Puzzle Soup APP695 Transgenic Mice Therapeutic Effect And Its Mechanism

Alzheimer disease(AD) is a kind of degenerative disease of central nervous system, the major pathological changes in AD are the formation of senile plaques(SP), neurofibrillary tangles(NFT) and loss of synapase and neurons in the cerebral cortex and subcortical structure, Which can severely impair the cognitive function and cause psychical and behavioral abnormality of the patient. The plaques are composed primarily of a small protein called amyloid-beta (Aβ). Aβhas toxicities to neurons and play important role in neurodegeneration and associated with cognitive and behavioral abnormalities. The evidence shows that intraparenchymal deposition of Aβis the major pathological hallmarks and plays a crucial part of the pathogenesis in AD. Traditional Chinese Medicine (TCM) considers that AD occurs in the brain and is closely connected with the deficiency of kidney and excess in superficiality in terms of its pathogenesis. Reinforcing the kidney to replenish marrow, removing blood and phlegm stasis to enrich the brain are the regular therapies. YiZhi fang is mainly composed of Herba Epimedii(Yinyanghuo), Panax quinquefolium(Xiyangshen), Barbary Wolfberry Fruit(GouQi), Rhizoma(Chuanxiong), Tall Gastrodia Tuber(Tianma) and Acorus tatarinowii Schott(Shichangpu), Which has the function of invigorating kidney to replenish marrow and removing phlegm and blood stasis to enrich the brain. The experiment focuses on the curative mechanism of YiZhi decoction to APP695 transgenic mice and provides a scientific ground and theoretical basis in clinical applications.Materials and Methods:1. Experimental study of the protective effect and its mechanism of YiZhi decoction on APP695 transgenic mice:APP695 transgenic mice (13 months old) were randomly divided into model group and YiZhidecoction group, and negative transgenic mice (C57BL/6J) were included into normal group. The mice in YiZhi decoction group were given intragastrically YiZhi decoction (0.1g/kg) for 3 months, and the model group and normal group were given equal quantity of distilled water. (1)The learning and memory behavior of mice were examined by using Y-type electric maze test and LashleyⅢwater maze test. (2) Applying immunohistochemical technique, the expressions of APP, Aβ1-40, Aβ1-42 and Caspase3 in mouse cerebral cortex and hippocampal CA1 were observed by light microscope, using Image analysis their integral optical density in all group. (3)The changes of cell morphology were detected by HE stain and were observed by light microscope, the ultrastructure in cerebral cortex of the mice was observed by the transmission electronic microscope. (4)The levels of Aβ1-42 in cerebrospinal fluid(CSF) by using ELISA Assay Kit. (5)Cerebral neurocyte apoptosis in all group were detected with Terminal deoxynucleotidel transferase mediated uridine nucleotide end labeling(TUNEL).2. Experimental study of the YiZhi decoction rat serum on primary cultured neural cells induced by Aβ25-35:The primary cultured fetal rat cortical neurons were growth for 8 days. The neurons were treated with aggregated Aβ25-35 with different concentration for 24h. The morphologic changes of fetal rat cortical neurons induced by Aβ25-35 were observed by light microscope. Using MTT assay detected the changes of vitality of cells and detected the leakage of LDH. And By using the same means, YiZhi decoction rat serum as a protective effect, we also detected the survival rate and the leakage of LDH in all groups of primary cultured fetal rat cortical neuron induced by Aβ25-35 and YiZhi decoction rat serum, to explore the protective effect of YiZhi decoction rat serum on damage induced by Aβ25-35.Objectives:1. YiZhi decoction can improve learning and memory capacity of the model mice:The results of Y-type electric maze test showed that the number used to indicate learning ability increased and the number used to indicate memory ability decreased in model group as compared with normal group (P<0.01), while the number used to indicate learning ability decreased and the number used to indicate memory ability increased in YiZhi decoction group as compared with model group. The results of Lashley III water maze test showed that the numbers of entering to blind-ending and lengthened latency in model group were more and longer than those in normal group, but those in YiZhi decoction group were considerably fewer and shorter than those in model group. The two means indicate that APP695 transgenic mice has learning and memory deficit and coincide with AD clinical feature, YiZhi decoction can improve learning and memory capacity of the model mice.2. YiZhi decoction can down-regulate the expressions of APP, Aβ1-40 and Aβ1-42 in APP695 transgenic mice:The number of APP, Aβ1-40 and Aβ1-42 positive cell and the degree of staining by their antibody in the cortex