| First of all, we screened 40 healthy SD rats. Then the rats were randomly divided into five groups as follows (8 in each group):normal control group, sham-operated group, Alzheimer disease (AD) model, AD model and low-dose traditional Chinese medicine group, AD model and high-dose traditional Chinese medicine group. In accordance with Reference and maps, by using of the Aβ25-35 and brain stereotaxic apparatus,According to the three-dimensional map of rat brian,the AD pathological model in rats were established. In order to demonstrate the ability of study and memory significantly decreased in the AD rats that induced by Aβ25-35 united D-galactose, and Tiantai NO.1 improved the ability of study and memory significantly, we studied the training of study, locational navigation, and behavior in rats, and we observed the pathologic change and apoptosis, meanwhile we detected important protein of ERS,the mechanism involved in anti-apoptosis, anti-endoplasmic reticulum stress. This research included two major parts:The first experiment was about the behavior of AD rats, and the second one was Histopathological observation of the hippocampus,apoptosis,and ERS important protein detection.(1) Experimental study about the behavior of AD rats. Morris water maze was used for screening rats. First, the rats were rearing in barrier environment and seeding the conventional food and water for three days. Later, the rats entered the experiment of Morris water maze. The experiment of Morris water maze includes two parts:training of study, locational navigation. A circular stainless steel tank with diameter 150cm was divided into four quadrants by software, namely, northeast (NE), southeast (SE), southwest (SW), and northwest (NW). Safe platform was placed in a fixed position (20cm away from the tank wall) in the SW quadrant. The water was injected into the stainless steel tank with the water 2cm higher than the safe platform and the temperature maintained at 24-25℃. The camera device was installed just above the tank, and the camera and computer was connected according to the instructions. All of the experimental equipments were installed in a room with a good light and fixed experimental environment. We started the experiment after running the computer and setting the parameters. First, the software confirmed the background. Second, the rat was place on the safe platform. Third, the software automatically recognized the rat as goals. Lastly, we started the experiment. The rats whose journey time was more than 30% to the average were removed, the remaining rats were used for experiments. The establishment of Alzheimer's Disease model in rats. The rats were anesthetized by intraperitoneal injection with 4% aqueous solution of sodium pentobarbital. Then these rats were pronely fixed in the brain three-dimensional positioning instruments. At first we conventionally opened the skin at the top of the head, the marker points was beside 2.5mm to the center line of the skull and behind 3.6mm to the bregma. We inserted the micro-syringe into the hippocampus 3.0mm depth under the parietal bone.lul of Aβ25-35 was slowly injected into the hippocampus within 5 minutes and the micro-syringe was removed for 10 minutes. After that 50000 units of penicillin was gived intramuscularly once a day. In addition, the normal control group of rats were subcutaneous injected 0.6ml/kg of 0.9% normal saline solution in the back and neck outside each rat, AD model group and sham-operated group of rats were subcutaneous injected 150mg/kg of D-galactose each rat. From the 26 after the operation, Morris water maze experiment was began, and the experiment lasted for 5 days. The behavioral results were analyzed statistically. At the end of the behavior experiment all rats were killed through the decapitation. The left brains were fixed with 4% paraformaldehyde. We evaluate the AD model in accordance with the behavioral results.The AD model rats were randomly divided into 3 groups,Tiantai NO.1 high-dose group,Tiantai NO.1 low-dose group, and AD model group, while there was the establishment of normal control group and sham-operated group. Each group had 8 rats, which were SD rats, SPF-class, male, weight 240-270g, were rearing in barrier environment and seeding the conventional food and water. Tiantai NO.1 high-dose group and Tiantai NO.1 low-dose group were given oral 10% and 5% of Tiantai NO.1, respectively. The volume was lOml/kg. The rats of the sham-operated group and the normal control group were given the same volume of normal saline in accordance with the manner above. The administration was once a day for 30 days continuously. The behavioral experiments were started from the 26th day of the administration, including the training of studying, locational navigation.(2)Histopathological observation of the hippocampus, apoptosis,and ERS important protein detection. At the end of behavior experiment all rats were killed through the decapitation. The left brain was fixed with 4% paraformaldehyde, and the right brain was stored in liquid nitrogen. The left brain was regularly stained by Nissl and Congo red staining and observed the morphology of organizational structure and neurons in the CA1, CA3 and DG area of hippocampus. Slices in each group were randomly selected five for pathological observation, and the average number of neurons in the dorsal DG area of hippocampus were calculated by automatic image analysis system. Slices in each group were randomly selected five for observation ofβ-AP and phosphorylated tau protein. Slices in each group were randomly selected five for microscopic observation (magnification×400), and per slice of CA1, CA3 and DG areas were randomly taken four visual field, then the average number of positive cells in the areas were calculated by automatic image analysis system. We studied the neuronal apoptosis in brain tissue, especially the tissue of hippocampus by TUNEL method. Per slice was randomly selected 5 high-power visual field for calculation of apoptosis index (AI). And We observed the expression of CHOP,PER,Bip protein in the tissue of hippocampus each group by immunohistochemistry and Analysis software Leica QWin V3.First,all the date is dealed with normality test and homogeneity of variance test.the two groups date obey normal distribution is used to compare by the(One-Way ANOVA)multiple comparisons between groups using LSD method(when variance is homogeneity)or Dunnett T3 method(when variance is not homogeneity)obey normal distribution and homogeneity of variance of the measurement date usin non-parametric test methods.multiple comparison use the Kruskal-Wallis H test;to compare the place navigation performance before and after modeling,wich does not meet the normal distribution and homogeneity of variance