【作者】 彭均华；

【导师】 王建枝；

【作者基本信息】 华中科技大学 ， 病理生理学， 2007， 博士

【摘要】 阿尔茨海默病(Alzheimer’s disease,AD)是老年人常见的一种慢性进行性神经退行性疾病,目前针对AD的药物治疗效果不明显,仅对轻中度AD患者的痴呆症状有所改善或延缓痴呆进展,不能治愈或阻止疾病发展。由于AD的发病原因及机制没有完全清楚,研究开发出真正具有预防或治疗作用的药物将有广阔的前景和深远的意义。AD的主要神经病理学特征是大量形成以过度磷酸化的微管相关蛋白tau为主要成分的神经原纤维缠结(neurofibrillary tangles, NFTs)及由β-淀粉样多肽(β-amyloid, Aβ)组成的神经细胞间的大量老年斑(senile plaques,SP),tau蛋白过度磷酸化被发现是AD的早期改变,而且AD患者的临床痴呆程度仅与NFTs的数量密切相关,因此研究针对以tau蛋白过度磷酸化及其机制为靶点的药物可能是防治AD的有效方法。去氢吴茱萸碱(dehydroevodiamine, DHED)是从吴茱萸未成熟果实中提取的单体化合物生物碱。本研究主要目的是探讨DHED在wotmanni(nWT)及GF-109203X(GFX)引起的AD样病理变化模型中的药理作用及其机制。我们尾静脉注射给予DHED(6.25 mg/kg/day或12.5 mg/kg/day)或生理盐水7天,同时进行Morris水迷宫训练,第8天在大鼠侧脑室联合注射10μl浓度为100μM的PI3K特异性抑制剂wortmannin (WT)及PKC的特异性抑制剂GF-109203X (GFX)。侧脑室注射24h后进行Morris水迷宫测试检测大鼠空间记忆保留功能,水迷宫测试后取材进行免疫印迹及免疫组化检测。结果显示:1.DHED可改善WT/GFX引起的空间记忆保留障碍侧脑室注射WT/GFX 24h后,大鼠搜寻水迷宫平台的时间延长,搜寻轨迹杂乱,大鼠表现空间记忆保留障碍,预先给予DHED 7天可以明显缩短大鼠寻找平台的时间,搜寻策略也得以改善,由杂乱无章变成趋向式搜寻。结果表明预先给予DHED能有效改善因WT/GFX所致的大鼠空间记忆障碍。2. DHED可减轻WT/GFX引起的大鼠海马tau蛋白过度磷酸化侧脑室注射WT/GFX 24h后,免疫印迹检测tau蛋白磷酸化的抗体PHF-1及PS396显色增强,而识别非磷酸化位点的tau-1显色减弱,表明WT/GFX可以引起tau蛋白在Ser396/404及Ser199/202位点发生过度磷酸化,12.5 mg/kg/day的DHED可以减轻tau蛋白在PHF-1、PS396及tau-1位点的磷酸化,而低浓度6.25 mg/kg/day的DHED仅可减轻PS396位点的磷酸化。用R134d检测了总tau蛋白水平,各个实验组均无明显变化。免疫组织化学检测tau蛋白磷酸化情况,结果显示侧脑室注射WT/GFX 24h后,PHF-1在大鼠海马CA3区的阳性染色明显增强,而tau-1在海马CA3区的阳性染色明显减弱,表示WT/GFX可导致tau蛋白过度磷酸化,用DHED处理后,WT/GFX引起的PHF-1及tau-1的染色改变均明显减轻,表明DHED能减轻WT/GFX导致的tau蛋白在PHF-1及tau-1位点的过度磷酸化。3. DHED可减轻WT/GFX诱导的大鼠海马GSK-3β过度激活通过免疫印迹检测大鼠海马GSK-3β的免疫反应,尽管其总GSK-3β水平在各组均无明显变化,与对照组相比,注射WT/GFX后其非活性形式Ser9磷酸化的GSK-3β降低,预先给予DHED可增加Ser9磷酸化GSK-3β的水平,表明DHED能抑制WT/GFX所致的GSK-3β过度激活。为了进一步研究在细胞水平激活GSK-3β对tau蛋白过度磷酸化的影响以及DHED的保护作用,我们用不同浓度的DHED预处理野生型鼠成神经瘤(N2a)细胞株24h,然后用浓度为1μM的WT/GFX处理细胞1h。结果显示:1. DHED可减轻WT/GFX引起的N2a细胞tau蛋白过度磷酸化DHED 5、10μM预处理细胞24h均能明显减轻WT/GFX所致的tau蛋白在Ser396/404及Ser199/202位点过度磷酸化(p<0.01),两个浓度的DHED降低tau蛋白磷酸化的作用没有差别;单独用DHED处理细胞对tau蛋白磷酸化没有明显影响。2. DHED可减轻N2a细胞的GSK-3β过度激活通过免疫印迹检测细胞GSK-3β的免疫反应,与对照组相比,总GSK-3β水平在各组均无明显变化,1μM的WT/GFX处理1h后其非活性形式Ser9磷酸化的GSK-3β降低到41%(p<0.01);5、10μM的DHED预处理细胞24h均可逆转WT/GFX所致的Ser9磷酸化GSK-3β下降,分别上升到81%(5μM)及87%(10μM)(p<0.01),表明DHED能减轻WT/GFX激活的GSK-3β。免疫荧光染色发现WT/GFX处理后细胞的Ser9磷酸化GSK-3β显色减弱,而PHF-1的显色加强,用DHED 10μM预处理细胞24h可以明显逆转WT/GFX所致的变化。瞬时转染wtGSK-3β到N2a细胞后用10μM DHED处理24h。DHED可以降低N2a细胞过表达外源性GSK-3β的活性及抑制其引起的tau蛋白过度磷酸化,进一步证明DHED可以抑制GSK-3β的活性。3. DHED可以增加N2a细胞的Akt(PKB)磷酸化水平Akt是使GSK-3β的Ser9发生磷酸化的最重要的磷酸激酶,其通过Ser473及Thr308两个位点磷酸化得以激活。我们检测了N2a细胞的Akt磷酸化水平,与对照组相比,总Akt水平在各组均无明显变化,但1μM的WT/GFX处理1h后Akt在Ser473及Thr-308位点磷酸化水平降低(p<0.01);DHED预处理细胞24h可增加Akt在Ser473及Thr-308位点的磷酸化水平,说明DHED可以促进Akt激活。根据上述结果,我们得出以下结论,WT/GFX激活GSK-3β,从而导致tau蛋白过度磷酸化,DHED可以通过抑制GSK-3β活性从而降低tau蛋白的磷酸化,其抑制GSK-3β可能是通过激活Akt来实现。更多还原

【Abstract】 Alzheimer’s disease is a common and progressive neurodegenerative disease in the eldly population. Current medications that have passed approval for treatment of AD seem to be able to produce modest symptomatic improvement in some of mild to moderate cases. None of the medications, however, appears to be able to cure AD or stop the disease progression. Because the etiology of AD is still unclear, the search for efficient drugs to prevent or treat AD is facing opportunity and will have nice prospect.Pathologically, AD is characterized by the formation of numerous intracellular neurofibrillary tangles (NFTs) and extracellular senile