the morphology of the occluded area was improved, most of neurons in the injured regions survived ischemia-reperfusion result, lactate dehydrogenase (LDH) and creatine phosphokinase (CK) in plasma were decreased by 60 mg·kg-1 dose of ASA. (2) With the use of 6 mg·kg-1 and 60 mg·kg-1 doses of ASA, the ratio of prostacyclin (PGI2)/ thromboxane (TXA2) in plasma and the ratio of bcl-2/bax in injured brain tissue were increased, the numbers of apoptotic cells were dramatically reduced. In plasma, nitric oxide (NO) content was significantly decreased and endothlin (ET) level was increased by 6 mg·kg-1 dose of aspirin. In brain tissue of occluded side, malondialdehyde (MDA) content <WP=6>was reduced and adenosine 5’-triphosphate (ATP) level was increased by the 60 mg·kg-1 dose of aspirin. No significant effect on superoxide dismutase (SOD), myeloperoxidase (MPO) and calcineurin (CaN) content were discovered.The protective effects and its mechanism of ASA on CIRI rats for 72 hCompare with CIRI 24 h, the brain injured area was reduced and the morphology of the occluded area was improved, no significant difference in cerebral edema of occluded side, mortality and neuron density were discovered on CIRI rats for 72 h. With the use of 6 mg·kg-1 and 60 mg·kg-1 doses of ASA, the brain injured area, cerebral edema of occluded side, and mortality were dramatically reduced, the morphology of the occluded area was improved, most of neurons in the injured regions survived. (2) Compare with CIRI 24 h, in brain tissue the ATP level decreased deeply, the ratio of bcl-2/bax in injured brain tissue increased, the numbers of apoptotic cells reduced, and CaN activity increased on CIRI rats for 72 h. The ATP level was increased by 6 mg·kg-1 and 60 mg·kg-1 doses of aspirin. The numbers of apoptotic cells were reduced, the ratio of bcl-2/bax in injured brain tissue was increased, and CaN activity was inhibited by 60 mg·kg-1 dose of aspirin. 3. The protective effects and its mechanism of ASA with different treatment time-window on CIRI rats for 24 hThe injured area of brain , and mortality in the three groups were dramatically reduced by 60 mg·kg-1 dose of aspirin. No significant difference was discovered among the three groups. The cerebral edema of occluded side in the three groups were dramatically reduced by 60 mg·kg-1 dose of aspirin, but that was more serious in the group of treatment after reperfusion 3 h than that in other two groups. The morphology and neuron density of the occluded area were improved by 60 mg·kg-1 dose of aspirin, but that in the 0 h group was the best in the three groups. The ATP level was improved by 60 mg·kg-1 dose of aspirin in the group of treatment at reperfusion onset, and no similar effect was discovered in other two groups.4. The protective effects of ASA on remote-organs with CIRI rats (1) With the use of 6 mg·kg-1 and 60 mg·kg-1 doses of ASA, morphologic c更多还原