天泰1号对Aβ_（25-35）联合D-半乳糖诱导老年性痴呆模型的干预作用研究
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摘要
通过筛选,将筛选合格的健康正常SD大鼠40只,随机分为正常对照组,假手术对照组、AD对照模型组、模型+中药低剂量组、模型+中药高剂量组,每组各8只。参照文献方法,应用Aβ25-35,依照大鼠脑立体图谱,通过脑立体定位仪,建立AD大鼠病理模型,通过对大鼠进行学习训练、定位航行、行为学、病理形态学观察、以及细胞凋亡、内质网应激(ERS)重要蛋白表达水平的检测,以期阐明本方法建立的阿尔茨海默病(AD)模型大鼠学习记忆功能的改变,以及天泰1号对Aβ25-35诱导的AD大鼠模型的学习记忆能力的影响及其药理药效机理。
本实验包括两部分：一、动物行为学实验研究；二、海马组织病理观察、细胞凋亡及ERS重要蛋白检测。
利用Morris水迷宫进行动物筛选,大鼠适应性饲养3天后进行Morris水迷宫实验,Morris水迷宫实验测试过程分学习训练、定位航行试验二部分。将安全平台放置在西南象限(直径150cm的圆形不锈钢水池被软件平均分为四个象限,即NE、SE、SW、NW)的固定位置(离池壁20cm),向Morris不锈钢水池中注入自来水,以水面高过安全平台2cm为宜,水池中水温保持在24-25℃。将摄像装置安装在水池的正上方,按仪器说明书连接好摄像机、计算机,整套实验设备安装在光线良好、实验环境固定的房间。先运行计算机,设定参数后进行实验。实验前将动物不放入水池,软件先确认背景,然后放入大鼠于安全台上,软件自动识别大鼠为目标后开始实验。将定位航行时间超过平均值30%的大鼠剔除,剩余大鼠作为实验用。
阿尔茨海默病(Alzheimer's Disease,AD)大鼠模型的建立：在质量分数为4%的戊巴比妥钠腹腔注射麻醉下,用脑立体定位仪固定大鼠,取平颅头位,根据大鼠脑立体定位图谱,确定前囟后3.6mm、中线旁2.5mm为颅骨表面标志点,颅骨表面下3.0mm为海马背侧部,用1ul微量注射器5min内缓慢注入凝聚态Aβ25-351ul,予以留针10min。术后给予青霉素钠盐5万单位肌注,每天一次。正常组大鼠颈背部注射0.9%生理盐水0.6ml/kg,AD模型组和假手术组大鼠均皮下注射D-半乳糖150mg/kg。第26天开始Morris水迷宫实验,连续实验5天,将行为学结果进行统计学分析。末次行为学实验结束后断头处死大鼠,取左半脑用4%多聚甲醛固定。根据行为学结果初步评价AD模型的成功与否。
在制备AD大鼠疾病模型成功的基础上,按以上方法制备大鼠AD疾病模型。将大鼠随机分为5组,即天泰1号方大、小剂量2个组, AD模型组与正常对照组和假手术组,每组8只大鼠(SD大鼠、SPF级、雄性、240-270g,均饲养在屏障环境中,常规给食物和水),天泰1号方大、小剂量组大鼠分别灌胃给予10%、5%的天泰1号方10ml/kg,假手术组和正常对照组则以相同的方式给予同体积的生理盐水,每日1次,连续给药30天。从给药第26天开始进行行为学实验,实验内容包括学习训练和定位航行实验二部分。
末次行为学实验结束后断头处死大鼠,取全脑。其右脑保存于液氮中,左脑用4%多聚甲醛固定。左脑进行常规尼氏和刚果红染色,观察脑组织结构、细胞的形态和海马CA1、CA3和DG区神经元数量；各组切片中随机选5张进行病理学观察,自动图像分析系统计算出海马DG背侧细胞带的平均神经元数；各组切片中随机选5张观察β-AP和磷酸化tau蛋白；各组切片中随机选5张镜下观察,统一放大倍数(×400)下,每张切片的海马CA1、CA3和DG区分别随机取4个视野,自动图像分析系统计算所要观察部位的平均阳性细胞数；取各组脑组织切片,用TUNEL法检测细胞凋亡,重点观察海马神经元凋亡情况,显微镜下观测,每张切片随机选取5个高倍视野,计算细胞凋亡指数(AI)；取各组脑组织切片,免疫组织化学法Leica QWin V3分析软件定量检测各组动物海马组织PERK、GRP78/Bip、CHOP蛋白表达水平。
