京尼平苷对链脲佐菌素诱导的AD动物模型学习记忆行为的保护作用，tau蛋白过磷酸化的缓解作用，抗凋亡作用及GSK3调节作用

发布时间：2018-03-27 18:48

本文选题：Alzheimer病　切入点：STZ侧脑室注射　出处：《山西医科大学》2013年硕士论文

【摘要】：虽然已经有五种FDA认证的药物（tacrine, rivastigmine,galantamine, donepezil, and memantine）可以有效的延缓Alzheimer病（AD）的发病进程，但是至今依然缺乏对AD这种中枢神经退行性疾病有效的治疗策略。目前认为，有效阻止AD的方法是依赖于早期的诊断和预防。对于中药治疗AD的研究不失为一种AD的早期预防与治疗有效策略。 AD与2型糖尿病（T2DM）之间的密切关系目前已经得到了广泛的证实。基于两者的共同发病机制，糖尿病治疗药物或许可以发挥神经保护作用。通过激活GLP-1受体，GLP-1类似物已经成为了一种临床上成熟的糖尿病治疗新药。同时，在动物实验中，GLP-1类似物则被证实具有对AD动物模型的神经保护效应。本实验所采用的中药单体京尼平苷（Geniposide）已被证明是一种GLP-1受体激动剂，同时在培养细胞水平已经被证实具有神经保护作用。本实验旨在从AD动物模型的水平证实京尼平苷的神经保护效应。采用链脲佐菌素（STZ）侧脑室注射形成AD大鼠模型。通过Morris水迷宫实验和免疫组织化学实验我们证实了单次侧脑室注射京尼平苷（50μM,10μl）有效的阻止了约40%的STZ诱导的空间认知损伤和约30%的STZ诱导的tau蛋白的过度磷酸化。与我们的预期相反，在免疫组化试验中，我们并没有在STZ诱导的大鼠脑内观察到AD的另一重要病理变化β-淀粉样蛋白（Aβ）的聚集，同时westernblot测定也显示Aβ的前体蛋白——淀粉样前体蛋白（APP）在STZ大鼠脑内的表达也没有明显升高。在治疗组的基础上，我们利用PI3K的特异性抑制剂wortmannin（WT）阻止PI3K活性从而引起其下游蛋白GSK3的去抑制，以此干预治疗组从而探究PI3K/GSK3信号通路在京尼平苷神经保护作用中扮演的角色。结果显示，，WT一定程度上阻止了京尼平苷对STZ诱导的大鼠行为学保护作用以及tau蛋白过度磷酸化的缓解作用，这一结果证实了PI3K/GSK3在京尼平苷的神经保护作用中扮演着关键角色。 GSK3是一种重要的参与tau蛋白磷酸化调节的激酶。GSK3的过度活化在AD的多种病理变化有着广泛的相关性。通过westernblot实验，我们发现STZ侧脑室注射引发了脑内颞叶皮层GSK3β的过度活化。同时，京尼平苷通过对GSK3β两个调控位点的调节——上调抑制位点Ser9的磷酸化，下调激活位点Tyr216的磷酸化——抑制了STZ诱导的GSK3β的过度活化。最后，我们通过透射电镜进行超微结构观察并发现京尼平苷阻止了STZ诱导的超微结构变化，包括双螺旋细丝（PHFs）样结构，突触前膜囊泡的过度聚集，内质网异常，以及以暗细胞为特征的早期凋亡。总之，我们的研究证明了京尼平苷对于STZ急性侧脑室注射诱导的AD大鼠模型的神经保护作用，这也提示京尼平苷可以作为一种AD预防或治疗的新药进行进一步的研究。
[Abstract]:Although there are already five FDA certified drugs, such as tacrine, rivastigmine galantamine, donepezil, and memantine, which can effectively delay the onset of Alzheimer, there is still a lack of effective treatment strategy for AD, a central nervous degenerative disease. The effective method of preventing AD depends on the early diagnosis and prevention. The research on the treatment of AD with traditional Chinese medicine is an effective strategy for early prevention and treatment of AD. The close relationship between AD and type 2 diabetes mellitus (T2DM) has been widely confirmed. Diabetes drugs may play a neuroprotective role. By activating the GLP-1 receptor GLP-1 analogue, it has become a clinically mature new drug for the treatment of diabetes. In animal experiments, GLP-1 analogues have been shown to have neuroprotective effects on AD animal models. Geniposide, a traditional Chinese medicine monomer, has been proved to be a GLP-1 receptor agonist. At the same time, neuroprotective effects of geniposide have been proved at the level of cultured cells. The purpose of this experiment was to confirm the neuroprotective effect of geniposide from the level of AD animal model. The rat model of AD was formed by injecting streptozotocin (STZ) into lateral ventricle. By Morris water maze test and immunohistochemistry, we have proved that a single intracerebroventricular injection of geniposide (50 渭 m ~ (10 渭 l)) can effectively prevent spatial cognitive impairment induced by about 40% STZ and tau protein hyperphosphorous induced by about 30% STZ. Acidification. Contrary to our expectations, In the immunohistochemical test, we did not observe another important pathological change of AD, 尾 -amyloid A 尾, in the brain of rats induced by STZ. Westernblot analysis also showed that the expression of amyloid precursor protein (app) of A 尾 in the brain of STZ rats was not significantly increased. We used the specific inhibitor of PI3K, wortmanningnan WTA, to block the activity of PI3K and induce the desuppression of its downstream protein GSK3. In this intervention group, the role of PI3K/GSK3 signaling pathway in the neuroprotection of genipin was explored. The results showed that WT prevented the behavioral protective effect of genipin on STZ induced rats and tau protein to some extent. The mitigation of hyperphosphorylation, The results confirm that PI3K/GSK3 plays a key role in the neuroprotective effect of genipin. The over-activation of GSK3, an important kinase involved in the phosphorylation of tau protein, has a broad correlation with various pathological changes in AD. We found that intracerebroventricular injection of STZ induced excessive activation of GSK3 尾 in the temporal cortex of the brain. At the same time, geniposide up-regulated the phosphorylation of Ser9, the inhibitory site, by regulating the two regulatory sites of GSK3 尾. Down-regulating the phosphorylation of Tyr216, the activation site, inhibited the overexpression of GSK3 尾 induced by STZ. Finally, we observed the ultrastructure of STZ by transmission electron microscopy and found that geniposide prevented the ultrastructural changes induced by STZ, including the hyperaggregation of presynaptic vesicles and abnormal endoplasmic reticulum (ER). In conclusion, our study demonstrated the neuroprotective effect of genipin on AD rat model induced by acute intracerebroventricular injection of STZ. This suggests that geniposide can be further studied as a new drug for AD prevention or treatment.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R749.1

【共引文献】