EGCG对APP/PS1转基因鼠保护作用及可能机制
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摘要
目的
阿尔茨海默病(Alzheimer's disease, AD)亦称老年性痴呆(senile dementia),是一种发生于中老年的以进行性认知障碍和记忆能力下降为主的退行性神经病变。该病起病隐袭,病程呈进行性进展,以渐进性学习记忆、认知功能减退、行为异常、日常生活能力下降及情感、思维障碍等为主要临床表现。其典型的病理改变是神经细胞间出现大量以p-淀粉样肽(β-Amyloid, Aβ)为核心的老年斑(senile plaques, SP)、神经元的胞体中出现神经原纤维缠结(neurofibrillary tangles, NFT)及神经元的丢失等。AD的发病机制目前尚未完全阐明,但自由基损伤学说和细胞凋亡学说备受人们的关注。自由基形成和氧化应激增强,进一步引起神经细胞凋亡,是导致AD的功能退化和神经变性的基础。p75神经营养因子受体(P75 neurotrophin receptor, P75NTR)是神经营养素的低亲和力受体,与AD神经元细胞凋亡有关。TrkA即酪氨酸激酶A (tyrosine kinase A)是神经营养素的特异性受体,与神经元细胞分化、生长、修复、存活密切有关。p75NTR诱导凋亡的信号通路目前仍不十分清楚,但已广泛公认的是其对c-jun氨基末端激酶(c-jun N-terminal kinase, JNK)的激活是介导神经元细胞凋亡的关键步骤,并进一步通过激活促凋亡蛋白P53、Bad等的表达,促进或引起细胞凋亡。此外,JNK还能通过诱导细胞色素C的释放进一步活化Caspase-3而导致细胞凋亡。TrkA对神经元发挥促进其分化、生长、存活功能的机制研究的相对比较透彻,其主要通过激活Ras-MAPK-ERK信号转导通路,特别是通过活化的Erkl/2信号转导通路来实现。
研究并建立可靠的AD动物模型对于探明AD的病因、发病机制及防治药物的研发均有重要的意义。到目前为止用于AD研究的动物模型很多,其中转基因模型是一种病因模型,过量表达人源性突变AD相关基因的转基因鼠,因其具有明确的病因,并能反映AD的部分病理与功能变化,有助于我们了解AD的发病机制,也逐渐成为研究AD的最为理想的整体模型。
防治AD的抗氧化剂及抗凋亡药物的研究与开发成为当今药理学研究领域的重要课题之一。(-)表没食子儿茶素没食子酸酯(epigallocatechin-3-gallate, EGCG)是绿茶的一种主要多酚成分,研究证实绿茶有效的铁螯和、抗氧化、抗炎、抗癌及神经保护等作用的发挥主要依赖于EGCG。Levites等人通过研究人类成神经细胞瘤SHSY5Y细胞和大鼠成纤维细胞瘤PC12细胞,发现EGCG能够减少具有神经毒性作用的Ap的产生。因此,本研究拟通过遗传性的APP/PS1转基因小鼠,采用Nissl、TUNEL染色、免疫组化、Western blot等方法研究APP/PS1转基因小鼠认知行为、病理改变、抗氧化能力、细胞凋亡、Aβ(1-40)、淀粉样前体蛋白(Amyloid precursor Protein, APP)、促凋亡蛋白caspase-3及P75-JNK-P53凋亡相关通路及抗凋亡蛋白Phospho-c-raf、Phospho-MEK1/2、Phospho-ERK1、Phospho-p90RSK、Phospho-MSK1相关通路的影响,即9月龄的APP/PS1转基因小鼠上述指标的改变,同时进一步探讨EGCG对APP/PS1转基因小鼠模型的神经保护作用及其作用机制。
材料与方法
APP/PS1转基因小鼠实验取C57小鼠10只及APP/PS1小鼠22只,C57小鼠为对照组(Control)、APP/PS1小鼠22只随机分为模型组(APP/PS1)和实验组(APP/PS1+2 mg/kg EGCG)。实验组APP/PS1转基因小鼠按2 mg/kg的剂量灌胃0.04%的EGCG,每日一次,连续灌胃4周；对照组及模型组小鼠灌胃等量的双蒸水,每日一次,连续灌胃4周。最后一周对小鼠进行水迷宫、避暗及自主活动等行为学检测,其间继续给药。行为学结束后,快速取一部分小鼠脑组织入-80℃低温冰箱中冻存,分别进行SOD、GSH-Px、AChE酶活性、MDA含量的测定及Western blot印迹分析APP、caspase-3、NGF、P75、JNK2、P53、Phospho-TrkA.Phospho-c-raf、Phospho-MEK1/2、Phospho-ERK1、Phospho-p90RSK、Phospho-MSK1、CREB等蛋白表达水平的实验。另一部分小鼠进行灌流固定,取脑后分别进行HE染色、Nissl染色、TUNEL染色及Aβ(1-40)、caspase-3、APP、CREB、TrkA等免疫组化的检测分析。
实验结果
1、EGCG对APP/PS1转基因小鼠记忆及判断能力的影响水迷宫、避暗、自主活动等行为学结果显示APP/PS1转基因小鼠出现明显的学习记忆及判断能力的下降,但活动能力未受影响。EGCG明显的改善了APP/PS1转基因小鼠的学习记忆障碍,提高了小鼠的记忆与判断能力。
2、EGCG对APP/PS1转基因小鼠脑组织病理改变的影响
HE染色及Nissl染色结果显示,APP/PS1转基因小鼠大脑皮层及海马区神经元数量明显减少,细胞排列松散,细胞核固缩、染色质边集和核碎裂,尼氏体分界不清,排列紊乱、松散,细胞周围出现间隙,其中许多细胞胞体缩小、胞浆呈淡红色、胞核固缩,可见坏死的神经元；EGCG明显的减轻了APP/PS1转基因小鼠脑内神经元损伤程度,细胞排列相对整齐,细胞形态规则,层次丰富,核仁清晰。
3、EGCG对APP/PS1转基因小鼠脑内Aβ(1-40)蛋白表达的影响
Aβ(1-40)蛋白免疫组化染色结果显示：APP/PS1转基因小鼠大脑皮层及海马区的Aβ(1-40)蛋白表达水平显著增高；EGCG能明显抑制小鼠大脑皮层及海马区的Aβ(1-40)蛋白表达水平。
4、EGCG对APP/PS1转基因小鼠海马区SOD、GSH-Px及AChE酶活性及MDA含量的影响
酶学结果显示APP/PS1转基因小鼠海马内SOD、GSH-Px活性明显降低,AchE酶活性及MDA含量明显升高；EGCG能明显的提高小鼠海马内SOD、GSH-Px酶活性,并明显的降低MDA含量和AchE酶的活性。
5、EGCG对APP/PS1转基因小鼠脑区神经元细胞凋亡的影响
TUNEL法染色结果显示APP/PS1转基因小鼠大脑皮层及海马区可见有较多的凋亡细胞,且细胞凋亡指数与空白对照组比明显增多；EGCG能明显的减少APP/PS1转基因小鼠大脑内神经元细胞的凋亡
6、EGCG对APP/PS1转基因小鼠脑内CREB蛋白表达的影响
CREB免疫组化染色及Western blot印迹分析结果显示APP/PS1转基因小鼠大脑皮层及海马区的CREB蛋白表达水平明显降低：EGCG明显的提高了APP/PS1转基因小鼠大脑皮层及海马区的CREB蛋白表达水平。
7、EGCG对APP/PS1转基因小鼠脑内APP蛋白表达的影响
APP免疫组化染色及Western blot印迹分析结果显示APP/PS1转基因小鼠大脑皮层及海马区的APP蛋白表达水平明显增高；EGCG明显的抑制了APP/PS1转基因小鼠大脑皮层及海马区的APP蛋白表达水平的增高。
8、EGCG对APP/PS1转基因小鼠脑内内P75NTR、JNK2及P53蛋白表达的影响
Western blot印迹分析结果显示APP/PS1转基因脑内海马内P75蛋白的表达水平增高,同时海马区JNK2及P53蛋白表达水平增加；EGCG可明显的抑制p75ICD、JNK2及P53蛋白表达水平。
9、EGCG对APP/PS1转基因小鼠脑内内Phospho-c-raf、Phospho-MEK1/2、Phospho-ERK1/2、Phospho-p90RSK、Phospho-MSK1蛋白表达的影响
Western blot印迹分析结果显示APP/PS1转基因小鼠脑内Phospho-c-raf、Phospho-MEK1/2、Phospho-ERK1/2、Phospho-p90RSK、Phospho-MSK1蛋白的表达水平降低；EGCG可明显的提高APP/PS1转基因小鼠脑内Phospho-c-raf、Phospho-MEK1/2、Phospho-ERK1/2、Phospho-p90RSK、Phospho-MSK1等蛋白表达水平。
结论
1、EGCG能够改善APP/PS1转基因小鼠的学习记忆障碍。
2、EGCG能够提高APP/PS1转基因小鼠脑内内的抗氧化酶活性,及降低AchE酶的活性,发挥有效的抗氧化作用及增强记忆能力的作用。
3、EGCG通过抑制APP/PS1转基因小鼠大脑皮层及海马内的APP及Aβ(1-40)蛋白水平,对神经细胞发挥一定的保护作用。
4、EGCG通过抑制APP/PS1转基因小鼠脑内内p75ICD、JNK2及P53凋亡通路相关蛋白表达水平,进一步抑制caspase-3蛋白活性,对神经细胞发挥有效的抗凋亡作用。
5、EGCG通过促进APP/PS1转基因小鼠大脑皮层及海马内的Trka及CREB蛋白水平,对神经细胞发挥一定的保护作用。
6、EGCG通过提高APP/PS1转基因小鼠脑内内Phospho-c-raf、Phospho-MEK1/2、Phospho-ERK1/2、Phospho-p90RSK、Phospho-MSK1抗凋亡通路相关蛋白表达水平,对神经细胞发挥有效的抑制凋亡的作用。
