Studies On The Innovative Complex Model Of Alzheimer's Disease Induced By Aβ25-35 United D-galactose And Intervening Effects Of Chinese Medicine

Alzheimer's disease (AD), which can affected people since the age of forty, also know as presenile dementia, is a neurodegenerative disease of central nervous system. However, the mechanism underlying it is still unknown, and the causes of AD are complex. It is characterized by progressive memory loss, cognitive deficit and other symptoms, which now can not be cured. Dementia has been the forth cause of death in developed countries afterheart disease, cancer and stroke, since the progress of aging population. Patients who have severe dementia couldn't take care of themselves because of psychosis and often died of infection or other diseases in 3 to 9 years after diagnose. AD is now the most common type of dementia, with heavy costs to society of family care giving for patients, has become a worldwide problem.AD is characterized by pathological changes such as neuritic plaques, neurofibrillary tangles and regional neuron loss. In recent years, evidence has accumulated that suggests that oligomeric species of amyloid and tau maybe major cause of AD. The severity of the cognitive defect in Alzheimer's disease correlates with levels of regional deposition of amyloid plaques in the brain, which is the early event of AD. An imbalance between production and clearance, and aggregation of peptides, causes Aβto accumulate, and this excess may be the initiating factor in Alzheimer's disease. This idea, called the "amyloid hypothesis". Accumulated experimental evidence indicates that Aβis neurotoxic. Animal models can be reproduced by encephalocoele or intra hippocampus injection of Aβ. Rats showed memory and study deficits, and the activity of ChAT in the hippocampus is reduced similar to patients with AD. It can be used in researches of mechanism underlying the deposition or aggregation of Aβin AD, as well as screening drugs for AD.Inhibitors of AChE are the main treatment of AD in clinic. Trials of small-molecule inhibitors of (3-amyloid and tau oxidation and aggregation are under way. Besides, BDNF replacement is another option for the treatment of Alzheimer's disease. Since the natural components of the traditional Chinese medicine (TCM), with poly-targets effect, it can be used for dealing with diseases which have complex etiopathogenisis. Experimental studies and clinical application of TCM have been paid closer attention gradually.In recent years, evidence has accumulated that suggests that oligomeric species of amyloid and tau maybe major cause of AD, which is now the focus of most researches, and the relationship between them comfused the most. The severity of the cognitive defect in Alzheimer's disease correlates with levels of regional deposition of amyloid plaques in the brain, which is the early event of AD. An imbalance between production and clearance, and aggregation of peptides, causes A(3 to accumulate, and this excess may be the initiating factor in Alzheimer's disease. This idea, called the "amyloid hypothesis". Accumulated experimental evidence indicates that Aβis neurotoxic. Animal models can be reproduced by encephalocoele or intra hippocampus injection of Aβ. Rats showed memory and study deficits, and the activity of ChAT in the hippocampus is reduced similar to patients with AD. It can be used in researches of mechanism underlying the deposition or aggregation of Aβin AD, as well as screening drugs for AD.Emotional disorders are thought to be one of the important causes of dementia in theory of traditional Chinese medicine, such as excessive unhappiness, depression and angry. Depression could be seen in all phages of dementia. Apathia, depression, apprehension and irritability are the common symptoms of AD patients. Thus stagnation of liver-QI could be the pathology mechanism of AD in theory of traditional Chinese medicine. Furthermore recent researches indicated that stagnation of liver-QI is based on the activities of the central nervous system.Endoplasmic reticulum (ER) is one of the most important organelle in the cell. In the ER, proteins fold into their native conformation and undergo a series of post-translational modifications. To maintain the stabilization of internal environment, proteins without normal conformation or undergoing wrong folding would be transported to the plasma through ER membrane, as to say ER associated degeneration (ERAD). Studies in vivo and in vitro have already indicated that ER stress is involved in the mechanisms of AD.Interestingly, to accomplish the precise quality control system of protein in the cell, the ER need to be unobstructed, either in morphosis nor in effectiveness or functional status. That is similar to the physiological nature of the liver in the traditional Chinese