统计学方法：先对所有数据进行正态性检验及方差齐性检验。服从正态分布计量资料组间比较采用单向方差分析(One-way ANOVA),组间多重比较采用LSD法(方差齐时)或Dunnett T3法(方差不齐时)。不服从正态分布及方差齐性的计量资料采用非参数检验方法,多组间比较用Kruskal-Wallis H检验,组间多重比较采用Nemenyi法检验；造模前后定位航行成绩比较不满足正态分布及方差齐性,采用Wilcoxon符号秩和检验。所有数据用SPSS13.0统计分析软件处理,显著性水准取a=0.05(双侧),P<0.05为差异有统计学意义。
1.Morris水迷宫是检测实验动物学习记忆水平的重要工具,学习测试有利于筛选具有正常学习记忆能力的动物,记忆测试可以判断动物记忆储存能力及提取再现能力。经过学习和训练可以把正常对照组和模型组明显区分开来,正常对照组学习记忆能力强,空间定位能力好,模型组学习记忆能力明显下降,逃避潜伏期明显延长,提示Aβ25-35造成大鼠学习记忆能力的损害。
2.通过Morris水迷宫测试,天泰1号方大、小剂量组与AD模型组比较,在学习训练、定位航行实验时达到安全平台的时间和距离缩短,提示天泰1号能显著改善AD大鼠的学习记忆能力。
3.常规尼氏染色各组切片中随机选5张进行病理学观察,自动图像分析系统计算出海马DG背侧细胞带的平均神经元数。方差分析结果显示,各组大鼠海马DG背侧细胞带的神经元数量比较差异有统计学意义(F=75.433,P<0.001)。其中,AD模型组较正常组及假手术组神经元数量减少,差异有显著性意义(P<0.001)；天泰高剂量组及天泰低剂量组较AD模型组神经元数量增加,差异有显著性意义(P<0.001)。
4.淀粉样蛋白刚果红染色发现,AD模型组与正常组、假手术组相比较淀粉样蛋白斑块显著增多,与AD模型组相比较,中药组淀粉样斑块减少,提示天泰1号具有抗淀粉样斑块形成的药效作用。
5.β-AP免疫组织化学染色,自动图像分析系统计算所要观察部位的平均阳性细胞数。各组大鼠海马CA1、CA3和DG区平均阳性细胞数比较：方差分析显示,各组大鼠海马CA1、CA3和DG区平均阳性细胞数比较,差异有统计学意义(F=20.065,P<0.001；F=24.007,P<0.001；F=22.987,P<0.001)。AD模型组较正常组、假手术组CA1、CA3和DG区平均阳性细胞数显著增多,差异有显著性意义(P<0.001)；天泰高剂量组、天泰低剂量组与AD模型组比较,CA1、CA3和DG区平均阳性细胞数明显减少,差异有统计学意义(P<0.05)。
6.磷酸化tau蛋白免疫组织化学染色各组大鼠海马CA1、CA3和DG区平均阳性细胞数比较：方差分析显示,各组大鼠海马CA1、CA3和DG区平均阳性细胞数比较,差异有统计学意义(F=16.982,P<0.001；F=21.510,P<0.001；F=15.971,P<0.001)。AD模型组较正常组、假手术组CA1、CA3和DG区平均阳性细胞数显著增多,差异有显著性意义(P<0.001)；天泰高剂量组、天泰低剂量组与AD模型组比较,CA1、CA3和DG区平均阳性细胞数明显减少,差异有统计学意义(P<0.05)。
7.左半脑组织TUNEL法检测细胞凋亡实验发现,方差分析显示各组凋亡率差异有统计学意义(F=123.478,P<0.001)。组间多重比较显示,AD模型组凋亡率较正常组及假手术组明显增高,差异有显著性意义(P<0.001)；天泰高、低剂量组较AD模型组神经元细胞凋亡率明显降低,差异有统计学意义(P<0.001：P<0.043)。