Alzheimer's disease (AD), also known as senile dementia, is a progressive neurodegenerative disease, with the characteristic of insidious onset, progressive memory loss, cognitive deterioration, behavioral disorders. And at last Decreased ability of life AD is pathologically characterized by deposition ofβ-amyloid (Aβ) peptides as senile plaques and neurofibrillary tangles in the brain. The precise mechanisms of AD are not yet clearly understood, but Free Radical Stress Theory and Cell Apoptosis Theory are paid close attention to. Oxidative stress and reactive oxygen species (ROS) have been proposed to be major cause of functional disorder and neurodegeneration in AD. P75 neurotrophin receptor is the low affinity receptor of neurotrophins and it is closely relative to neuronal apoptosis in AD. The apoptosis signaling pathway P75NTR involved in is not yet clearly understood, but it is well known that P75NTR can activate c-jun N-terminal kinase (JNK) and pro-apoptotic protein p53 and Bad, and induce cell apoptosis. In addition, JNK can also promote cytochrome c release and activation of caspase-3. TrkA (tyrosine kinase A) is the high affinity receptor of neurotrophins and it is closely relative to neuronal anti-apoptosis in AD.when NGFand TrkA are binded together, though MAPK-ERKpathway TrkA performs its effects.
It is very important to study and make suitable animal models for evaluating the cause, pathogenesy of AD and developing drugs to cure AD. So far different animal models have been developed to study the etiology, evolution and new therapeutic alternatives for the illnesses, among which the mice transgenic mouse models have been created with mutations in genes related to AD. Moreover, double transgenics, such as APP/PS1 transgenic mice, provide a valuable model for evaluating the pathogenesy of AD.
(-)-Epigallocatechin-3-gallate(EGCG), which is classified the catechin family and is one of the major polyphenol constituents of green tea. It has been reported that EGCG possess potent iron-chelating, antioxidant, anti-inflammatory, anticancer and neuroprotective activities. EGCG has been shown to have neuroprotective effects by elevatingα-secretase activity of amyloid precursor protein (APP) to soluble APP-alpha (sAPP-α) and reducing Amyloid beta (Aβ)-induced neurotoxicity in human SH-SY5Y neuroblastoma and rat pheochromocytoma PC12 cells. In the present study, we used APP/PS1 transgenic mice, and observed whether EGCG (2 mg/kg.d,4 weeks, ig) had the potent antioxidant and anti-apoptotic neuroprotective effects on the AD mice and APP/PS1 transgenic by behavioral and pathological testing, measurements of the activities of total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px), contents of malondialdehyde (MDA) and activation and expression of pro-apoptotic protein caspase-3, P75, JNK, P53and anti-apoptotic protein Phospho-c-raf Phospho-MEK1/2, Phospho-ERK1/2, Phospho-p90RSK, Phospho-MSK1 in the hippocampus of mice by immunohistochemical staining and Western blot analysis.
Materials and Methods
Ten C57 mice and twenty two APP/PS1 mice were randomly divided into three groups (n=10 each group):control group, APP/PS1 group and APP/PS1+2 mg/kg EGCG group. The mice of APP/PS1+EGCG (2 mg/kg) group were intragastricly given with EGCG at the dose of 2 mg/kg.d for 4 weeks. The mice of control group and APP/PS1 group were administered with same volume vehicle distilled water. After finishing all treatments, animals were evaluated by behavioral testing, and then immediately sacrificed to dissect hippocampus and stored at-80℃for the examinations of expression of pro-apoptotic protein P75, JNK, P53 and anti-apoptotic Phospho-c-raf, Phospho-MEK1/2, Phospho-ERK1/2, Phospho-p90RSK, Phospho-MSK1 in the hippocampus of mice by Western blot analysis. Other mice (n=4) were transcardially perfused with normal saline followed by 4% paraformaldehyde solution. The hippocampus were removed, post-fixed in 4% paraformaldehyde and used for HE and TUNEL and immunohistochemical staining of Aβ(1-40) and CREB.
Results
1. The effects of EGCG on learning and memory impairment in APP/PS1 Transgenic Mouse
The results by water maze, step-through and spontaneous activity test showed APP/PS1 Transgenic Mouse could significantly impair learning and memory of mice, but could not affect their locomotor activity. And EGCG (2 mg/kg. d,4 weeks, ig) could evidently improve the learning and memory impairment in this APP/PS1 Transgenic Mouse.