plaques (SPs) in the brain, which are respectively consisted of hyperphosphorylated tau andβ-amyloid (Aβ) peptide. Hyperphosphorylated tau is believed to be an early pathological event of AD and only the amount of NFTs is closely correlated with the clinical dementia degree of AD patients. Thus target the underlying pathogenic mechanisms of tau hyperphosphorylation in AD might be a potential therapeutic strategy for prevention or treatment of the diseae.Dehydroevodiamine (DHED) is a constituent of alkaloids isolated from the unripe fruit of a Chinese herb, namely Evodia rutaecarpa Bentham. In our present study, we have investigated the pharmacological effect of DHED on WT/GFX induced AD-like pathological models and its involved mechanism of the effection.The rats were injected through vena caudalis 6.25 mg/kg/day or 12.5 mg/kg/day of DHED or NS for 7 days. At the same time, the rats received water maze training. At the day 8, wortmannin (WT, a specific PI3K inhibitor) and GF-109203X (GFX, a specific PKC inhibitor) (100μM of each, total volume of 10μl) were co-injected into the left ventricle of the rats and the spatial memory retention was tested at 24 h after the brain injection. The samples were prepared for western blotting and immunocytochemistry research after the spatial memory retention test. The results are shown as follows.1. Effects of DHED on WT/GFX-induced spatial memory retention deficits of rats.It was shown that the latency increased significantly after the injection of WT/GFX, and pre-injection of DHED at both dosages significantly shortened the WT/GFX-induced increase of the latency. Instead of taking a tortuous swimming path to find the hidden platform as seen in WT/GFX-injected group, the pre-injection of DHED at both dosages significantly improved the searching strategy of the rats. These results suggest that pre-administration of DHED can effectively prevent the rats from WT/GFX-induced spatial memory deficits.2. Effects of DHED on WT/GFX-induced tau hyperphosphorylation in rat hippocampus.The immunoreactivity of PHF-1 and PS396 was enhanced and the immunoreactivity of tau-1 was decreased in WT/GFX-injected rats, suggesting that WT/GFX induces tau hyperphosphorylation at Ser396/404 (PHF-1 and PS396) and Ser199/202 (tau-1 reacts with non-phosphorylated tau). Pre-injection of DHED at 12.5 mg/Kg/day attenuated the WT/GFX-induced tau hyperphosphorylation at PHF-1, PS396 and tau-1 epitopes. When lower concentration of DHED (6.25 mg/Kg/day) was used, the attenuation of tau hyperphosphorylation was only seen at PS396 site but not at PHF-1 and tau-1 epitopes. We also tested the level of total tau using antibody R134d, but no significant difference was observed.The effect of DHED on WT/GFX-induced tau hyperphosphorylation was also studied by immunocytochemistry. In WT/GFX-injected rats, the immunoreaction of PHF-1 mainly in the mossy fibers of CA3 region of the hippocampus was enhanced, while the immunoreactivity of tau-1 in the mossy fibers of CA3 region was decreased, suggesting that WT/GFX induces tau hyperphosphorylation. Pre-injection of DHED significantly arrested the WT/GFX-induced tau hyperphosphorylation at PHF-1 and tau-1 epitopes.3. Effects of DHED on WT/GFX-induced overactivation of