and hippocampal CA1 in the model group were higher than those in the normal group, using Image analysis their integral optical density (IOD). The levels of APP, Aβ1-40 and Aβ1-42 expression in the cortex and hippocampal CA1 of the model group obviously increased as compared with the normal group(P<0.01). the expression level of APP, Aβ1-40 and Aβ1-42 in YiZhi dectaion group were significantly lower than those in the model group(P<0.05). The levels of Aβ1-42 in cerebrospinal fluid(CSF) of model group were significantly higher than that in the normal group and YiZhi decoction group by using ELISA assay kit((P<0.05). The results indicate YiZhi decoction can down-regulate the level of APP, Aβ1-40 and Aβ1-42 in APP695 transgenic mice.3. YiZhi decotaion can relieve brain tissue morphological changes in APP695 transgenic mice:By the light microscope the modality configuration of cells in the Cerebral cortex and hippocampal CA1 in the normal group were normal, the neurons had regular arrangement with clear edges and obvious nucleus. But in model group the neurons had irregular arrangement and the edge of many cells was unclear, and there were infiltration of inflammatory cells in blood vessel walls, some neurons were atrophied; and those changes in the YiZhi decoction group were better than those in the model group, YiZhi decoction could improve brain tissue morphological changes in APP695 transgenic mice.By the electron microscope in normal group mice the cell in cerebral cortex were normal, the nucleus was round and big, the normal and abnormal chromatin was obvious, the configurations of endoplasmic reticulum, mitochondrion and synapses were also normal, both presynaptic and postsynaptic membranes were clear. In model group, the cells were atrophied and dwindled with obvious borderline, the swollen, disrupted mitochondria, active microglia, degenerative synaptic structure and disarranged microtubule were frequently found, the presynaptic and postsynaptic membranes were illegible; cell organelles reduced but the deposition of lipofuscin granules increased, the basement membranes of capillaries was irregular and had some deposits; Myelin sheaths were loose, part of myelin fragments completely separated, microtubule and microfilaments showed particle dissolution. These pathological changes in the brain of YiZhi decoction treat group mice were not so apparent and were similar to model group.4. YiZhi decoction can decrease cerebral neurocyte apoptosis in APP695 transgenic mice to protect cerebral neurocyte:The number of Caspase3 positive cell and the degree of staining by Caspase3 antibody increased in the brain of model group as compared with the normal group(P<0.01). The expression level of Caspase3 in YiZhi decotaion group was significantly lower than that in the model group (P<0.05). The TUNEL positive cells in model group increased than those in normal group(P<0.01). The TUNEL positive cell decreased in YiZhi dectoaion group than that in model group. The results indicate YiZhi deoctaion can decrease cerebral neurocyte apoptosis in APP695 transgenic mice to protect cerebral neurocyte.5 YiZhi decoction rat serum can protect the primary cultured neural cells from the damage induced by Aβ25-35:The primary cultured fetal rat cortical neuron were performed with Aβ25-35 (5,10,20, 40μmol/L) for 24 hours, the growth state is bad. The activity of cultured neurons were decreased and LDH leakage increased in culture solution, and the cell viability decreased gradually when the concentration of Aβ25-35 raised up, there was significantly difference between model group and normal control group(P＜0.01). using 10μmol/L Aβ25-35 to made AD model cells, added YiZhi decoction rat serum, The activity of cultured neurons were higher in YiZhi decoction group than that in Aβ25-35 group, the growth state is good. The activity of nerve cells increased and LDH activity decreased by comparison with the experimental control group, which indicate that the YiZhi decoction rat serum could protect the cells from the damage induced by Aβ25-35 through abatting Aβnerotoxicity.Conclusion: APP695 transgenic mice has learning and memory deficit, increase the level of APP and Aβexpression, and has cerebral pathological changes and neurocyte apoptosis in brain, so it is a good animal model for AD. YiZhi decoction can regulate the level of APP and Aβ, can relieve cerebral pathological changes and decrease cerebralneurocyte apoptosis to protect cerebral neurocyte. That is the basement to improve learning and memory ability. YiZhi decoction rat serum could protect the cells from the damage induced by Aβ25-35. So YiZhi decoction can antagonize toxicity of Aβon neuron and protect nerve cell. The study provides experimental support for reinforcing the kidney to replenish marrow, removing blood and phlegm stasis to provide a scientific ground for clinical treatment of AD.