Protective Effects And Mechanisms Of EGCG On Alzheimer's Disease Mice

ObjectiveAlzheimer's disease(AD) is a progressive neurodegenerative disease,with the characteristic of progressive memory loss,cognitive deterioration and behavioral disorders.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.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 continuously injected with D-galactose(D-gal) have been extensively used for pharmacological research on AD. 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 AD mouse model induced by D-gal(150mg/kg.d,6 weeks,sc.) and APP/PS1 transgenic mice,and observed whether EGCG(2 mg/kg.d or 6 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 and P53 in the hippocampus of mice by immunohistochemical staining and Western blot analysis.Materials and MethodsNinety-six healthy Kunming mice were randomly divided into six groups(n=16 each group):control group,D-gal+ddH₂O group,D-gal+oil group,D-gal+VE (280IU/kg) group,D-gal+EGCG(2 mg/kg) group and D-gal+EGCG(6mg/kg) group.The mice of D-gal+ddH₂O group,D-gal+oil group,D-gal+VE(280IU/kg) group,D-gal+EGCG(2 mg/kg.d) group and D-gal+EGCG(6 mg/kg.d) group were subcutaneously injected with 3%D-gal at the dose of 150 mg/kg body weight once daily for 6 weeks,while those of control group were treated with same volume normal saline.From the third week,the mice of D-gal+VE(280IU/kg) group,D-gal+EGCG (2 mg/kg.d) group and D-gal+EGCG(6 mg/kg.d) group were intragastricly given with 5.6%VE at the dose of 280IU/kg and EGCG at the dose of 2 mg/kg.d or 6 mg/kg.d respectively after injection of D-gal.The mice of control group,D-gal+ddH₂O group and D-gal+oil group were administered with same volume vehicle distilled water and soybean oil respectively.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 activities of SOD and GSH-Px,contents of MDA, and activation and expression of pro-apoptotic protein caspase-3,P75,JNK and P53 in the hippocampus of mice by Western blot analysis.Other mice(n=6) were transcardially perfused with normal saline followed by 4%paraformaldehyde solution. The hippocampus were removed,post-fixed in 4%paraformaldehyde and used for HE, Nissl and TUNEL staining and immunohistochemical staining of Aβ(1-40),caspase-3 and APP.Ten C57 mice and twenty 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 and P53 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 immunohistochemical staining of Aβ(1-40).Results1.The effects of EGCG on learning and memory impairment in AD model mice induced by D-gal.The results by water maze,step-through and spontaneous activity test showed D-gal (150 mg/kg.d,6 weeks,sc.) could significantly impair learning and memory of mice, but could not affect their locomotor activity.And EGCG(2 mg/kg.d or 6 mg/kg.d,4 weeks,ig) and VE(280IU/kg,4 weeks,ig) could evidently improve the learning and memory impairment in AD model mice induced by D-gal.2.The histopathological effects of EGCG on the brains of AD model mice induced by D-gal.The results by HE and Nissl staining showed the number of neuron was significantly decreased,the arrangement of neurons in cortex and hippocampus of D-gal model mice was sparse and Nissl body was decreased and dissolved.EGCG(2 mg/kg.d or 6 mg/kg.d,4 weeks,ig) and VE(280IU/kg,4 weeks,ig) could evidently release neuronal injury induced by D-gal.3.The effects of EGCG on express of Aβ(1-40) in brains of AD model mice induced by D-galThe results by immunohistochemical staining of Aβ(l-40) showed D-gal (150 mg/kg.d,6 weeks,sc.) induced an obvious increase of Aβ(1-40) in the cortex and hippocampus of mice,and EGCG(2 mg/kg.d or 6 mg/kg.d,4 weeks,ig) and VE (280IU/kg,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 AD model mice induced by D-gal.The results by biological analysis showed D-gal(150 mg/kg.d,6 weeks,sc.) decreased activities of SOD and GSH-Px,increased contents of MDA in