2. The histopathological effects of EGCG on the brains of APP/PS1 Transgenic Mouse
The results by HE and Nissl staining showed the number of neuron was significantly decreased, the arrangement of neurons in cortex and hippocampus of APP/PS1 Transgenic Mouse was sparse and Nissl body was decreased and dissolved. EGCG (2 mg/kg,4weeks, ig) and could evidently release neuronal injury in APP/PS1 Transgenic Mouse
3. The effects of EGCG on expression of Aβ(1-40) in brains of APP/PS1 Transgenic Mouse
The results by immunohistochemical staining of Aβ(1-40) showed APP/PS1 Transgenic Mouse an obvious increase of Aβ(1-40) in the cortex and hippocampus of mice, and EGCG (2 mg/kg.d,4 weeks, ig) significantly reversed the effect.
4. The effects of EGCG on activities of SOD, GSH-Px and contents of MDA in the hippocampus of APP/PS1 Transgenic Mouse
The results by biological analysis showed APP/PS1 Transgenic Mouse decreased activities of SOD and GSH-Px, increased contents of MDA in mouse hippocampus, and EGCG (2 mg/kg.d,4 weeks, ig) significantly elevated the activities of SOD and GSH-Px, decreased the contents of MDA in hippocampus of APP/PS1 Transgenic Mouse.
5. The effects of EGCG on neuronal apoptosis in brains of APP/PS1 Transgenic Mouse
TUNEL staining results showed TUNEL positive neurons were highly widespread in the cerebral cortex and hippocampus in APP/PS1 Transgenic Mouse and the cell apoptosis index was obviously increased in the hippocampus of mice in model group, compared with control group. EGCG (2 mg/kg.d,4 weeks, ig) and significantly decreased cell apoptosis index in the hippocampus of APP/PS1 Transgenic Mouse
6. The effects of EGCG on activation of CREB in the brains of APP/PS1 Transgenic Mouse
The results by immunohistochemical staining and western blot analysis of CREB showed APP/PS1 Transgenic Mouse significantly reduced in the activation of CREB in the cerebral cortex and hippocampus of mice. EGCG (2 mg/kg.d,4 weeks, ig) significantly increase the activation of CREB in the cerebral cortex and hippocampus of APP/PS1 Transgenic Mouse.
7. The effects of EGCG on expresstion of APP in the brains of APP/PS1 Transgenic Mouse
The results by immunohistochemical staining and western blot analysis of APP showed APP/PS1 Transgenic Mouse significantly increased in the expresstion of APP in the cerebral cortex and hippocampus of mice. EGCG(2 mg/kg.d,4 weeks, ig) significantly reduced the express of APP in the cerebral cortex and hippocampus of APP/PS1 Transgenic Mouse.
8. The effects of EGCG on express of P75NTR, JNK2 and P53 in the hippocampus of APP/PS1 Transgenic Mouse
The results by western blot analysis showed APP/PS1 Transgenic Mouse significantly promoted the shearing of P75 in the hippocampus of mice and increased the displacement of p75ICD to the nucleus, and APP/PS1 Transgenic Mouse also significantly increased the express of JNK2 and P53 in the hippocampus of mice. EGCG (2 mg/kg. d,4 weeks, ig) significantly decreased the expresstion of p75ICD, JNK2 and P53 in the hippocampus of APP/PS1 Transgenic Mouse.
9. The effects of EGCG on expresstion of Phospho-c-raf, Phospho-MEK1/2, Phospho-ERK1/2, Phospho-p90RSK, Phospho-MSK1 in the hippocampus of APP/PS1 Transgenic Mouse
The results by western blot analysis showed APP/PS1 Transgenic Mouse significantly decreased Phospho-c-raf, Phospho-MEKl/2, Phospho-ERK1/2, Phospho-p90RSK、Phospho-MSK1 in the hippocampus of mice, and APP/PS1 Transgenic Mouse also significantly decreased the express of TrkA in the hippocampus of mice. EGCG (2 mg/kg.d,4 weeks, ig) significantly increased the express of Phospho-c-raf, Phospho-MEK1/2, Phospho-ERK1/2, Phospho-p90RSK, Phospho-MSK1 and TrkA in the hippocampus of APP/PS1 Transgenic Mouse.
Conclusion
1. EGCG can improve the learning and memory impairment of APP/PS1 transgenic mice.
2. EGCG has a potent antioxidant effect by elevating the activities of SOD and GSH-Px and decreasing the contents of MDA and the activities of AChE in hippocampus of APP/PS1 Transgenic Mouse.
3. EGCG has a protective effect on APP/PS1 Transgenic Mouse by decreasing the express of APP andβ-Amyloid (1-40) in the cerebral cortex and hippocampus of mice.
4. EGCG has a potent anti-apoptotic effect by reducing the express of p75ICD, JNK2 and P53, and decreasing the activation of caspase-3 in the hippocampus of APP/PS1 transgenic mice.
5. EGCG has a potent anti-apoptotic effect by increase the express of Phospho-c-raf, Phospho-MEK1/2, Phospho-ERK1/2, Phospho-p90RSK, Phospho-MSK1, in the hippocampus of APP/PS1 transgenic mice.
6. EGCG has a potent anti-apoptotic effect by increase the expresstion of TrkA and CREB in the hippocampus of APP/PS1 transgenic mice.