Medicine theory. So it was supposed that the physical fundament of liver controlling dispersion is the function of ER. Our advisor, Professor Wu Zhengzhou proposed the hypothesis of "hepatic depressed vital energy-ER stress" as to be the pathology mechanism of AD in the integrated traditional and western medicine theory. The key view of this hypothesis is that hepatic depressed vital energy is the onset and process of AD, while ER stress is the subcellular mechanism. So disperse the depressed liver-energy with counteract luxuriate could alleviate ER stress in AD.To observe the improvements of spatial study and memory performance and apoptosis of neurons of the Alzheimer's disease (AD) model rats by intragastric administration of Bupleurum Liver-Soothing Powder and Fructus Broussonetiaein, the animal models were induced by Aβ25-35 and D-galactose. To investigate the mechanisms underlying those improvements.40 normal male SD rats were randomly divided into 5 groups with 8 rats in each group, the normal control group, the sham operation (SO) group, the AD model group, the Chaihu shugan san group and the Fructus Broussonetiae group (see table 1). The AD model of rats were established by bilateral hippocampus stereotacticly injection of Aβ25-35 in 5 minutes, while hypodermic injection with D-gal in 50 days.Morris water maze with computer system were used to detect the differences of spatial study and memory performance of rats between groups. The probe navigation experiment and the spatial exploration experiment were used to assess the spatial study and memory performance.Immunohistochemistry (IHC) were used to detect the expression of Aβ, phosphorylated tau, marker proteins of ER stress and apoptosis, which including PERK, BiP and GADD153/CHOP. HE, Congo red stain and toluidine blue O stain were used to observe the pathology changes of rats brains between groups. TdT-mediated dUTP-biotin nick end labeling (TUNEL) technique was used to detect mean apoptosis index of neurons in the hippocampus of all rats.The statistical analysis of all the experimental data was accomplished by SPSS13.0 for windows. The data were all expressed in X±S, and P<0.05 is statistical significant. The experimental data included escape latency, swimming distance, searching time percent, searching distance percent, mean Aβpositive cell number, mean gray of IHC of Bopper and GADD153/CHOP, mean neuron numbers in DG of rats and mean apoptosis index, which were compared means between groups. One-way ANOVA was used when data is homoscedastic and LSD for multiple comparisons. While data is not homoscedastic, Welch was used for statistics analysis and Dennett's T3 for multiple comparisons.1. The effects of Chaihu shugan san and Fructus Broussonetiae on performance of probe navigation experiment of rats:the results of performance of probe navigation experiment showed that in escape latency, there were significant differences between the groups (F=5.004, P=0.007). The escape latency of AD model group were longer than that in normal control group and SO group (P=0.017, P=0.041). The escape latency of Chaihu shugan san group and Fructus Broussonetiae group were longer than that in AD model group (P=0.013, P=0.010). The results of performance of probe navigation experiment showed that in swimming distance, there were significant differences between the groups (F=3.256, P=0.037). The swimming distance of Fructus Broussonetiae group were smaller than that in AD model group (P=0.046).2. The effects of Chaihu shugan san and Fructus Broussonetiae on performance of spatial experiment of rats:the results of performance of spatial exploration experiment showed that in searching time percent, there were significant differences between the groups (F=44.487, P<0.001). The searching time percent of AD model group were less than that in normal control group and SO group (P<0.001, P <0.001). The searching time percent of Chaihu shugan san group and Fructus Broussonetiae group were longer than that in AD model group (P<0.001, P< 0.001). The results of performance of probe navigation experiment showed that in searching distance percent, there were significant differences between the groups (F=37.828, P<0.001). The searching distance percent of AD model group were less than that in normal control group and SO group(P<0.001, P<0.001). The searching distance percent of Chaihu shugan san group and Fructus Broussonetiae group were longer than that in AD model group (P<0.001, P< 0.001).3. The effects of Chaihu shugan san and Fructus Broussonetiae on expression of Aβand phosphorylated tau of AD model rats:the results of performance of IHC showed that in mean Aβpositive cell number, there were significant differences between the groups (F=46.824, P<0.001). The mean Aβpositive cell number of AD model group were higher than that in normal control