银思维对拟SAD大鼠脑内tau磷酸化及其O-GlcNAc修饰的调节作用及相关研究

【作者】 第五永长；

【导师】 田金洲；

【作者基本信息】 北京中医药大学 ， 中西医结合临床， 2011， 博士

【摘要】 目的：基于胰岛素信号转导与散发性老年性痴呆(SAD)的关系及SAD发病的脑能量代谢障碍学说与tau蛋白异常修饰学说,以大鼠侧脑室注射链脲佐菌素(ICV-STZ)建立SAD动物模型,研究具有升发阳气,祛逐阴邪,健脾开郁化痰、平衡机体阴阳功效的中药复方银思维提取物对SAD模型大鼠的行为学、海马神经元超微结构、脑能量代谢及胆碱能系统相关酶的活性、脑组织tau蛋白磷酸化与O-G1cNAc糖基化修饰的影响及调节作用,探讨复方银思维防治SAD的可能作用机制。方法：将SPF级健康雄性SD大鼠随机分为假手术组、模型组、多奈哌齐对照组、银思维小、中、大剂量组。除假手术组外,其余动物双侧侧脑室重复注射STZ造模,第二次造模后21天开始治疗。治疗组分别以盐酸多奈哌齐、银思维大、中、小不同剂量浓度灌胃,模型组及假手术组给予等体积的双蒸水,各组均每日1次,共灌胃2个月。治疗结束后,以MOrris水迷宫试验法测试大鼠的空间学习记忆能力,以透射电镜观察大鼠海马CA1区微管等超微结构的变化,以生化方法测定大鼠大脑皮层能量代谢与线粒体功能相关酶以及胆碱能系统酶的活性,以免疫组化及蛋白印迹方法检测大鼠脑组织tau蛋白O-G1cNAc糖基化修饰水平及重要位点Thr231、Ser422的磷酸化水平,同时检测O-G1cNAc糖基化修饰过程中OGT、O—G1cNAcase酶表达的变化。结果：1.ICV-STZ拟SAD模型大鼠与假手术组相比,在定向航行试验中的平均逃避潜伏期及总游泳距离显著延长(P<0.01),空间探索试验中在目标象限的游泳时间明显缩短(P<0.01)。复方银思维能明显缩短SAD模型大鼠在定向航行试验中的平均逃避潜伏期及总游泳距离,组间比较差异有统计学意义(P<0.05,P<0.01),且能延长SAD大鼠空间探索试验中在目标象限的游泳时间(P<0.05,P<0.01)。银思维小、中、大剂量组与多奈哌齐组组间比较无显著性差异(P>0.05)2.SAD模型组大鼠大脑皮层SDH、COX、Na+-K+-ATPase活性明显低于假手术组(P<0.01),而复方银思维能显著提高SAD大鼠皮层SDH、COX、Na+-K+-ATPase的活性(P<0.05,P<0.01),多奈哌齐对照组与SAD模型组相比则无显著性差异(P>0.05)3.SAD模型组大鼠大脑皮层ChAT活性明显低于假手术组(P<0.01),而AchE活性高于假手术组(P<0.01),复方银思维能够提高大鼠大脑皮层ChAT活性,同时使得AchE活性下降,组间差异有统计学意义(P<0.05,P<0.01),多奈哌齐组与银思维小、中、大剂量组组间比较无显著性差异(P>0.05)4.SAD模型大鼠海马组织以sWGA富集的O-G1cNAc糖基化蛋白质明显低于假手术组(P<0.01),且用RL2、CTD110.6抗体检测的O-G1cNAc糖基化tau蛋白表达明显低于假手术组(P<0.01)；而复方银思维能明显提高SAD大鼠海马组织以sWGA富集的O-G1cNAc糖基化蛋白质以及用RL2、CTD110.6检测的O-G1cNAc糖基化tau蛋白表达,组间差异有统计学意义(P<0.05,P<0.01)。多奈哌齐对照组与SAD模型组相比则无显著性差异(P>0.05)。5.SAD模型大鼠海马组织OGT的表达明显低于假手术组(P<0.01)O-G1cNAcase表达明显高于假手术组(P<0.01),而复方银思维能显著提高SAD大鼠海马OGT的表达,降低O-G1cNAcase的表达,组间差异有统计学意义(P<0.05,P<0.01)。多奈哌齐对照组与SAD模型组相比无显著性差异(P>0.05)6.SAD模型大鼠海马组织tau蛋白在重要位点Thr231和Ser422的磷酸化P-Thr231、P-Ser422表达高于假手术组(P<0.01),而复方银思维能减少SAD模型大鼠海马组织P-Thr231、P-Ser422的表达(P<0.05,P<0.01)。多奈哌齐对照组与SAD模型组相比则无显著性差异(P>0.05)结论：1. ICV-STZ模型大鼠存在脑皮层能量代谢与线粒体功能障碍,存在胆碱能系统紊乱,有明显的学习记忆障碍,有脑组织tau蛋白重要位点的异常磷酸化,且模型重复性好,稳定可靠,可以作为SAD模型。2.复方银思维能明显改善SAD大鼠的学习记忆能力,其作用机制可能是：能够保护微管结构和功能,改善皮层神经元线粒体功能及能量代谢,改善皮层胆碱能系统功能,调节海马组织tau蛋白O-G1cNAc糖基化修饰与tau磷酸化之间的平衡,抑制tau蛋白重要位点的过度磷酸化及其tau毒性,防止SAD的病理进展。更多还原