阿尔茨海默病(Alzheimer's disease, AD)亦称老年性痴呆(senile dementia),是一种发生于中老年的以进行性认知障碍和记忆能力下降为主的退行性神经病变。该病起病隐袭,病程呈进行性进展,以渐进性学习记忆、认知功能减退、行为异常、日常生活能力下降及情感、思维障碍等为主要临床表现。其典型的病理改变是神经细胞间出现大量以p-淀粉样肽(β-Amyloid, Aβ)为核心的老年斑(senile plaques, SP)、神经元的胞体中出现神经原纤维缠结(neurofibrillary tangles, NFT)及神经元的丢失等。AD的发病机制目前尚未完全阐明,但自由基损伤学说和细胞凋亡学说备受人们的关注。自由基形成和氧化应激增强,进一步引起神经细胞凋亡,是导致AD的功能退化和神经变性的基础。p75神经营养因子受体(P75 neurotrophin receptor, P75NTR)是神经营养素的低亲和力受体,与AD神经元细胞凋亡有关。TrkA即酪氨酸激酶A (tyrosine kinase A)是神经营养素的特异性受体,与神经元细胞分化、生长、修复、存活密切有关。p75NTR诱导凋亡的信号通路目前仍不十分清楚,但已广泛公认的是其对c-jun氨基末端激酶(c-jun N-terminal kinase, JNK)的激活是介导神经元细胞凋亡的关键步骤,并进一步通过激活促凋亡蛋白P53、Bad等的表达,促进或引起细胞凋亡。此外,JNK还能通过诱导细胞色素C的释放进一步活化Caspase-3而导致细胞凋亡。TrkA对神经元发挥促进其分化、生长、存活功能的机制研究的相对比较透彻,其主要通过激活Ras-MAPK-ERK信号转导通路,特别是通过活化的Erkl/2信号转导通路来实现。
研究并建立可靠的AD动物模型对于探明AD的病因、发病机制及防治药物的研发均有重要的意义。到目前为止用于AD研究的动物模型很多,其中转基因模型是一种病因模型,过量表达人源性突变AD相关基因的转基因鼠,因其具有明确的病因,并能反映AD的部分病理与功能变化,有助于我们了解AD的发病机制,也逐渐成为研究AD的最为理想的整体模型。
防治AD的抗氧化剂及抗凋亡药物的研究与开发成为当今药理学研究领域的重要课题之一。(-)表没食子儿茶素没食子酸酯(epigallocatechin-3-gallate, EGCG)是绿茶的一种主要多酚成分,研究证实绿茶有效的铁螯和、抗氧化、抗炎、抗癌及神经保护等作用的发挥主要依赖于EGCG。Levites等人通过研究人类成神经细胞瘤SHSY5Y细胞和大鼠成纤维细胞瘤PC12细胞,发现EGCG能够减少具有神经毒性作用的Ap的产生。因此,本研究拟通过遗传性的APP/PS1转基因小鼠,采用Nissl、TUNEL染色、免疫组化、Western blot等方法研究APP/PS1转基因小鼠认知行为、病理改变、抗氧化能力、细胞凋亡、Aβ(1-40)、淀粉样前体蛋白(Amyloid precursor Protein, APP)、促凋亡蛋白caspase-3及P75-JNK-P53凋亡相关通路及抗凋亡蛋白Phospho-c-raf、Phospho-MEK1/2、Phospho-ERK1、Phospho-p90RSK、Phospho-MSK1相关通路的影响,即9月龄的APP/PS1转基因小鼠上述指标的改变,同时进一步探讨EGCG对APP/PS1转基因小鼠模型的神经保护作用及其作用机制。
材料与方法
APP/PS1转基因小鼠实验取C57小鼠10只及APP/PS1小鼠22只,C57小鼠为对照组(Control)、APP/PS1小鼠22只随机分为模型组(APP/PS1)和实验组(APP/PS1+2 mg/kg EGCG)。实验组APP/PS1转基因小鼠按2 mg/kg的剂量灌胃0.04%的EGCG,每日一次,连续灌胃4周；对照组及模型组小鼠灌胃等量的双蒸水,每日一次,连续灌胃4周。最后一周对小鼠进行水迷宫、避暗及自主活动等行为学检测,其间继续给药。行为学结束后,快速取一部分小鼠脑组织入-80℃低温冰箱中冻存,分别进行SOD、GSH-Px、AChE酶活性、MDA含量的测定及Western blot印迹分析APP、caspase-3、NGF、P75、JNK2、P53、Phospho-TrkA.Phospho-c-raf、Phospho-MEK1/2、Phospho-ERK1、Phospho-p90RSK、Phospho-MSK1、CREB等蛋白表达水平的实验。另一部分小鼠进行灌流固定,取脑后分别进行HE染色、Nissl染色、TUNEL染色及Aβ(1-40)、caspase-3、APP、CREB、TrkA等免疫组化的检测分析。
实验结果
1、EGCG对APP/PS1转基因小鼠记忆及判断能力的影响水迷宫、避暗、自主活动等行为学结果显示APP/PS1转基因小鼠出现明显的学习记忆及判断能力的下降,但活动能力未受影响。EGCG明显的改善了APP/PS1转基因小鼠的学习记忆障碍,提高了小鼠的记忆与判断能力。
2、EGCG对APP/PS1转基因小鼠脑组织病理改变的影响
HE染色及Nissl染色结果显示,APP/PS1转基因小鼠大脑皮层及海马区神经元数量明显减少,细胞排列松散,细胞核固缩、染色质边集和核碎裂,尼氏体分界不清,排列紊乱、松散,细胞周围出现间隙,其中许多细胞胞体缩小、胞浆呈淡红色、胞核固缩,可见坏死的神经元；EGCG明显的减轻了APP/PS1转基因小鼠脑内神经元损伤程度,细胞排列相对整齐,细胞形态规则,层次丰富,核仁清晰。
3、EGCG对APP/PS1转基因小鼠脑内Aβ(1-40)蛋白表达的影响
Aβ(1-40)蛋白免疫组化染色结果显示：APP/PS1转基因小鼠大脑皮层及海马区的Aβ(1-40)蛋白表达水平显著增高；EGCG能明显抑制小鼠大脑皮层及海马区的Aβ(1-40)蛋白表达水平。
4、EGCG对APP/PS1转基因小鼠海马区SOD、GSH-Px及AChE酶活性及MDA含量的影响
酶学结果显示APP/PS1转基因小鼠海马内SOD、GSH-Px活性明显降低,AchE酶活性及MDA含量明显升高；EGCG能明显的提高小鼠海马内SOD、GSH-Px酶活性,并明显的降低MDA含量和AchE酶的活性。
5、EGCG对APP/PS1转基因小鼠脑区神经元细胞凋亡的影响
TUNEL法染色结果显示APP/PS1转基因小鼠大脑皮层及海马区可见有较多的凋亡细胞,且细胞凋亡指数与空白对照组比明显增多；EGCG能明显的减少APP/PS1转基因小鼠大脑内神经元细胞的凋亡
6、EGCG对APP/PS1转基因小鼠脑内CREB蛋白表达的影响
CREB免疫组化染色及Western blot印迹分析结果显示APP/PS1转基因小鼠大脑皮层及海马区的CREB蛋白表达水平明显降低：EGCG明显的提高了APP/PS1转基因小鼠大脑皮层及海马区的CREB蛋白表达水平。
7、EGCG对APP/PS1转基因小鼠脑内APP蛋白表达的影响
APP免疫组化染色及Western blot印迹分析结果显示APP/PS1转基因小鼠大脑皮层及海马区的APP蛋白表达水平明显增高；EGCG明显的抑制了APP/PS1转基因小鼠大脑皮层及海马区的APP蛋白表达水平的增高。
8、EGCG对APP/PS1转基因小鼠脑内内P75NTR、JNK2及P53蛋白表达的影响
Western blot印迹分析结果显示APP/PS1转基因脑内海马内P75蛋白的表达水平增高,同时海马区JNK2及P53蛋白表达水平增加；EGCG可明显的抑制p75ICD、JNK2及P53蛋白表达水平。
9、EGCG对APP/PS1转基因小鼠脑内内Phospho-c-raf、Phospho-MEK1/2、Phospho-ERK1/2、Phospho-p90RSK、Phospho-MSK1蛋白表达的影响
Western blot印迹分析结果显示APP/PS1转基因小鼠脑内Phospho-c-raf、Phospho-MEK1/2、Phospho-ERK1/2、Phospho-p90RSK、Phospho-MSK1蛋白的表达水平降低；EGCG可明显的提高APP/PS1转基因小鼠脑内Phospho-c-raf、Phospho-MEK1/2、Phospho-ERK1/2、Phospho-p90RSK、Phospho-MSK1等蛋白表达水平。
结论
1、EGCG能够改善APP/PS1转基因小鼠的学习记忆障碍。
2、EGCG能够提高APP/PS1转基因小鼠脑内内的抗氧化酶活性,及降低AchE酶的活性,发挥有效的抗氧化作用及增强记忆能力的作用。
3、EGCG通过抑制APP/PS1转基因小鼠大脑皮层及海马内的APP及Aβ(1-40)蛋白水平,对神经细胞发挥一定的保护作用。
4、EGCG通过抑制APP/PS1转基因小鼠脑内内p75ICD、JNK2及P53凋亡通路相关蛋白表达水平,进一步抑制caspase-3蛋白活性,对神经细胞发挥有效的抗凋亡作用。
5、EGCG通过促进APP/PS1转基因小鼠大脑皮层及海马内的Trka及CREB蛋白水平,对神经细胞发挥一定的保护作用。
6、EGCG通过提高APP/PS1转基因小鼠脑内内Phospho-c-raf、Phospho-MEK1/2、Phospho-ERK1/2、Phospho-p90RSK、Phospho-MSK1抗凋亡通路相关蛋白表达水平,对神经细胞发挥有效的抑制凋亡的作用。
Alzheimer's disease (AD), also known as senile dementia, is a progressive neurodegenerative disease, with the characteristic of insidious onset, progressive memory loss, cognitive deterioration, behavioral disorders. And at last Decreased ability of life AD is pathologically characterized by deposition ofβ-amyloid (Aβ) peptides as senile plaques and neurofibrillary tangles in the brain. The precise mechanisms of AD are not yet clearly understood, but Free Radical Stress Theory and Cell Apoptosis Theory are paid close attention to. Oxidative stress and reactive oxygen species (ROS) have been proposed to be major cause of functional disorder and neurodegeneration in AD. P75 neurotrophin receptor is the low affinity receptor of neurotrophins and it is closely relative to neuronal apoptosis in AD. The apoptosis signaling pathway P75NTR involved in is not yet clearly understood, but it is well known that P75NTR can activate c-jun N-terminal kinase (JNK) and pro-apoptotic protein p53 and Bad, and induce cell apoptosis. In addition, JNK can also promote cytochrome c release and activation of caspase-3. TrkA (tyrosine kinase A) is the high affinity receptor of neurotrophins and it is closely relative to neuronal anti-apoptosis in AD.when NGFand TrkA are binded together, though MAPK-ERKpathway TrkA performs its effects.