GSK-3βin rat hippocampus.we measured the total level and the activity-dependent Ser9-phosphorylated level of GSK-3βin the hippocampal extracts by western blotting. No obvious change was seen in total level of GSK-3βin WT/GFX-injected or DHED-preinjected rats. However, the level of Ser9-phosphorylated GSK-3β(p-S9-GSK-3β, representing the inactivated form of the kinase) was significantly decreased in WT/GFX-injected control group, and preinjection of DHED increased the level of the Ser9-phosphorylated GSK-3β. suggesting that the DHED could prevent GSK-3βfrom WT/GFX-induced overactivation.To further study the effect of DHED on tau phosphorylation and GSK-3βactivation in cell line, we pretreated the neuro2A (N2a) cells with different dose of DHED for 24h, and then treated 1μM each of WT and GFX for 1h. The main results are as followings:1. Effect of DHED on tau hyperphosphorylation induced by WT/GFX in N2a cells.Western blotting demonstrated that the immunoreactivity of tau Ser396/404 and Ser199/202 was significantly attenuated by 5 and 10μM DHED (p<0.01). No significant difference was observed between the two doses of DHED. The application of DHED alone did not change of tau phosphorylation level.2. Effect of DHED on overactivation of GSK-3βin N2a cells. We measured the total level and the activity-dependentSer9-phosphorylated level of GSK-3βin N2a cells by western blotting. No obvious change was seen in total GSK-3βwith or without DHED. However, the level of Ser9-phosphorylated GSK-3β(p-S9-GSK-3β) was significantly decreased to 41% after 1μM WT/GFX treatment 1h(p<0.01), and pretreatment of DHED 5μM and 10μM for 24h increased the level of the p-S9-GSK-3βto 81% and 87% respectively (p<0.01). Suggesting that the DHED could prevent GSK-3βfrom WT/GFX-induced overactivation.By immunofluorescence staining, a significantly decreased immunoreaction of p-S9-GSK-3βwith an enhanced staining of the phosphorylated tau at PHF-1 was seen after WT/GFX treatment, and pretreatment of the cells with 10μM DHED for 24h reversed the changes.We transiently transfected wtGSK-3βin N2a cells and treated the cells with DHED for 24h. The overexpression of the exogenous GSK-3βwas suppressed by DHED. Tau hyperphosphorylation following with overexpression of wtGSK-3βalso attenuated by add with DHED. These data confirmed that DHED down-regulating the activity of GSK-3β.3. DHED increases the phosphorylation level of Akt in N2a cells.The most important kinase responsible for Ser9-phosphorylation of GSK-Sβis Akt. It is to believe that Akt requires phosphorylation at Ser473 and Thr308 to be activated. We tested the activity-dependent Ser473- and Thr308-phosphorylated level of Akt in the N2a cells. No obvious change was seen in total level of Akt with or without DHED. However, the level of Ser473 and Thr308-phosphorylated Akt was significantly decreased after 1μM WT/GFX treatment 1h, and pretreatment of DHED for 24h increased the level of the Ser473 and Thr308-phosphorylated Akt.In summary, we have demonstrated in the present study that WT/GFX induced tau hyperphosphorylation through GSK-3βactivation in N2a cell line and rats. DHED attenuates tau hyperphosphorylation via GSK-3βinhibition. The effect of DHED on GSK-3βmay be involves Akt activation.更多还原