Aβ25-35联合D-半乳糖诱导老年性痴呆复合模型的建立及中药干预研究
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摘要
阿尔茨海默病(Alzheimer's disease, AD)又称早老性痴呆,是一种病因未明的中枢神经系统退行性病变。该病以记忆力减退、认知功能障碍为特征。由于病因复杂,发病机制不明,临床尚无有效治疗方法。随着人均寿命的延长,人口老龄化发展,痴呆已经成为发达国家继心脏病、癌症和脑卒中之后第四位死因。痴呆病人晚期丧失劳动能力,生活不能自理,心理能力急剧下降,通常在诊断后3-9年内死于感染及其它系统疾病,给家庭和社会带来沉重的经济负担。作为痴呆最常见类型的阿尔茨海默病,因其发病年龄较早(可早至40岁),也称为早老性痴呆,已经成为全球范围的世纪性难题。
AD特征性病理改变是β-淀粉样蛋白沉积形成的老年斑(senile plaque)、tau蛋白异常磷酸化聚集形成神经原纤维缠结(neurofibrillary tangles)以及区域选择性神经元死亡。近年来,逐渐积累的证据显示低聚物形式的Aβ和tau可能是阿尔茨海默病主要的罪魁祸首。由于脑内特定区域的淀粉样斑块与认知损害的程度相关,且Aβ的蓄积是阿尔茨海默病的早期事件。有人认为当Aβ的产生与清除之间出现不平衡,加上肽段间的自我聚集作用,导致Aβ开始蓄积。这种现象超过一定限度,可能是AD的启动因子。支持这种观点者称之为淀粉样蛋白级联假说("amyloid cascade hypothesis")。不断积累的证据也显示,Aβ具有神经毒性。利用Aβ脑室内或者海马内注射可以复制阿尔茨海默病动物模型。可致大鼠学习记忆障碍,海马胆碱乙酰转移酶(ChAT)活性降低等。该类模型适用于研究Aβ聚集或沉积在AD发病中的作用机制,并可作为评价药物对AD治疗作用的模型。
目前已经用于临床治疗阿尔茨海默病的药物主要是胆碱酯酶抑制剂,正在进行研究的还有抗Ap的单克隆抗体、抵抗tau氧化和聚集作用的小分子试剂以及脑源性神经营养因子(brain-derived neurotrophic factor, BDNF)替代疗法等。同时因为中药的成分天然,作用靶点多,对解决病因复杂的疾病有独特优势,相关实验研究及临床应用逐渐受到重视。
中医传统理论认为,情志所伤、所愿不遂、肝气郁滞是本病的重要病因和基本病机。现代临床医学研究表明,抑郁发生在老年性痴呆的各个阶段,AD患者最常见的行为异常是表情淡漠、情感低落、焦虑不安、易激惹等情绪相关症状。因此,AD的中医病理属性当属肝气郁滞。同时,中医现代研究表明,肝郁证的主要病理基础在大脑高级中枢。
内质网是重要的细胞器之一,蛋白质合成和修饰的场所。为保持内环境的稳态,内质网会将错误折叠的分子跨膜转运到细胞浆中降解。这种机制称为内质网相关性蛋白降解机制。体内外研究证明,内质网应激是AD发病机制之一。
基于内质网是细胞内精密的质量控制系统,在调控和维持细胞内环境稳定中发挥极其重要的作用,其形态结构、生理作用与功能状态均具有疏通的特点,且以调和畅达为至关紧要,与中医肝藏疏泄、调达的生理特性十分吻合,因此我们认为,内质网的结构和功能是肝主疏泄的亚细胞生理基础。基于此,导师吴正治教授结合传统理论与现代分子机制研究进展,提出老年性痴呆“肝气郁滞-内质网应激关联”中西医结合病理假说,该假说的核心观点是：肝气郁滞是老年性痴呆的基本病机,内质网应激为本病肝气郁滞的亚细胞病理基础,疏肝解郁可以缓解和拮抗老年性痴呆内质网应激。
建立Aβ25-35与D-半乳糖复合老年性痴呆模型；观察疏肝解郁代表方剂柴胡疏肝散、补肝益髓中药楮实子对Aβ25-35与D-半乳糖复合老年性痴呆模型大鼠空间学习记忆能力及病理学改变的干预作用;探讨AD内质网应激及细胞凋亡的相关发病机制,验证肝气郁滞-内质网应激关联新假说。