8.免疫组织化学法检测各组动物海马组织ERS重要相关蛋白,各组PERK、GRP78/Bip、CHOP蛋白平均灰度值比较：方差分析显示,各组PERK、GRP78/Bip、CHOP蛋白平均灰度值比较,差异有统计学意义(F=186.104,P<0.001；F=312.133,P<0.001；F=2765.613,P<0.001)。AD模型组与正常组、假手术组比较,PERK、GRP78/Bip、CHOP蛋白平均灰度值显著降低,差异有统计学意义(P<0.001)；天泰高剂量组、天泰低剂量组与AD模型组比较,PERK、CHOP蛋白平均灰度值明显增高,差异有统计学意义(P<0.05),天泰高剂量组、天泰低剂量组与AD模型组比较,GRP78/Bip蛋白平均灰度值明显降低,差异有统计学意义(P<0.05),提示天泰1号方在抗AD机理可能与其影响细胞的ERS有关。
本研究以Aβ25-35联合D-半乳糖建立AD动物模型,并观察天泰1号对该病理模型认知障碍的影响和海马组织病理观察、细胞凋亡及ERS重要蛋白检测。结果显示Aβ25-35联合D-半乳糖建立的AD病理模型学习记忆认知功能显著障碍,天泰1号对该病理模型的认知功能显著障碍具有改善作用,研究表明Aβ25-35联合D-半乳糖可以成功建立复合性AD动物病理模型,该模型既模拟了老年性痴呆的认知行为障碍,又具有整体衰老的体内生理病理环境,为深入开展老年性痴呆的发病机制及药效学靶点研究提供了可行的在体模型；PERK为ERS感应和保护性信号通路调控蛋白、GRP78/Bip为内质网应激早期标志和保护性蛋白、CHOP为凋亡促进因子,天泰1号能够显著减少PERK、CHOP蛋白的表达水平,提高GRP78/Bip的表达水平,提示,天泰1号的抗AD作用机理涉及其抗凋亡、抗内质网应激等多重途径。
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 difference
1.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.
本实验包括两部分：一、动物行为学实验研究；二、海马组织病理观察、细胞凋亡及ERS重要蛋白检测。
利用Morris水迷宫进行动物筛选,大鼠适应性饲养3天后进行Morris水迷宫实验,Morris水迷宫实验测试过程分学习训练、定位航行试验二部分。将安全平台放置在西南象限(直径150cm的圆形不锈钢水池被软件平均分为四个象限,即NE、SE、SW、NW)的固定位置(离池壁20cm),向Morris不锈钢水池中注入自来水,以水面高过安全平台2cm为宜,水池中水温保持在24-25℃。将摄像装置安装在水池的正上方,按仪器说明书连接好摄像机、计算机,整套实验设备安装在光线良好、实验环境固定的房间。先运行计算机,设定参数后进行实验。实验前将动物不放入水池,软件先确认背景,然后放入大鼠于安全台上,软件自动识别大鼠为目标后开始实验。将定位航行时间超过平均值30%的大鼠剔除,剩余大鼠作为实验用。
阿尔茨海默病(Alzheimer's Disease,AD)大鼠模型的建立：在质量分数为4%的戊巴比妥钠腹腔注射麻醉下,用脑立体定位仪固定大鼠,取平颅头位,根据大鼠脑立体定位图谱,确定前囟后3.6mm、中线旁2.5mm为颅骨表面标志点,颅骨表面下3.0mm为海马背侧部,用1ul微量注射器5min内缓慢注入凝聚态Aβ25-351ul,予以留针10min。术后给予青霉素钠盐5万单位肌注,每天一次。正常组大鼠颈背部注射0.9%生理盐水0.6ml/kg,AD模型组和假手术组大鼠均皮下注射D-半乳糖150mg/kg。第26天开始Morris水迷宫实验,连续实验5天,将行为学结果进行统计学分析。末次行为学实验结束后断头处死大鼠,取左半脑用4%多聚甲醛固定。根据行为学结果初步评价AD模型的成功与否。