It is very important to study and make suitable animal models for evaluating the cause, pathogenesy of AD and developing drugs to cure AD. So far different animal models have been developed to study the etiology, evolution and new therapeutic alternatives for the illnesses, among which the mice transgenic mouse models have been created with mutations in genes related to AD. Moreover, double transgenics, such as APP/PS1 transgenic mice, provide a valuable model for evaluating the pathogenesy of AD.
(-)-Epigallocatechin-3-gallate(EGCG), which is classified the catechin family and is one of the major polyphenol constituents of green tea. It has been reported that EGCG possess potent iron-chelating, antioxidant, anti-inflammatory, anticancer and neuroprotective activities. EGCG has been shown to have neuroprotective effects by elevatingα-secretase activity of amyloid precursor protein (APP) to soluble APP-alpha (sAPP-α) and reducing Amyloid beta (Aβ)-induced neurotoxicity in human SH-SY5Y neuroblastoma and rat pheochromocytoma PC12 cells. In the present study, we used APP/PS1 transgenic mice, and observed whether EGCG (2 mg/kg.d,4 weeks, ig) had the potent antioxidant and anti-apoptotic neuroprotective effects on the AD mice and APP/PS1 transgenic by behavioral and pathological testing, measurements of the activities of total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px), contents of malondialdehyde (MDA) and activation and expression of pro-apoptotic protein caspase-3, P75, JNK, P53and anti-apoptotic protein Phospho-c-raf Phospho-MEK1/2, Phospho-ERK1/2, Phospho-p90RSK, Phospho-MSK1 in the hippocampus of mice by immunohistochemical staining and Western blot analysis.
Materials and Methods
Ten C57 mice and twenty two APP/PS1 mice were randomly divided into three groups (n=10 each group):control group, APP/PS1 group and APP/PS1+2 mg/kg EGCG group. The mice of APP/PS1+EGCG (2 mg/kg) group were intragastricly given with EGCG at the dose of 2 mg/kg.d for 4 weeks. The mice of control group and APP/PS1 group were administered with same volume vehicle distilled water. After finishing all treatments, animals were evaluated by behavioral testing, and then immediately sacrificed to dissect hippocampus and stored at-80℃for the examinations of expression of pro-apoptotic protein P75, JNK, P53 and anti-apoptotic Phospho-c-raf, Phospho-MEK1/2, Phospho-ERK1/2, Phospho-p90RSK, Phospho-MSK1 in the hippocampus of mice by Western blot analysis. Other mice (n=4) were transcardially perfused with normal saline followed by 4% paraformaldehyde solution. The hippocampus were removed, post-fixed in 4% paraformaldehyde and used for HE and TUNEL and immunohistochemical staining of Aβ(1-40) and CREB.
Results
1. The effects of EGCG on learning and memory impairment in APP/PS1 Transgenic Mouse
The results by water maze, step-through and spontaneous activity test showed APP/PS1 Transgenic Mouse could significantly impair learning and memory of mice, but could not affect their locomotor activity. And EGCG (2 mg/kg. d,4 weeks, ig) could evidently improve the learning and memory impairment in this APP/PS1 Transgenic Mouse.
2. The histopathological effects of EGCG on the brains of APP/PS1 Transgenic Mouse
The results by HE and Nissl staining showed the number of neuron was significantly decreased, the arrangement of neurons in cortex and hippocampus of APP/PS1 Transgenic Mouse was sparse and Nissl body was decreased and dissolved. EGCG (2 mg/kg,4weeks, ig) and could evidently release neuronal injury in APP/PS1 Transgenic Mouse
3. The effects of EGCG on expression of Aβ(1-40) in brains of APP/PS1 Transgenic Mouse
The results by immunohistochemical staining of Aβ(1-40) showed APP/PS1 Transgenic Mouse an obvious increase of Aβ(1-40) in the cortex and hippocampus of mice, and EGCG (2 mg/kg.d,4 weeks, ig) significantly reversed the effect.
4. The effects of EGCG on activities of SOD, GSH-Px and contents of MDA in the hippocampus of APP/PS1 Transgenic Mouse
The results by biological analysis showed APP/PS1 Transgenic Mouse decreased activities of SOD and GSH-Px, increased contents of MDA in mouse hippocampus, and EGCG (2 mg/kg.d,4 weeks, ig) significantly elevated the activities of SOD and GSH-Px, decreased the contents of MDA in hippocampus of APP/PS1 Transgenic Mouse.
5. The effects of EGCG on neuronal apoptosis in brains of APP/PS1 Transgenic Mouse
TUNEL staining results showed TUNEL positive neurons were highly widespread in the cerebral cortex and hippocampus in APP/PS1 Transgenic Mouse and the cell apoptosis index was obviously increased in the hippocampus of mice in model group, compared with control group. EGCG (2 mg/kg.d,4 weeks, ig) and significantly decreased cell apoptosis index in the hippocampus of APP/PS1 Transgenic Mouse
6. The effects of EGCG on activation of CREB in the brains of APP/PS1 Transgenic Mouse
The results by immunohistochemical staining and western blot analysis of CREB showed APP/PS1 Transgenic Mouse significantly reduced in the activation of CREB in the cerebral cortex and hippocampus of mice. EGCG (2 mg/kg.d,4 weeks, ig) significantly increase the activation of CREB in the cerebral cortex and hippocampus of APP/PS1 Transgenic Mouse.
7. The effects of EGCG on expresstion of APP in the brains of APP/PS1 Transgenic Mouse
The results by immunohistochemical staining and western blot analysis of APP showed APP/PS1 Transgenic Mouse significantly increased in the expresstion of APP in the cerebral cortex and hippocampus of mice. EGCG(2 mg/kg.d,4 weeks, ig) significantly reduced the express of APP in the cerebral cortex and hippocampus of APP/PS1 Transgenic Mouse.