本研究通过双侧海马内脑立体定向注射Aβ25-35联合长期皮下注射D-半乳糖的方法建立AD大鼠模型。将大鼠随机分为5组,即正常对照组、AD模型组、假手术组、中药1组(柴胡疏肝散)组、中药2组(楮实子提取物)组(每组8只,共40只),见表1。通过Morris水迷宫实验检测各组大鼠空间探索实验逃避潜伏期及定位航行试验相关指标的变化,比较不同处理组别间大鼠空间学习记忆能力。观察各组大鼠脑组织病理学改变：HE染色用以观察病理学改变、甲苯胺蓝染色用以计数大鼠海马区神经元数目及刚果红染色法用以观察各组大鼠脑组织淀粉样蛋白沉积。采用免疫组织化学(immunohistochemical, IHC)法检测β淀粉样肽(Aβ)、磷酸化tau蛋白表达情况,以及内质网应激标志蛋白PERK、BiP、GADD153/CHOP的表达情况。原位末端凋亡法(TUNEL)用以检测各组海马区细胞平均凋亡率。
实验结果用SPSS 13.0 for windows统计软件分析,均用均数±标准差(X±S)表示,P<0.05为差异有统计学意义。对实验数据大鼠逃避潜伏期、游泳距离、搜索时间百分比、搜索距离百分比、Aβ、tau平均阳性细胞数、BiP、PERK、GADD153/CHOP蛋白免疫组化平均灰度值、DG区平均神经元数目、平均凋亡率,分别进行各组间多个样本均数比较。若方差齐性,采用单因素方差分析方法(one-way ANOVA),用LSD法进行多重比较；若方差不齐,用Welch近似方差分析法进行检验,用Dunnett's T3法进行多重比较。
1、Aβ与D-半乳糖复合老年性痴呆模型的行为变化：Aβ25-35与D-半乳糖复合老年性痴呆模型大鼠较正常对照组大鼠活动及打斗减少,对抓触逃避反应明显减弱,游泳速度较其余组别大鼠减低。
2、药物对老年性痴呆模型大鼠定位航行实验的影响：实验结果经统计学分析表明,大鼠搜索并找到安全平台的逃避潜伏期及游泳距离各组间有显著差异(分别为F=5.004,P=0.007；F=3.256,P=0.037),结果见表2。AD模型组逃避潜伏期较正常对照组及假手术组均延长(分别为P=0.017,P=0.041),中药1组组、中药2组组较AD模型组逃避潜伏期均缩短(分别为P=0.013,P=0.010),中药2组组游泳距离较AD模型组缩短(P=0.046)。
3、药物对老年性痴呆模型大鼠空间探索实验的影响：实验结果经统计学分析表明,大鼠在原平台所在象限搜索时间及搜索距离所占总时间和轨迹长度的百分比各组间有显著差异(分别为F=44.487,P<0.001；F=37.828,P<0.001),结果见表3。AD模型组原平台象限搜索时间百分比较正常对照组及假手术组均缩短(分别为P<0.001,P<0.001),中药1组组、中药2组组较AD模型组原平台象限搜索时间百分比均延长(分别为P<0.001,P<0.001),AD模型组原平台象限搜索距离百分比较正常对照组及假手术组均缩短(分别为P<0.001,P<0.001),中药1组组、中药2组组较AD模型组原平台象限搜索距离百分比均延长(分别为P<0.001,P<0.001)。
4、常规HE染色显示正常对照组大鼠海马神经细胞呈锥形,有长的突起,胞核明显,为圆形或椭圆形,着色均匀,为浅蓝紫色,核浆比值大,可见深染的核仁。在CA1区锥体细胞排列为3-4层。模型组大鼠海马CA各区及齿状回(DG区)神经元数量较其余各组明显减少,排列不规则,可见神经细胞胞体缩小,胞浆浓缩,核固缩,CA1区细胞排列层数减少。
5、药物对海马大鼠DG区平均神经元数目的影响：实验结果经统计学分析表明,DG区平均神经元数目各组间有显著差异(F=10.560,P<0.001),结果见表6。AD模型组DG区平均神经元数目比正常对照组及假手术组均降低(分别为P=0.002,P=0.012),中药1组和中药2组组DG区平均神经元数比AD模型组均升高(分别为P=0.001,P<0.001)。
6、淀粉样蛋白刚果红染色显示：AD模型组大鼠海马区可见明显嗜刚果红淀粉样斑块着色,正常组及假手术组大鼠海马区未见明显嗜刚果红淀粉样斑块着色,中药干预组大鼠海马区嗜刚果红淀粉样斑块着色较少。
7、药物对老年性痴呆模型大鼠海马Aβ和磷酸化tau蛋白阳性表达细胞数目的影响：实验结果经统计学分析表明,Aβ、tau平均阳性细胞数各组间有显著差异(分别为F=46.824,P<0.001；F=30.553,P<0.001),结果见表4。AD模型组Aβ、tau平均阳性细胞数比正常对照组及假手术组高(分别为P<0.001,P<0.001,P<0.001,P<0.001),中药1组和中药2组组Aβ、tau平均阳性细胞数均比AD模型组低(分别为P<0.001,P<0.001,P<0.001,P<0.001)。