在制备AD大鼠疾病模型成功的基础上,按以上方法制备大鼠AD疾病模型。将大鼠随机分为5组,即天泰1号方大、小剂量2个组, AD模型组与正常对照组和假手术组,每组8只大鼠(SD大鼠、SPF级、雄性、240-270g,均饲养在屏障环境中,常规给食物和水),天泰1号方大、小剂量组大鼠分别灌胃给予10%、5%的天泰1号方10ml/kg,假手术组和正常对照组则以相同的方式给予同体积的生理盐水,每日1次,连续给药30天。从给药第26天开始进行行为学实验,实验内容包括学习训练和定位航行实验二部分。
末次行为学实验结束后断头处死大鼠,取全脑。其右脑保存于液氮中,左脑用4%多聚甲醛固定。左脑进行常规尼氏和刚果红染色,观察脑组织结构、细胞的形态和海马CA1、CA3和DG区神经元数量；各组切片中随机选5张进行病理学观察,自动图像分析系统计算出海马DG背侧细胞带的平均神经元数；各组切片中随机选5张观察β-AP和磷酸化tau蛋白；各组切片中随机选5张镜下观察,统一放大倍数(×400)下,每张切片的海马CA1、CA3和DG区分别随机取4个视野,自动图像分析系统计算所要观察部位的平均阳性细胞数；取各组脑组织切片,用TUNEL法检测细胞凋亡,重点观察海马神经元凋亡情况,显微镜下观测,每张切片随机选取5个高倍视野,计算细胞凋亡指数(AI)；取各组脑组织切片,免疫组织化学法Leica QWin V3分析软件定量检测各组动物海马组织PERK、GRP78/Bip、CHOP蛋白表达水平。
统计学方法：先对所有数据进行正态性检验及方差齐性检验。服从正态分布计量资料组间比较采用单向方差分析(One-way ANOVA),组间多重比较采用LSD法(方差齐时)或Dunnett T3法(方差不齐时)。不服从正态分布及方差齐性的计量资料采用非参数检验方法,多组间比较用Kruskal-Wallis H检验,组间多重比较采用Nemenyi法检验；造模前后定位航行成绩比较不满足正态分布及方差齐性,采用Wilcoxon符号秩和检验。所有数据用SPSS13.0统计分析软件处理,显著性水准取a=0.05(双侧),P<0.05为差异有统计学意义。
1.Morris水迷宫是检测实验动物学习记忆水平的重要工具,学习测试有利于筛选具有正常学习记忆能力的动物,记忆测试可以判断动物记忆储存能力及提取再现能力。经过学习和训练可以把正常对照组和模型组明显区分开来,正常对照组学习记忆能力强,空间定位能力好,模型组学习记忆能力明显下降,逃避潜伏期明显延长,提示Aβ25-35造成大鼠学习记忆能力的损害。
2.通过Morris水迷宫测试,天泰1号方大、小剂量组与AD模型组比较,在学习训练、定位航行实验时达到安全平台的时间和距离缩短,提示天泰1号能显著改善AD大鼠的学习记忆能力。
3.常规尼氏染色各组切片中随机选5张进行病理学观察,自动图像分析系统计算出海马DG背侧细胞带的平均神经元数。方差分析结果显示,各组大鼠海马DG背侧细胞带的神经元数量比较差异有统计学意义(F=75.433,P<0.001)。其中,AD模型组较正常组及假手术组神经元数量减少,差异有显著性意义(P<0.001)；天泰高剂量组及天泰低剂量组较AD模型组神经元数量增加,差异有显著性意义(P<0.001)。
4.淀粉样蛋白刚果红染色发现,AD模型组与正常组、假手术组相比较淀粉样蛋白斑块显著增多,与AD模型组相比较,中药组淀粉样斑块减少,提示天泰1号具有抗淀粉样斑块形成的药效作用。
5.β-AP免疫组织化学染色,自动图像分析系统计算所要观察部位的平均阳性细胞数。各组大鼠海马CA1、CA3和DG区平均阳性细胞数比较：方差分析显示,各组大鼠海马CA1、CA3和DG区平均阳性细胞数比较,差异有统计学意义(F=20.065,P<0.001；F=24.007,P<0.001；F=22.987,P<0.001)。AD模型组较正常组、假手术组CA1、CA3和DG区平均阳性细胞数显著增多,差异有显著性意义(P<0.001)；天泰高剂量组、天泰低剂量组与AD模型组比较,CA1、CA3和DG区平均阳性细胞数明显减少,差异有统计学意义(P<0.05)。