8. The effects of EGCG on express of P75NTR, JNK2 and P53 in the hippocampus of APP/PS1 Transgenic Mouse
The results by western blot analysis showed APP/PS1 Transgenic Mouse significantly promoted the shearing of P75 in the hippocampus of mice and increased the displacement of p75ICD to the nucleus, and APP/PS1 Transgenic Mouse also significantly increased the express of JNK2 and P53 in the hippocampus of mice. EGCG (2 mg/kg. d,4 weeks, ig) significantly decreased the expresstion of p75ICD, JNK2 and P53 in the hippocampus of APP/PS1 Transgenic Mouse.
9. The effects of EGCG on expresstion of Phospho-c-raf, Phospho-MEK1/2, Phospho-ERK1/2, Phospho-p90RSK, Phospho-MSK1 in the hippocampus of APP/PS1 Transgenic Mouse
The results by western blot analysis showed APP/PS1 Transgenic Mouse significantly decreased Phospho-c-raf, Phospho-MEKl/2, Phospho-ERK1/2, Phospho-p90RSK、Phospho-MSK1 in the hippocampus of mice, and APP/PS1 Transgenic Mouse also significantly decreased the express of TrkA in the hippocampus of mice. EGCG (2 mg/kg.d,4 weeks, ig) significantly increased the express of Phospho-c-raf, Phospho-MEK1/2, Phospho-ERK1/2, Phospho-p90RSK, Phospho-MSK1 and TrkA in the hippocampus of APP/PS1 Transgenic Mouse.
Conclusion
1. EGCG can improve the learning and memory impairment of APP/PS1 transgenic mice.
2. EGCG has a potent antioxidant effect by elevating the activities of SOD and GSH-Px and decreasing the contents of MDA and the activities of AChE in hippocampus of APP/PS1 Transgenic Mouse.
3. EGCG has a protective effect on APP/PS1 Transgenic Mouse by decreasing the express of APP andβ-Amyloid (1-40) in the cerebral cortex and hippocampus of mice.
4. EGCG has a potent anti-apoptotic effect by reducing the express of p75ICD, JNK2 and P53, and decreasing the activation of caspase-3 in the hippocampus of APP/PS1 transgenic mice.
5. EGCG has a potent anti-apoptotic effect by increase the express of Phospho-c-raf, Phospho-MEK1/2, Phospho-ERK1/2, Phospho-p90RSK, Phospho-MSK1, in the hippocampus of APP/PS1 transgenic mice.
6. EGCG has a potent anti-apoptotic effect by increase the expresstion of TrkA and CREB in the hippocampus of APP/PS1 transgenic mice.
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30 Jean-Francois Lavoie TrkA Induces Apoptosis of Neuroblastoma Cells and Does Sovia a p53-dependent Mechanism2005; 280(32):29199-29207
31 Nicholson DW, Ali A, Thornberry NA, et al. Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature.1995; 376:37-43.
32 Lu J, Zheng YL, Wu DM, et al. Ursolic acid ameliorates cognition deficits and attenuates oxidative damage in the brain of senescent mice induced by d-galactose. Biochemical Pharmacology.2007; 74:1078-1090.
33 Silva A, Montague JR, Lopez TF, et al. Growth factor effects on survival and development of calbindin immunopositive cultured septal neurons. Brain Res Bull.2000; 51:35-42.
34 Jung KM, Tan S, Landman N, Petrova K et al. Regulated intramembrane proteolysis of the p75 neurotrophin receptor modulates its association with the TrkA receptor. J Biol Chem.2003; 278: 42161-9.
35 Costantini C, Scrable H, Puglielli L. An aging pathway controls the TrkA to p75NTR receptor switch and amyloid beta-peptide generation. EMBO J.2006; 25:1997-2006.
36 Hashimoto Y, Kaneko Y, Tsukamoto E, et al. Molecular characterization of neurohybrid cell death induced by Alzheimer's amyloid-beta peptides via p75NTR/PLAIDD. J Neurochem.2004; 90:549-558.
37 Jaffar S, Counts SE, Ma SY, et al. Neuropathology of mice carrying mutant APP (swe) and/or PS1 (M146L) transgenes:alterations in the p75 (NTR) cholinergic basal forebrain septohippocampal pathway. Exp. Neurol.2001; 170:227-243.
38 Chauhan NB, Siegel GJ. Effect of PPF and ALCAR on the induction of NGF-and p75-mRNA and on APP processing in Tg2576 brain. Neurochem Int.2003; 43:225-233.
39 Angelika. Eggert Molecular dissection of TrkA signal transduction pathways mediating differentiation in human neuroblastoma cells. Oncogene 2000; 19:2043-2051.
40 Keramaris E, Ruzhynsky VA, Callaghan SM, et al. Required roles of Bax and JNKs in central and peripheral nervous system death of retinoblastoma-deficient mice. J Biol Chem.2008; 283: 405-15.
41 Lauricella M, Emanuele S, D'Anneo A, et al. JNK and AP-1 mediate apoptosis induced by bortezomib in HepG2 cells via FasL/caspase-8 and mitochondria-dependent pathways. Apoptosis.2006; 11:607-25.
42 Aloyz RS, Bamji SX, Pozniak CD, et al. p53 is essential for developmental neuron death as regulated by the TrkA and p75 neurotrophin receptors. J Cell Biol.1998; 143:1691-703.
43 Hastak K, Gupta S, Ahmad N, et al. Role of p53 and NF-kappaB in epigallocatechin-3-gallate-induced apoptosis of LNCaP cells. Oncogene.2003; 22:4851-4859.
44 Choi JS, Choi YJ, Shin SY, et al. Dietary flavonoids differentially reduce oxidized LDL-induced apoptosis in human endothelial cells:role of MAPK-and JAK/STAT-signaling. J Nutr.2008; 138:983-90.
45 Cotman CW. Apoptosis decision cascades and neuronal degeneration in Alzheimer's disease. Neurobiol Aging.1998; 19:S29-32.
46 Ferrer I, Planas AM. Signaling of cell death and cell survival following focal cerebral ischemia: life and death struggle in the penumbra. J Neuropathol Exp Neurol.2003; 62(4):329-339.
47 Claudio COSTANTINI.A TrkA-to-p75NTR molecular switch activates amyloid β-peptide generation during aging. Biochem. J.2005; 391:59-67.
48 Koh SH, Lee SM, Kim HY, et al. The effect of epigallocatechin gallate on suppressing disease progression of ALS model mice. Neurosci Lett.2006; 395:103-107.
49 Kang J, Lemaire HG, Unterbeck A, et al. The precursor of Alzheimer's disease amyloid A4 protein resembles a cell-surface receptor. Nature.1987; 325:733-736.
50 Lichtenthaler SF, Haass C. Amyloid at the cutting edge:activation of alpha-secretase prevents amyloidogenesis in an Alzheimer disease mouse model. J. Clin. Invest.2004; 113:1384-1387.
51 Pike CJ, Burdick D, Walencewicz AJ, et al. Neurodegeneration induced by beta-amyloid peptides in vitro:the role of peptide assembly state. J. Neurosci.1993; 13:1676-1687.