【Abstract】 Objective:Based on the relation of insulin-signaling transmission and sporadic Alzheimer disease (SAD) and also on the theory of energy metabolism disorder in brain and the theory of tau normal modification, this paper aims at studying the effects of YSW-compound on the ethology of model rats with SAD, ultrastructure of hippocampal neurons, energy metabolism in brain, the activity of enzyme related cholinergic system, phosphorylation of tau protein in brain t issue and glycosylated modification of 0-GlcNAc, and also discussing the possible mechanism of protection and treatment of YSW-compound on SAD. YSW-compound is characterized by ascending yang qi, dispelling pathogenic yin, expelling stagnation, removing phlegm and balancing yin and yang. The dementia model (ICV-SAD) was established by intracerebroventricular injection of streptozotocin (ICV-STZ).Methods:SPF healthy male Sprague-Dawley (SD) rats were divided into six groups randomly:sham-operation group, model group, donepezil control group, YSW low dose, medium dose and high dose groups. The dementia model was established by bilateral intracerebroventricular injection of streptozotocin and their treatment was started on 21st after the second modeling except sham operation group and model group. The treatment groups were given the corresponding drugs respectively through gavage. Meanwhile, model group and sham operation group were given the same volume of distilled water through gavage once per day for two months. After the treatment, the Morris water maze was adopted to test spatial learning and memory ability of the rats. The ultrastructural changes of microtubes in hippocampal CA1 of rats were observed by electron microscope. The energy metabolism of cerebral cortex and the activity of enzyme related mitochondrial function and cholinergic system enzyme were tested by biochemical technique. The immunohistochemistry and western-blot methods were used to test the glycosylated modification levels of tau protein 0-G1 cNAc in brain tissue and the phosphorylated levels in the Thr231 and Ser422 sites, along with the expression changes of OGT and 0-G1 cNAcase enzyme in the process of 0-GlcNAc glycosylated modification.Results:1. Compared with sham operation group, the mean escape latency and total swimming distance of rats with SAD (by ICV-STZ) during place navigation test was longer (P<0.01), but the swimming time of rats with SAD in target quadrant was obviously shorter during space probe test (P<0.01). YSW-compound could significantly shorten the mean escape latency and total swimming distance of rats with SAD during place navigation test, with statistical significance (P<0.05, P<0.01) and could extend the swimming time of rats with SAD in target quadrant during space probe test (P<0.05, P<0.01). Donepezil group, compared with YSW low dose, medium dose and high dose groups respectively, had no significant difference (P>0.05).2. The activity of SDH, COX and Na+-K+-ATPase in cerebral cortex of rats with SAD in model group was lower than sham operation group (P<0.01), while YSW-compound could increase the activity of SDH, COX and Na+-K+-ATPase in cerebral cortex of rats(P<0.05, P<0.01). Donepezil group had no obvious difference in comparison with model group (P>0.05).3. Compared with sham operation group, the activity of ChAT in cerebral cortex of rats with SAD in model group was lower (P<0.01), but the activity of AchE was higher (P<0.01). YSW-compound could increase the activity of ChAT in cerebral cortex of rats and reduce the activity of AchE with statistical significance (P<0.05, P<0.01). Donepezil group, compared with YSW low dose,medium dose and high dose groups respectively, had no significant difference (P>0.05).4. Compared with sham operation group, the glycoproteomics of O-GlcNAc in hippocampal tissue with sWGA enrichment of rats with SAD was lower (P<0.01) and the expression of glycosylated tau protein tested by RL2 and CTD110.6 was also lower (P<0.01). However, YSW-compound could obviously improve the glycoproteomics of O-GlcNAc in hippocampal tissue with sWGA enrichment of rats with SAD and the expression of glycosylated tau protein tested by RL2 and CTD110.6, with statistical significance (P<0.05, P<0.01). Donepezil group had no significant difference from the model group (rats with SAD) (P>0.05).5. Compared with sham operation group, the expression of OGT in hippocampal tissue of rats with SAD was obviously lower (P<0.01) but the expression of O-GlcNAcase was remarkably higher (P<0.01). However, YSW-compound could significantly improve the expression of OGT in hippocampal tissue of rats with SAD and reduce the expression of O-GlcNAcase, with statistical meaning (P<0.05, P<0.01). Donepezil group had no significant difference from the model group (rats with SAD) (P>0.05).6. Compared with sham operation group, the expression of phosphorylated P-Thr231 and P-Ser422 of tau protein in hippocampal tissue in the Thr231 and Ser422 sites in model group was significantly higher (P<0.01), while YSW-compound could remarkably reduce the expression of P-Thr231 and P-Ser422 of hippocampal tissue of rats with SAD (P<0.05, P<0.01). Donepezil group had no significant difference from the model group (rats with SAD) (P>0.05).Conclusion:1. The ICV-STZ model rats with SAD is characterized by disorders with the energy metabolism of cerebral cortex, mitochondrial function and cholinergic system, along with the impairment of learning and memory and the abnormal phosphorylat ion of tau protein in the key sites of brain tissue. Moreover, the model has good repeatability, stability and reliability, and also is very appropriate to be used to do basic research and drug screening on rats with SAD.2. YSW-compound could significantly improve the learning and memory ability of rats with SAD and the function of neuron mitochondria in cerebral cortex of rats with SAD, and also adjust the balance between the glycosylated mod if icat ion of tau protein 0-G1 cNAc and the phosphorylat ion in hippocampal tissue. YSW-compound could inhibit the hyperphosphorylation of tau protein in the key sites and the toxicity of tau, and protect the structure and function of microtubes as well as prevent axons from injury更多还原