mouse hippocampus,and EGCG(2 mg/kg.d or 6 mg/kg.d,4 weeks,ig) and VE(280IU/kg,4 weeks,ig) significantly elevated the activities of SOD and GSH-Px,decreased the contents of MDA in hippocampus of AD model mice induced by D-gal.5.The effects of EGCG on neuronal apoptosis in brains of AD model mice induced by D-gal.TUNEL staining results showed TUNEL positive neurons were highly widespread in the cerebral cortex and hippocampus in AD model mice induced by D-gal 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 or 6 mg/kg.d,4 weeks,ig) and VE(280IU/kg,4 weeks,ig) significantly decreased cell apoptosis index in the hippocampus of mice induced by D-gal.6.The effects of EGCG on activation of caspase-3 in the brains of AD model mice induced by D-gal.The results by immunohistochemical staining and western blot analysis of caspase-3 showed D-gal(150 mg/kg.d,6 weeks,sc.) induced significantly increased in the activation of caspase-3 in the cerebral cortex and hippocampus of mice.EGCG(2 mg/kg.d or 6 mg/kg.d,4 weeks,ig) significantly reduced the activation of caspase-3 in the cerebral cortex and hippocampus of AD model mice induced by D-gal,but VE (280IU/kg,4 weeks,ig) was not observed the effect.7.The effects of EGCG on express of APP in the brains of AD model mice induced by D-gal. The results by immunohistochemical staining and western blot analysis of APP showed D-gal(150mg/kg.d,6 weeks,sc.) induced significantly increased in the express of APP in the cerebral cortex and hippocampus of mice.EGCG(2 mg/kg.d or 6 mg/kg.d,4 weeks,ig) and VE(280IU/kg,4 weeks,ig) significantly reduced the express of APP in the cerebral cortex and hippocampus of AD model mice induced by D-gal.8.The effects of EGCG on express of P75NTR,JNK2 and P53 in the hippocampus of AD model mice induced by D-gal.The results by western blot analysis showed D-gal(150 mg/kg.d,6 weeks,sc.) significantly promoted the shearing of P75 in the hippocampus of mice and increased the displacement of p75ICD to the nucleus,and D-gal(150 mg/kg.d,6 weeks,sc.) also significantly increased the express of JNK2 and P53 in the hippocampus of mice. EGCG(2 mg/kg.d or 6 mg/kg.d,4 weeks,ig) significantly decreased the express of p75ICD,JNK2 and P53 in the hippocampus of AD model mice induced by D-gal,and VE(280IU/kg,4 weeks,ig) was not found obviously inhibitory effect.9.The effects of EGCG on learning and memory of APP/PS1 transgenic mice.The results by step-through test showed APP/PS1 transgenic mice had obvious memory impairment compared with C57 mice.EGCG(2 mg/kg.d,4 weeks,ig) could significantly improve the memory impairment of APP/PS1 transgenic mice.10.The effects of EGCG on the express ofβ-Amyloid(1-40) in brains of APP/PS1 transgenic mice.The results by immunohistochemical staining ofβ-Amyloid(1-40) showed an obvious increase in the express ofβ-Amyloid(1-40) in the cortex and hippocampus of APP/PS1 transgenic mice,and EGCG(2 mg/kg.d,4 weeks,ig) significantly decreased the express ofβ-Amyloid(1-40) in the cortex and hippocampus of APP/PS1 transgenic mice.11.The effects of EGCG on the express of P75NTR,JNK2 and P53 in the hippocampus of APP/PS1 transgenic mice.The results by western blot analysis showed the express of p75ICD,JNK2 and P53 in the hippocampus of APP/PS1 transgenic mice were significantly increased, compared with C57 mice.EGCG(2 mg/kg.d,4 weeks,ig) significantly decreased the express of p75ICD,JNK2 and P53 in the hippocampus of APP/PS1 transgenic mice.Conclusion1.EGCG can improve the learning and memory impairment of mice AD model induced by D-gal and 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 MD A in hippocampus of AD model induced by D-gal.3.EGCG has a protective effect on AD model mice induced by D-gal by decreasing the express of APP and P-Amyloid(l-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 AD model induced by D-gal and APP/PS1 transgenic mice.