Apelin-13对大鼠局灶性脑缺血—再灌注损伤的保护作用及其机制

发布时间：2019-02-16 12:24

【摘要】：目的：观察Apelin-13对大鼠局灶性脑缺血-再灌注损伤的保护作用并探讨其可能的机制方法：采用线栓法建立S-D雄性大鼠局灶性缺血-再灌注损伤模型,缺血2小时再灌注72小时。进行侧脑室埋管,Apelin-13(0.1、1.0和10.0μg/kg)再灌注前15分钟进行侧脑室注射。术后在不同时间点进行神经功能缺损评分。测量并计算大鼠脑组织含水量。采用2,3,5-氯化三苯基四氮唑(2,3,5-three phenyl tetrazolium chloride, TTC)染色观察并计算脑梗死的体积和比例。H-E染色观察大鼠脑组织切片的病理学改变。取缺血周边脑组织,制备脑组织匀浆上清液,采用分光光度法检测脑组织匀浆上清液中活性氧(reactive oxygen species, ROS)、丙二醛(malonaldehyde, MDA)含量和超氧化物歧化酶(superoxide dismutase, SOD)的活性；采用双抗体夹心法测定脑组织匀浆上清液中总抗氧化力(total antioxidant capacity, T-AOC)的水平；采用ELISA法测定脑组织匀浆上清液中还原型谷胱甘肽(reduced glutathione hormon, GSH)的水平。采用实时定量PCR和Western blot检测缺血周边脑组织中脑源性神经营养因子(brain derived neurotrophic factor, BDNF)和酪氨酸激酶B (tyrosine kinase B, TrkB) mRNA和蛋白的表达。结果：(1)与缺血-再灌注损伤模型组比较,Apelin-13(1.0和10.0μg/kg)组大鼠神经功能缺损明显改善,肌力明显增强,神经功能评分显著性降低(均P0.05)。(2)与缺血-再灌注损伤模型组比较,Apelin-13(1.0和10.0μg/kg)处理显著降低了脑组织的含水量、脑梗死体积和比例,呈现剂量依赖性(均P0.05)。(3)假手术组大鼠脑组织结构正常,神经元的细胞核居中,没有神经细胞的变性和坏死。而与假手术组比较,模型组大鼠脑组织中神经细胞的数目明显减少,可见明显的组织水肿、神经细胞变性和坏死,神经元的细胞核出现不同程度的核固缩和溶解。与模型组相比,Apelin-13(1.0和10.0μg/kg)处理均可明显改善缺血-再灌注后大鼠脑组织中神经细胞的形态,减轻核固缩和胞体肿胀程度,抑制神经细胞的变性和坏死。(4)与缺血-再灌注损伤模型组比较,Apelin-13(1.0和10.0μg/kg)处理显著降低了缺血周边脑组织匀浆上清液中ROS水平和MDA含量(均P0.05),显著增加了缺血周边脑组织匀浆上清液中T-AOC水平、SOD的活性和GSH水平,呈现剂量依赖性(均P0.05)。(5)与缺血-再灌注损伤模型组比较,Apelin-13(1.0和10.0μg/kg)处理显著增加了缺血周边脑组织中BDNF和TrkB mRNA和蛋白的表达,呈现剂量依赖性(P0.05)。结论：Apelin-13对大鼠局灶性脑缺血-再灌注损伤有保护作用,其机制可能与Apelin-13抑制氧化应激、上调BDNF及受体TrkB的表达有关。图12幅,表1个,参考文献73篇。
[Abstract]:Objective: to observe the protective effect of Apelin-13 on focal cerebral ischemia-reperfusion injury in rats and explore its possible mechanism. Ischemia for 2 hours and reperfusion for 72 hours. Apelin-13 (0.1 渭 g/kg and 10.0 渭 g/kg) was injected 15 minutes before reperfusion. Neurological impairment scores were evaluated at different time points after operation. The water content of brain tissue was measured and calculated. The volume and proportion of cerebral infarction were observed and calculated by using the phenyl tetrazolium chloride, TTC) staining and H-E staining to observe the pathological changes of brain sections in rats. The supernatant of brain homogenate was prepared from the ischemic peripheral brain tissue. The content of reactive oxygen species (reactive oxygen species, ROS),) malondialdehyde (malonaldehyde, MDA) and the activity of superoxide dismutase (superoxide dismutase, SOD) in the supernatant of brain tissue homogenate were detected by spectrophotometry. The level of total antioxidant activity (total antioxidant capacity, T-AOC) in brain tissue homogenate supernatant was determined by double antibody sandwich method, and the level of reduced glutathione (reduced glutathione hormon, GSH) in brain tissue homogenate supernatant was measured by ELISA method. The expression of brain-derived neurotrophic factor (brain derived neurotrophic factor, BDNF) and tyrosine kinase (B (tyrosine kinase B, TrkB) mRNA) and protein in peripheral ischemic brain tissues were detected by real-time quantitative PCR and Western blot. Results: (1) compared with the model group of ischemia-reperfusion injury, Apelin-13 (1.0 渭 g/kg and 10.0 渭 g/kg) group significantly improved the nerve function defect and enhanced the muscle strength. Compared with the model group of ischemia-reperfusion injury, Apelin-13 (1.0 渭 g/kg and 10.0 渭 g/kg) significantly decreased the water content of brain tissue, the volume and proportion of cerebral infarction, compared with the model group of ischemia-reperfusion injury (P0.05). (2). In a dose-dependent manner (P0.05). (3), the brain structure of the rats in the sham-operation group was normal, the nucleus of the neurons was in the middle, and there was no degeneration and necrosis of the neurons. Compared with the sham-operated group, the number of nerve cells in the brain tissue of the model group was significantly reduced, and obvious tissue edema, degeneration and necrosis of the neurons and pyknosis and dissolution of the nucleus of the neurons were observed in the model group. Compared with the model group, Apelin-13 (1.0 渭 g/kg and 10.0 渭 g/kg) significantly improved the morphology of neurons in the brain tissue after ischemia-reperfusion, and alleviated the degree of nuclear pyknosis and somatic swelling. Inhibition of degeneration and necrosis of nerve cells. (4) compared with the model group of ischemia-reperfusion injury, Apelin-13 (1.0 渭 g/kg and 10.0 渭 g/kg) significantly decreased the level of ROS and MDA in the supernatant of ischemic brain tissue homogenate (P0.05), and significantly increased the T-AOC level in the supernatant of ischemic peripheral brain tissue homogenate. The activity of SOD and the level of GSH in a dose-dependent manner (P0.05). (5) were compared with those in the model group of ischemia-reperfusion injury. Apelin-13 (1.0 渭 g/kg and 10.0 渭 g/kg) significantly increased the expression of BDNF, TrkB mRNA and protein in the ischemic peripheral brain tissue in a dose-dependent manner (P0.05). Conclusion: Apelin-13 has protective effect on focal cerebral ischemia-reperfusion injury in rats, and its mechanism may be related to the inhibition of oxidative stress and up-regulation of BDNF and receptor TrkB expression by Apelin-13. There are 12 figures, 1 table and 73 references.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743.3

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