脑心通对急性缺血性脑组织神经保护机制的研究

发布时间：2018-02-22 17:23

本文关键词： 脑保护脑缺血脑心通凋亡 Akt　出处：《河北医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：脑梗死对人类的健康已然造成严重威胁，通过探讨其病理生理学机制，来寻求有效治疗是当前慢病防治研究的重点之一。近几年研究发现，细胞凋亡是缺血后脑损伤的重要病理生理机制之一。调控脑缺血后凋亡相关基因已成为临床治疗新的靶点。大量的基础体内外实验和临床对照研究发现，脑心通（NXT）具有抗炎、抗氧化、抗凋亡等多种作用，因而，本实验设计在小鼠大脑中动脉闭塞（middle cerebral arteryocclusion，MCAO）所致局灶脑缺血模型上，观察脑心通对缺血急性期脑组织是否具有保护作用及其对局部脑缺血后大脑皮层Akt、CREB以及Bcl-2表达是否有影响。方法：采用雄性成年CD-1小鼠，应用改良Longa线栓法建立小鼠右侧MCAO模型。实验动物随机分为假手术组（Sham）、模型组（MCAO）、溶剂对照组（Vehicle）、脑心通小剂量组（MCAO+NXT360mg/kg, NXT-L）、脑心通大剂量组（MCAO+NXT540mg/kg, NXT-H）、抑制剂组（MCAO+NXT540mg/kg+LY294002，NXT-LY）。术后24h对小鼠进行神经功能评分、测定脑组织的含水量，测定脑梗死体积，应用免疫组化、蛋白印记、实时定量逆转录多聚酶链反应和免疫荧光共定位技术测定Akt、CREB和Bcl-2的表达。应用PI3-K抑制剂LY294002观察其对上述指标表达的影响。结果：模型组、溶剂对照组、脑心通组小鼠均出现左侧肢体偏瘫。脑心通大剂量组的神经功能评分明显低于溶剂对照组（P 0.05），脑心通小剂量组神经功能评分减低，但与溶剂对照组相比无明显差异。在脑缺血后24h，脑心通大剂量组与溶剂对照组相比差异有统计学意义（35.28%±5.92%v.s.46.54%±4.24%, P 0.05）。脑心通小剂量组与溶剂对照组差异无统计学意义。正常组无梗死灶。脑心通大剂量组梗死体积比溶剂对照组明显减小，差异有统计学意义（81.35%±1.75%vs.85.55%±0.93%,P 0.05）。与溶剂对照组相比，脑心通小剂量组梗死体积无明显减小。脑缺血24h后，模型组Akt、CREB及Bcl-2的活性形式表达较正常组减低（P 0.05）。脑心通大剂量组p-Akt、p-CREB、Bcl-2蛋白及Akt、CREB、Bcl-2信使RNA表达升高，与溶剂对照组相比有统计学意义（P 0.05）。脑心通小剂量组Akt、CREB及Bcl-2表达也升高，但与溶剂对照组相比，结果无统计学意义。与脑心通大剂量组相比，抑制剂组p-Akt、p-CREB蛋白表达明显下降，结果有统计学意义（P 0.05）。抑制剂组Bcl-2蛋白表达下降，与脑心通大剂量组相比，结果无统计学意义。结论：脑缺血后脑组织中Akt、CREB及Bcl-2的活性形式表达降低。大剂量脑心通可以改善神经症状，，减轻脑水肿，减轻脑梗死体积，保护局灶性缺血脑组织。应用PI3-k抑制剂LY294002后，缺血区脑组织p-Akt、p-CREB蛋白表达下调，可部分逆转脑心通的保护作用。脑心通对缺血性脑血管病的保护作用可能与上调Akt、CREB、Bcl-2的活性形式表达有关。
[Abstract]:Objective: cerebral infarction has posed a serious threat to human health. It is one of the key points in the study of prevention and treatment of chronic diseases to seek effective treatment by exploring its pathophysiological mechanism. Apoptosis is one of the important pathophysiological mechanisms of brain injury after ischemia. Regulation of apoptosis-related genes after cerebral ischemia has become a new target of clinical therapy. Therefore, this experiment was designed on the model of focal cerebral ischemia induced by middle cerebral artery occlusion (MCAO) in mice. To observe the protective effect of Naoxintong on cerebral tissue in acute phase of ischemia and its effect on the expression of CREB and Bcl-2 in cerebral cortex after local cerebral ischemia. Methods: male adult CD-1 mice were used. The right side MCAO model of mice was established by modified Longa thread embolization method. The experimental animals were randomly divided into sham-operated group, model group, control group, Naoxintong small dose group, MCAO NXT 360 mg / kg, NXT-LJ, Naoxintong large dose group MCAO NXT 540 mg / kg, NXT-HU, inhibitor group, MCAO NXT540mg/kg LY294002NXT-LY. after operation, MCAO NXT 360mg / kg, NXT-LT 540mg / kg, NXT-HU, MCAO NXT540mg/kg LY294002NXT-LY. The neurological function of mice was evaluated at 24 hours. To measure the water content of brain tissue and the volume of cerebral infarction, immunohistochemistry and protein imprinting were used. The expression of Bcl-2 and CREB was detected by real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and immunofluorescence co-localization technique. The effect of LY294002 on the expression of these indexes was observed by PI3-K inhibitor LY294002. Results:. The model group, solvent control group and Naoxintong group all showed left limb hemiplegia, the scores of nerve function in the high dose group of Naoxintong group were significantly lower than those in the solvent control group (P 0.05), and the scores of nerve function in the small dose group of Naoxintong group were lower than those in the control group. But there was no significant difference compared with the solvent control group at 24 hours after cerebral ischemia, the difference between the high dose group and the solvent control group was 35.28% 卤5.92v.s.46.54% 卤4.240.There was no significant difference between the small dose group of Naoxintong and the solvent control group. The infarct volume in the high dose group of Naoxintong group was significantly smaller than that in the solvent control group. The difference was statistically significant (81.35% 卤1.75 vs 85.55% 卤0.93%, P 0.05). Compared with the solvent control group, the infarct volume in the small dose group of Naoxintong did not decrease significantly. After 24 hours of cerebral ischemia, the active forms of Bcl-2 and Bcl-2 in the model group were lower than those in the normal group (P 0.05), and the expression of p-Akttou p-CREBN Bcl-2 protein and the RNA expression of the RNA in the large dose of Naoxintong group were higher than those in the normal group. Compared with the solvent control group, the expression of Bcl-2 and Bcl-2 in the low dose group of Naoxintong was significantly higher than that in the control group, but there was no significant difference between the two groups. Compared with the high dose group of Naoxintong, the protein expression of p-Aktna-p-CREB was significantly decreased in the inhibitor group. Results the expression of Bcl-2 protein in the inhibitor group was significantly lower than that in the high dose group of Naoxintong. Conclusion: after cerebral ischemia, the expression of Bcl-2 and CREB in brain tissue is decreased. High dose Naoxintong can improve the neurological symptoms, reduce the cerebral edema, reduce the volume of cerebral infarction, protect the focal ischemic brain tissue. After the application of LY294002, the PI3-k inhibitor can be used. The down-regulation of p-Aktna-p-CREB protein expression in ischemic brain tissue may partially reverse the protective effect of Naoxintong on ischemic cerebrovascular disease, which may be related to the up-regulation of the active form of Akton-CREB-Bcl-2.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743.31

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