52 Selkoe DJ. Alzheimer's disease:genes, proteins, and therapy. Physiol. Rev.2001; 81:741-766.
53 Kanning KC, Hudson M, Amieux PS, et al. Proteolytic processing of the p75 neurotrophin receptor and two homologs generates C-terminal fragments with signaling capability.J Neurosci. 2003; 23:5425-5436.
54 Herrmann JL, Menter DG, Hamada J, et al. Mediation of NGF-stimulated extracellular matrix invasion by the human melanoma low-affinity p75 neurotrophin receptor:melanoma p75 functions independently of trkA. Mol Biol Cell.1993; 4:1205-1216.
55 Barker PA. p75NTR is positively promiscuous:novel partners and new insights.Neuron.2004; 42:529-33.
56 Bhakar AL, Howell JL, Paul CE, et al. Apoptosis induced by p75NTR overexpression requires Jun kinase-dependent phosphorylation of Bad. J Neurosci.2003; 23:11373-11381.
57 Cenini G, Sultana R, Memo M, et al. Elevated levels of pro-apoptotic p53 and its oxidative modification by the lipid peroxidation product, HNE, in brain from subjects with amnestic mild cognitive impairment and Alzheimer's disease. J. Cell. Mol. Med.2008; 12:987-94.
58 Yardin C. [Histopathology of Alzheimer's disease]. Morphologie 2007; 91:199-201.
59 Wang XP, Ding HL. Alzheimer's disease:epidemiology, genetics, and beyond. Neurosci Bull. 2008; 24:105-109.
60 Hang-Seng Liu. Inhibitory effect of green tea (-)-epigallocatechin gallate on resistin gene expression in 3T3-L1 adipocytes depends on the ERK pathway. Am J Physiol Endocrinol Metab 2006; 290:E273-E281
61 Schliebs R, Arendt T. The significance of the cholinergic system in the brain during aging and in Alzheimer's disease. J Neural Transm.2006; 113:1625-1644.
62 Lalonde R, Kim HD, Maxwell JA, et al. Exploratory activity and spatial learning in 12-month-old APP(695)SWE/co+PS1/DeltaE9 mice with amyloid plaques. Neurosci Lett.2005; 390:87-92.
63 Garcia-Alloza M, Robbins EM, Zhang-Nunes SX, et al. Charaterization of amyloid deposition in the APPswe/PS1dE9 mouse model of Alzheimer disease. Neurobiol Dis.2006; 24:516-524.
64 Duff K, Eckman C, Zehr C, et al. Increased amyloid-beta42(43) in brains of mice expressing mutant presenilin 1. Nature.1996; 383:710-713.
65 Lalonde R, Kim HD, Fukuchi K. Exploratory activity, anxiety, and motor coordination in bigenic APPswe+PS1/DeltaE9 mice. Neurosci Lett.2004; 369:156-161.
66 Qiu-Lan Ma. Evidence of Aβ-and transgene-dependent defects in ERK-CREB signaling in Alzheimer's models.J Neurochem.2007; 103(4):1594-1607
67 Petar Podlesniy.Pro-NGF from Alzheimer's Disease and Normal Human Brain Displays Distinctive Abilities to InduceProcessing and Nuclear Translocation of Intracellular Domain of p75NTR and Apoptosis. American Journal of Pathology; 2006:169(1) 119-131
68 Jose Morales-Corraliza, et al. In Vivo Turnover of Tau and APP Metabolites in the Brains of Wild-Type and Tg2576 Mice:Greater Stability of sAPP in the b-Amyloid Depositing Mice. In Vivo APP and Tau Turnove.2009; 4(9):1-8
69 Maria Manczak, et al. Neutralization of granulocyte macrophage colonystimulating factor decreases amyloid beta 1-42 and suppresses microglial activity in a transgenic mouse model of Alzheimer's disease. Human Molecular Genetics.2009; 18(20):3876-3893.
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4 Yardin C. [Histopathology of Alzheimer's disease]. Morphologie 2007; 91:199-201.
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7 Onyango IG, Khan Sm. Oxidative stress, mitochondrial dysfunction, and stress signaling in Alzheimer's disease. Curr Alzheimer Res.2006; 3:339-349.
8 Schliebs R, Arendt T. The significance of the cholinergic system in the brain during aging and in Alzheimer's disease. J Neural Transm.2006; 113:1625-1644.
9 Naghma Khan Targeting Multiple Signaling Pathways by Green Tea Polyphenol(-)-Epigallocatechin-3-Gallate Cancer Res 2006; 66:(5).2500-2505
10 Lovell MA, Markesbery WR. Amyloid beta peptide,4-hydroxynonenal and apoptosis. Curr Alzheimer Res.2006; 3:359-364.
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14 Lu J, Zheng YL, Wu DM, et al. Ursolic acid ameliorates cognition deficits and attenuates oxidative damage in the brain of senescent mice induced by d-galactose. Biochemical Pharmacology.2007; 74:1078-1090.
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17 Cui X, Zuo P, Zhang Q, et al. Chronic systemic d-galactose exposure induces memory loss, neurodegeneration, and oxidative damage in mice:protective effects of R-alpha-lipoic acid. J Neurosci Res.2006; 83:1584-1590.
18 Reznichenko L, Amit T, Zheng H, et al. Reduction of iron-regulated amyloid precursor protein and beta-amyloid peptide by (-)-epigallocatechin-3-gallate in cell cultures:implications for iron chelation in Alzheimer's disease. J Neurochem.2006; 97:527-536.
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23 Rezai-Zadeh K, Shytle D, Sun N, et al. Green tea epigallocatechin-3-gallate (EGCG) modulates amyloid precursor protein cleavage and reduces cerebral amyloidosis in Alzheimer transgenic mice. Neurosci.2005; 25:8807-8814.
24 Song X, Bao M, Li D, et al. Advanced glycation in d-galactose induced mouse aging model. Mech Ageing Dev.1999; 108:239-251.
25 Shen YX, Xu SY, Wei W, et al. Melatonin reduces memory changes and neural oxidative damage in mice treated with d-galactose. J Pineal Res.2002; 32:173-178.
26 Jeong JH, Kim HJ, Lee TJ, et al. Epigallocatechin 3-gallate attenuates neuronal damage induced by 3-hydroxykynurenine. Toxicology.2004; 195:53-60.
27 Levites Y, Youdim MBH, Maor G, et al. Attenuation of 6-hydroxydopamine (6-OHDA)-induced nuclear factor-kappaB (NF-kappaB) activation and cell death by tea extracts in neuronal cultures. Biochem Pharmacol.2002; 63:21-29.
28 Senthil Kumaran V, Arulmathi K, Srividhya R, et al. Repletion of antioxidant status by EGCG and retardation of oxidative damage induced macromolecular anomalies in aged rats. Exp Gerontol.2008; 43:176-183.
29 Lovell MA, Markesbery WR. Amyloid beta peptide,4-hydroxynonenal and apoptosis. Curr Alzheimer Res.2006; 3:359-364.
30 Jean-Francois Lavoie TrkA Induces Apoptosis of Neuroblastoma Cells and Does Sovia a p53-dependent Mechanism2005; 280(32):29199-29207
31 Nicholson DW, Ali A, Thornberry NA, et al. Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature.1995; 376:37-43.
32 Lu J, Zheng YL, Wu DM, et al. Ursolic acid ameliorates cognition deficits and attenuates oxidative damage in the brain of senescent mice induced by d-galactose. Biochemical Pharmacology.2007; 74:1078-1090.