using the Wilcoxon test.all date use the statistical analysis software SPSS13.0 to deal with,significant level takea=0.05(Bilateral),P<0.05 as statistically significant difference1.Morris water maze is an important tool to test the ability of leaning and memory in experimental animals. leaning test is conducive to screening animals with normal ability of leaning and memory, and memory test can determine the memory storage ability and reproduction ability of animal. After investigating the training of study in rats, we can separate the normal control group and the model group obviously with statistical significance (P<0.05):The normal control group had better ability of leaning, memory and spatial orientation. The ability of leaning and memory was significantly decreased, and escaping latency was significantly longer in the model group, suggesting that Aβ25-35 caused damage to the ability of leaning and memory in rats.2.Through the Morris water maze experiment, we found the distance to reach safe platform and the positioning time were shorter in the AD model group compared with others. It suggested that Tiantai NO.1 could improve significantly the ability of learning and memory in AD rats.3.Conventional Nissl's staining sections in each group was randomly selected five for pathological observation, and the average number of neurons in the dorsal DG area of hippocampus was calculated by automatic image analysis system. Analysis of variance show that the number of neurons in each group of DG dorsal hippocampus of rats,the difference is statistically significant(F=75.433,P<0.001).the number of cells in ADmodel group are significantly decreased than the normal group and sham operation group,the difference is statistically significant (P<0.001); compared with the AD model group Tiantai NO.1 high-does group and Tiantai NO.1 low-dose group are significantly decreased,the difference is statistically significant (P<0.001)4.Through amyloid Congo red staining, we found the amyloid plaques was significantly increased in the AD model group than the normal group and sham-operated group. Compared with the AD model group, the amyloid plaques was decreased in traditional Chinese medicine groups, suggesting that Tiantai NO.1 could prevent the formation of amyloid plaques.5.Stainingβ-AP by immunohistochemistry, and calculated the average number of positive cells in certain area by automatic image analysis system.comparing The average number of positive cells In each group rats of the hppocampal CA1, CA3 and DG areas, the difference was statistically significant (F=20.065, P<0.001; F=24.007, P<0.001; F=22.987, P<0.001).The positive cells of the AD model group CA1,CA3 and DG areas were significantly increased Than normal group and sham-operated group, the difference was statistically significant (P<0.001); comparing to the AD model group, the Tiantai NO.1 high-dose group and Tiantai NO.1 low-dose group, the positive cells in CA1, CA3 and DG areas were statistically significant (P<0.05); Tiantai NO.1 low-dose group compared to the Tiantai NO.1 high-dose group, the difference was statistically significant(P<0.001).6.Staining the Phosphorylated tau protein by immunohistochemistry, and calculating the average number of positive cells in certain area by automatic image analysis system.comparing The average number of positive cells In each group rats of the hppocampal CA1, CA3 and DG areas, the difference was statistically significant (F=16.982,P<0.001; F=21.510, P<0.001; F=15.971, P<0.001) The positive cells of the AD model group CA1,CA3 and DG areas were significantly increased Than normal group and sham-operated group,the difference was statistically significant (P<0.001); comparing to the AD model group, the Tiantai NO.1 high-dose group and Tiantai NO.1 low-dose group, the positive cells in CA1, CA3 and DG areas was statistically significant (P<0.05).7.From the experiment by TUNEL method on the tissue of left brain we found the neurons apoptosis were Statistically significant by the ONE-WAY ANOVA (F=123.478,P<0.001);multiple comparison between the two groups showed that the rate of apotosis of AD model group was higher than normal group and sham-operated group,the difference was significant(P<0.001);the rate of apoptosis of Tiantai NO.1 high-dose group and Tiantai NO.1 low-dose group was lower than in AD model group,the difference was significant(P<0.001;P<0.043).8.through detecting important associated proteins of ERS by immunehisto-chemistry quantification, comparison the average gray value of each group PERK,GRP78/Bip,CHOP protein:variance analysis showed that comparison the average gray value of each group PERK,GRP78/Bip,CHOP protein, the difference was statistically significant (F=186.104, P<0.001; F=312.133, P<0.001; F=2765.613, P<0.001). AD model group compared to the normal group and sham-operated group the average gray value of the PERK, GRP78/Bip, CHOP protein was decreased,the difference was statistically significantl(P<0.001); Tiantai NO.1 high-dose group and Tiantai NO.1 low-dose group compared to the AD model group, the average gray value of the PERK,CHOP protein was increased, Tiantai NO.1 high-dose group and Tiantai NO.1 low-dose group compared to the AD model group, the average gray value of the GRP78/Bip was decreased the difference was statistically significant (P<0.05), suggesting that Tiantai NO.1 affected ERS of cells when it resisted AD.in this study, Aβ25-35 United D-galactose to establish animal models of AD and to observe the effects of Tiantai NO.1 on the pathological model of cognitive impairment,and hippocampal pathology observed, apoptosis, and ERS important protein detection The results showed that Aβ25-35 United D-galactose established AD pathological model of cognitive learning and memory in a significant obstacle, Tiantai NO.1 on the pathological model of cognitive disorders have a significant improvement. Studies have shown that Aβ25-35 United D-galactose compound can be successfully establish the AD pathological animal model, the AD pathological animal model not only simulated senile dementia cognitive behavioral disorder, but also had the overall physiological and pathological environment, the AD pathological animal model for the depth of Alzheimer's disease pathogenesis and pharmacodynamics study provided a Feasible in vivo models; PERK as ERS sensors and protective signaling pathway regulatory protein, GRP78/Bip for endoplasmic reticulum stress Early signs and protective proteins, CHOP for the apoptosis-promoting factor, and Tiantai NO.1 can significantly reduce the PERK, CHOP protein expression level and improve the expression of GRP78/Bip, its role in the mechanism involved in anti-apoptosis, anti-endoplasmic reticulum stress, etc.
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