Study Of The Effects Of Grape Seed Proanthocyanidin On Alzheimer’s Disease In Vitro And In Vivo Model

ObjectiveUsing the APP/PS1 double transgenic mice and the PC12 cell oxidative damage model with AP25-35 induced, to establish the model of Alzheimer’s disease in vitro and in vivo, respectively from the overall and cell level to detect the effect of grape seed proanthocyanidin on the AD and its possible mechanism.MethodThe APP/PS1 double transgenic mice as AD animal model, from the behavior, gene and protein expression level to research the effect of grape seed proanthocyanidin in mice. The APP/PS1 double transgenic mice were randomly divided into APP/PS1 model group, donepezil group, grape seed proanthocyanidin low and high group, and with the same strain and age of C57BL/6 as normal control group. Each dosage group was intragastric administration respectively donepezil and grape seed proanthocyanidin, the APP/PS1 model group and normal control group were intragastric administration the same amount of saline solution. After 2 months, using the morris water maze to detect the ability of spatial learning and memory in mice; then cervical dislocation executed in mice and dissected its hippocampus tissues, HE and Congo red staining method were used respectively to research the mice hippocampus tissues pathhhological morphology and CA1 area beta amyloid deposits; at the same time, respectively by using Real time PCR and Western blot method determination the PS1 mRNA and APP, Tau protein expression of hippocampus in mice.After PC 12 cell culture and identification, the oxidative damage model with AP25-35 induced was established. After 24 hours, the cell was intervented by grape seed proanthocyanidin at 12.5μg/ml,25^ig/ml,50μg/ml,100μg/ml concentration, and compared with normal cultured cells.24 hour later, using CCK-8 and lactate dehydrogenase kit respectively to detect the cell vitality and LDH leakage, the apoptosis rate and mitochondrial membrane potential were evaluated by flow cytometry method.ResultMorris water maze method results show that compared with model group, grape seed proanthocyanidin can obviously enhance the ability of spatial learning in mice, performance in significantly shorten the escape latency and the distance to go to the platform, increase platform crossing numbers, the distance and time percentage of the central activity area. According to the results of HE staining, the model group mice hippocampal nerve cells arranged loosely, disorder, the cellular structure is not clear, part of the cell vacuoles degeneration, most of the cell body and nuclear pyknosis hyperchromatic. The cell morphology was regular and the cells were arranged more neatly and tightly, cell nuclei stained clear as well in grape seed proanthocyanidin high group. According to the results of Congo red staining, the model group mice hippocampal have many orange plaque deposits, however, the grape seed proanthocyanidin high group of mice CA1 area orange plaque deposition quantity and area were significantly reduced. At the same time, compared with model group, Real time PCR and Western blot results show that the grape seed proanthocyanidin high doses can significantly reduce the PS1 mRNA, APP and Tau protein expression in mice hippocampus.PC 12 cell were colony growth, having strongly colony stero sense, forming dense cell clusters, and irregular polygon or fusiform, with typical characteristics of the nerve cells after cluture 3 days. CCK-8 method results show that compared with model group, grape seed proanthocyanidin each dose group can increase the optical density value, it is suggests that the cell vitality were significantly increased. LDH kits results show that compared with model group, grape seed proanthocyanidin each dose group can decrease the optical density value, it is suggests that the cells LDH leakage rate were significantly decreased. Flow cytometry assay results show that compared with model group, grape seed proanthocyanidin each dose group can significantly reduce the apoptosis rate, cell survival rate and mitochondrial transmembrane potential were significantly increased.ConclusionIn AD pharmacodynamice experiment, comparaed with model group, the APP/PS1 double transgenic mice of escape latency and the distance to go to the platform were obviously shortened, platform crossing numbers, the distance and time percentage of the central activity area were significantly increased. Above results suggest that the model of APP/PS1 double transgenic mice were successful, and grape seed proanthocyanidin can advance the alility of learning and memory in mice, improve the symptoms of learning and memory obstacle.In mechanism research experiments, comparaed with model group, the APP/PS1 double transgenic mice hippocampal cells arranged neatly, closely, orange plaque deposition quantity and area reduction, PS1 mRNA, APP and Tau protein expression decreased. In the meantime, comparaed with control group, the oxidative damage of cell activity and mitochondrial transmembrane potential were increased, LDH leakage and apoptosis rate were declined. Above results suggest that grape seed proanthocyanidin can protect cells oxidative damage, reduce the leakage of LDH, improve mitochondrial transmembrane potential, so as to decrease the rate of cell apoptosis, while down regulateing the PS1 mRNA, APP and Tau protein expression, and maintain the mice hippocampal structure integrity, protect nerve cells and reduce the deposits of amyloid.This experiment research results show that the grape seed proanthocyanidin has improved APP/PS1 double transgenic mice spatial learning and memory ability, and its mechanism may be related to increased hippocampal cells vigor and mitochondrial transmembrane potential, decrease the rate of LDH leakage, derease the rate of apoptosis, also related with PS1 mRNA, APP and Tau protein expression, a beta deposition and so on.