33 Silva A, Montague JR, Lopez TF, et al. Growth factor effects on survival and development of calbindin immunopositive cultured septal neurons. Brain Res Bull.2000; 51:35-42.
34 Jung KM, Tan S, Landman N, Petrova K et al. Regulated intramembrane proteolysis of the p75 neurotrophin receptor modulates its association with the TrkA receptor. J Biol Chem.2003; 278: 42161-9.
35 Costantini C, Scrable H, Puglielli L. An aging pathway controls the TrkA to p75NTR receptor switch and amyloid beta-peptide generation. EMBO J.2006; 25:1997-2006.
36 Hashimoto Y, Kaneko Y, Tsukamoto E, et al. Molecular characterization of neurohybrid cell death induced by Alzheimer's amyloid-beta peptides via p75NTR/PLAIDD. J Neurochem.2004; 90:549-558.
37 Jaffar S, Counts SE, Ma SY, et al. Neuropathology of mice carrying mutant APP (swe) and/or PS1 (M146L) transgenes:alterations in the p75 (NTR) cholinergic basal forebrain septohippocampal pathway. Exp. Neurol.2001; 170:227-243.
38 Chauhan NB, Siegel GJ. Effect of PPF and ALCAR on the induction of NGF-and p75-mRNA and on APP processing in Tg2576 brain. Neurochem Int.2003; 43:225-233.
39 Angelika. Eggert Molecular dissection of TrkA signal transduction pathways mediating differentiation in human neuroblastoma cells. Oncogene 2000; 19:2043-2051.
40 Keramaris E, Ruzhynsky VA, Callaghan SM, et al. Required roles of Bax and JNKs in central and peripheral nervous system death of retinoblastoma-deficient mice. J Biol Chem.2008; 283: 405-15.
41 Lauricella M, Emanuele S, D'Anneo A, et al. JNK and AP-1 mediate apoptosis induced by bortezomib in HepG2 cells via FasL/caspase-8 and mitochondria-dependent pathways. Apoptosis.2006; 11:607-25.
42 Aloyz RS, Bamji SX, Pozniak CD, et al. p53 is essential for developmental neuron death as regulated by the TrkA and p75 neurotrophin receptors. J Cell Biol.1998; 143:1691-703.
43 Hastak K, Gupta S, Ahmad N, et al. Role of p53 and NF-kappaB in epigallocatechin-3-gallate-induced apoptosis of LNCaP cells. Oncogene.2003; 22:4851-4859.
44 Choi JS, Choi YJ, Shin SY, et al. Dietary flavonoids differentially reduce oxidized LDL-induced apoptosis in human endothelial cells:role of MAPK-and JAK/STAT-signaling. J Nutr.2008; 138:983-90.
45 Cotman CW. Apoptosis decision cascades and neuronal degeneration in Alzheimer's disease. Neurobiol Aging.1998; 19:S29-32.
46 Ferrer I, Planas AM. Signaling of cell death and cell survival following focal cerebral ischemia: life and death struggle in the penumbra. J Neuropathol Exp Neurol.2003; 62(4):329-339.
47 Claudio COSTANTINI.A TrkA-to-p75NTR molecular switch activates amyloid β-peptide generation during aging. Biochem. J.2005; 391:59-67.
48 Koh SH, Lee SM, Kim HY, et al. The effect of epigallocatechin gallate on suppressing disease progression of ALS model mice. Neurosci Lett.2006; 395:103-107.
49 Kang J, Lemaire HG, Unterbeck A, et al. The precursor of Alzheimer's disease amyloid A4 protein resembles a cell-surface receptor. Nature.1987; 325:733-736.
50 Lichtenthaler SF, Haass C. Amyloid at the cutting edge:activation of alpha-secretase prevents amyloidogenesis in an Alzheimer disease mouse model. J. Clin. Invest.2004; 113:1384-1387.
51 Pike CJ, Burdick D, Walencewicz AJ, et al. Neurodegeneration induced by beta-amyloid peptides in vitro:the role of peptide assembly state. J. Neurosci.1993; 13:1676-1687.
52 Selkoe DJ. Alzheimer's disease:genes, proteins, and therapy. Physiol. Rev.2001; 81:741-766.
53 Kanning KC, Hudson M, Amieux PS, et al. Proteolytic processing of the p75 neurotrophin receptor and two homologs generates C-terminal fragments with signaling capability.J Neurosci. 2003; 23:5425-5436.
54 Herrmann JL, Menter DG, Hamada J, et al. Mediation of NGF-stimulated extracellular matrix invasion by the human melanoma low-affinity p75 neurotrophin receptor:melanoma p75 functions independently of trkA. Mol Biol Cell.1993; 4:1205-1216.
55 Barker PA. p75NTR is positively promiscuous:novel partners and new insights.Neuron.2004; 42:529-33.
56 Bhakar AL, Howell JL, Paul CE, et al. Apoptosis induced by p75NTR overexpression requires Jun kinase-dependent phosphorylation of Bad. J Neurosci.2003; 23:11373-11381.
57 Cenini G, Sultana R, Memo M, et al. Elevated levels of pro-apoptotic p53 and its oxidative modification by the lipid peroxidation product, HNE, in brain from subjects with amnestic mild cognitive impairment and Alzheimer's disease. J. Cell. Mol. Med.2008; 12:987-94.
58 Yardin C. [Histopathology of Alzheimer's disease]. Morphologie 2007; 91:199-201.
59 Wang XP, Ding HL. Alzheimer's disease:epidemiology, genetics, and beyond. Neurosci Bull. 2008; 24:105-109.
60 Hang-Seng Liu. Inhibitory effect of green tea (-)-epigallocatechin gallate on resistin gene expression in 3T3-L1 adipocytes depends on the ERK pathway. Am J Physiol Endocrinol Metab 2006; 290:E273-E281
61 Schliebs R, Arendt T. The significance of the cholinergic system in the brain during aging and in Alzheimer's disease. J Neural Transm.2006; 113:1625-1644.
62 Lalonde R, Kim HD, Maxwell JA, et al. Exploratory activity and spatial learning in 12-month-old APP(695)SWE/co+PS1/DeltaE9 mice with amyloid plaques. Neurosci Lett.2005; 390:87-92.
63 Garcia-Alloza M, Robbins EM, Zhang-Nunes SX, et al. Charaterization of amyloid deposition in the APPswe/PS1dE9 mouse model of Alzheimer disease. Neurobiol Dis.2006; 24:516-524.
64 Duff K, Eckman C, Zehr C, et al. Increased amyloid-beta42(43) in brains of mice expressing mutant presenilin 1. Nature.1996; 383:710-713.
65 Lalonde R, Kim HD, Fukuchi K. Exploratory activity, anxiety, and motor coordination in bigenic APPswe+PS1/DeltaE9 mice. Neurosci Lett.2004; 369:156-161.
66 Qiu-Lan Ma. Evidence of Aβ-and transgene-dependent defects in ERK-CREB signaling in Alzheimer's models.J Neurochem.2007; 103(4):1594-1607
67 Petar Podlesniy.Pro-NGF from Alzheimer's Disease and Normal Human Brain Displays Distinctive Abilities to InduceProcessing and Nuclear Translocation of Intracellular Domain of p75NTR and Apoptosis. American Journal of Pathology; 2006:169(1) 119-131
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