6.磷酸化tau蛋白免疫组织化学染色各组大鼠海马CA1、CA3和DG区平均阳性细胞数比较：方差分析显示,各组大鼠海马CA1、CA3和DG区平均阳性细胞数比较,差异有统计学意义(F=16.982,P<0.001；F=21.510,P<0.001；F=15.971,P<0.001)。AD模型组较正常组、假手术组CA1、CA3和DG区平均阳性细胞数显著增多,差异有显著性意义(P<0.001)；天泰高剂量组、天泰低剂量组与AD模型组比较,CA1、CA3和DG区平均阳性细胞数明显减少,差异有统计学意义(P<0.05)。
7.左半脑组织TUNEL法检测细胞凋亡实验发现,方差分析显示各组凋亡率差异有统计学意义(F=123.478,P<0.001)。组间多重比较显示,AD模型组凋亡率较正常组及假手术组明显增高,差异有显著性意义(P<0.001)；天泰高、低剂量组较AD模型组神经元细胞凋亡率明显降低,差异有统计学意义(P<0.001：P<0.043)。
8.免疫组织化学法检测各组动物海马组织ERS重要相关蛋白,各组PERK、GRP78/Bip、CHOP蛋白平均灰度值比较：方差分析显示,各组PERK、GRP78/Bip、CHOP蛋白平均灰度值比较,差异有统计学意义(F=186.104,P<0.001；F=312.133,P<0.001；F=2765.613,P<0.001)。AD模型组与正常组、假手术组比较,PERK、GRP78/Bip、CHOP蛋白平均灰度值显著降低,差异有统计学意义(P<0.001)；天泰高剂量组、天泰低剂量组与AD模型组比较,PERK、CHOP蛋白平均灰度值明显增高,差异有统计学意义(P<0.05),天泰高剂量组、天泰低剂量组与AD模型组比较,GRP78/Bip蛋白平均灰度值明显降低,差异有统计学意义(P<0.05),提示天泰1号方在抗AD机理可能与其影响细胞的ERS有关。
本研究以Aβ25-35联合D-半乳糖建立AD动物模型,并观察天泰1号对该病理模型认知障碍的影响和海马组织病理观察、细胞凋亡及ERS重要蛋白检测。结果显示Aβ25-35联合D-半乳糖建立的AD病理模型学习记忆认知功能显著障碍,天泰1号对该病理模型的认知功能显著障碍具有改善作用,研究表明Aβ25-35联合D-半乳糖可以成功建立复合性AD动物病理模型,该模型既模拟了老年性痴呆的认知行为障碍,又具有整体衰老的体内生理病理环境,为深入开展老年性痴呆的发病机制及药效学靶点研究提供了可行的在体模型；PERK为ERS感应和保护性信号通路调控蛋白、GRP78/Bip为内质网应激早期标志和保护性蛋白、CHOP为凋亡促进因子,天泰1号能够显著减少PERK、CHOP蛋白的表达水平,提高GRP78/Bip的表达水平,提示,天泰1号的抗AD作用机理涉及其抗凋亡、抗内质网应激等多重途径。
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 difference
1.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.
引文
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综上所述,AD的发病机制仍不清楚,尚无一假说可较完整的解释AD的发病机制。但随着科学的发展,一些新的诊断工具和治疗药物的不断出现,将会给AD的确诊和治疗效果带来极大的希望。
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综上所述,AD的发病机制仍不清楚,尚无一假说可较完整的解释AD的发病机制。但随着科学的发展,一些新的诊断工具和治疗药物的不断出现,将会给AD的确诊和治疗效果带来极大的希望。
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