group and SO group (P <0.001, P<0.001). The mean Aβpositive cell number of Chaihu shugan san group and Fructus Broussonetiae group were lower than that in AD model group (P<0.001, P<0.001). There was no definite phosphorylated tau positive cell in all groups.4. The effects of Chaihu shugan san and Fructus Broussonetiae on expression of BiP of AD model rats:the results of IHC showed that in mean gray of IHC of BiP, there were significant differences between the groups (F=174.042, P< 0.001). The mean gray of IHC of BiP of AD model group were lower than that in normal control group and SO group (P<0.001, P<0.001). The mean gray of IHC of BiP of Chaihu shugan san group and Fructus Broussonetiae group were lower than that in AD model group (P<0.001, P<0.001). The mean gray of IHC of BiP of Chaihu shugan san group and Fructus Broussonetiae group were lower than that in normal control group (P<0.001, P<0.001). The mean gray of IHC of BiP of Chaihu shugan san group and Fructus Broussonetiae group were lower than that in SO group (P<0.001, P<0.001).5. The effects of Chaihu shugan san and Fructus Broussonetiae on expression of PERK of AD model rats:the results of IHC showed that in mean gray of IHC of PERK, there were significant differences between the groups (F=144.556, P <0.001). The mean gray of IHC of PERK of AD model group were lower than that in normal control group and SO group(P<0.001, P<0.001). The mean gray of IHC of PERK of Chaihu shugan san group and Fructus Broussonetiae group were higher than that in AD model group (P<0.001, P<0.001). The mean gray of IHC of PERK of Chaihu shugan san group and Fructus Broussonetiae group were lower than that in normal control group (P<0.001, P<0.001). The mean gray of IHC of PERK of Chaihu shugan san group and Fructus Broussonetiae group were lower than that in SO group (P=0.001, P<0.001).6. The effects of Chaihu shugan san and Fructus Broussonetiae on expression of GADD153/CHOP of AD model rats:the results of IHC showed that in mean gray of IHC of GADD 153/CHOP, there were significant differences between the groups (F=48.703, P<0.001). The mean gray of IHC of GADD153/CHOP of AD model group were lower than that in normal control group and SO group (P< 0.001, P<0.001). The mean gray of IHC of GADD153/CHOP of Chaihu shugan san group and Fructus Broussonetiae group were higher than that in AD model group (P<0.001, P<0.001). The mean gray of IHC of GADD153/CHOP of Chaihu shugan san group and Fructus Broussonetiae group were lower than that in normal control group (P<0.001, P<0.001). The mean gray of IHC of GADD153/CHOP of Chaihu shugan san group and Fructus Broussonetiae group were lower than that in SO group (P=0.001, P<0.001).7. HE stain showed that the neurons in the hippocampus of normal control group are cone shape, with even amethyst color and long neuritis, clear round or oval-shaped nuclei. Nucleoli were in deep amethyst. There are 3 to 4 layers of pyramidal neurons in CA1 region of hippocampus in normal control group, while less of that in AD model group. In AD model group, the neurons are smaller than normal control group, with condense plasma and nucleoli.8. The effects of Chaihu shugan san and Fructus Broussonetiae on mean neuron numbers in DG of AD model rats:the results of toluidine blue O stain showed that in mean neuron numbers in DG, there were significant differences between the groups (F=10.560, P<0.001). The mean neuron numbers in DG of AD model group were lower than that in normal control group and SO group (P=0.002, P=0.012). The mean neuron numbers in DG of Chaihu shugan san group and Fructus Broussonetiae group were higher than that in AD model group (P=0.001, P<0.001).9. Congo red stain showed that the vessel of the rats brain were Congo red positive, while there were no obvious Congo red positive plaques.10. The effects of Chaihu shugan san and Fructus Broussonetiae on mean apoptosis index of AD model rats:the results of TUNEL showed that in mean apoptosis index, there were significant differences between the groups (F=240.155, P<0.001). The mean apoptosis index of AD model group were higher than that in normal control group and SO group (P<0.001, P<0.001). The mean apoptosis index of Chaihu shugan san group and Fructus Broussonetiae group were lower than that in AD model group (P<0.001, P<0.001).Chaihu shugan san and Fructus Broussonetiae can improve study and memory disorders and neuron apoptosis of the model rats induced by aβ25-35 and D-galactose. The mechanism underlying it maybe involved in ER stress, such as up-regulation of BiP, down-regulation of PERK and CHOP. Disperse the depressed liver-energy with counteract luxuriate could alleviate ER stress in AD and improve the symptoms of AD rat models. In the other way, hypothesis "hepatic depressed vital energy-ER stress" had been supported with study data.