Protective Effects And Mechanisms Of EGCG On APP/PS1 Transgenic Mice

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 MethodsTen 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.Results1. The effects of EGCG on learning and memory impairment in APP/PS1 Transgenic MouseThe 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 MouseThe 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 Mouse3. The effects of EGCG on expression of Aβ(1-40) in brains of APP/PS1 Transgenic MouseThe 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 MouseTUNEL 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 Mouse6. The effects of EGCG on activation of CREB in the brains of APP/PS1 Transgenic MouseThe 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 MouseThe 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 MouseThe 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 MouseThe 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.Conclusion1. 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.