8、药物对大鼠海马DG区平均凋亡率的影响：实验结果经统计学分析表明,平均凋亡率各组间有显著差异(F=240.155,P<0.001),结果见表7。AD模型组平均凋亡率正常对照组及假手术组均升高(分别为P<0.001,P<0.001),中药1组和中药2组组平均凋亡率比AD模型组均降低(分别为P<0.001,P<0.001)。
9、药物对老年性痴呆模型大鼠脑组织BiP蛋白表达的影响：实验结果经统计学分析表明,BiP蛋白免疫组化平均灰度值各组间有显著差异(F=174.042,P<0.001),结果见表5。AD模型组BiP蛋白免疫组化平均灰度值比正常对照组及假手术组均降低(分别为P<0.001,P=0.001),中药1组和中药2组组BiP蛋白免疫组化平均灰度值比AD模型组均降低(分别为P<0.001,P<0.001),中药1组和中药2组组BiP蛋白免疫组化平均灰度值比正常对照组均降低(分别为P<0.001,P<0.001),中药1组和中药2组组BiP蛋白免疫组化平均灰度值比假手术组均降低(分别为P<0.001,P<0.001)。
10、药物对老年性痴呆模型大鼠脑组织PERK蛋白表达的影响：实验结果经统计学分析表明,PERK蛋白免疫组化平均灰度值各组间有显著差异(F=144.556,P<0.001),结果见表5。AD模型组PERK蛋白免疫组化平均灰度值比正常对照组及假手术组均降低(分别为P<0.001,P<0.001),中药1组和中药2组组PERK蛋白免疫组化平均灰度值比AD模型组均升高(分别为P<0.001,P<0.001),中药1组和中药2组组PERK蛋白免疫组化平均灰度值比正常对照组均降低(分别为P<0.001,P<0.001),中药1组和中药2组组PERK蛋白免疫组化平均灰度值比假手术组均降低(分别为P=0.001,P<0.001)。
11、药物对老年性痴呆模型大鼠脑组织GADD153/CHOP蛋白表达的影响：实验结果经统计学分析表明,GADD153/CHOP蛋白免疫组化平均灰度值各组间有显著差异(F=48.703,P<0.001),结果见表5。AD模型组GADD153/CHOP蛋白免疫组化平均灰度值比正常对照组及假手术组均降低(分别为P<0.001,P<0.001),中药1组和中药2组组GADD153/CHOP蛋白免疫组化平均灰度值比AD模型组均升高(分别为P<0.001,P<0.001),中药1组和中药2组组GADD153/CHOP蛋白免疫组化平均灰度值比正常对照组均降低(分别为P<0.001,P<0.001),中药1组和中药2组组GADD153/CHOP蛋白免疫组化平均灰度值比假手术组均降低(分别为P<0.001,P<0.001)。
Aβ25-35联合D-半乳糖所致AD模型大鼠出现明显的空间学习记忆能力障碍,脑内出现嗜刚果红淀粉样斑块,磷酸化tau与Aβ免疫组化阳性细胞数目显著增多,海马神经元凋亡显著增多,内质网应激标志蛋白表达显著增多；在柴胡疏肝散浓缩液与楮实子浓缩液的干预下,海马神经元凋亡减少,大鼠空间学习记忆能力改善。柴胡疏肝散浓缩液与楮实子浓缩液通过上调保护性内质网分子伴侣GRP78/BiP蛋白,下调PERK蛋白,阻断细胞内质网应激所致凋亡信号通路,减少神经元凋亡,有效改善AD大鼠症状及病理学改变。通过疏肝解郁、补肝益髓,能够有效改善AD模型大鼠行为学改变,肝气郁滞-内质网应激关联假说得到了实验支持。
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.
AD特征性病理改变是β-淀粉样蛋白沉积形成的老年斑(senile plaque)、tau蛋白异常磷酸化聚集形成神经原纤维缠结(neurofibrillary tangles)以及区域选择性神经元死亡。近年来,逐渐积累的证据显示低聚物形式的Aβ和tau可能是阿尔茨海默病主要的罪魁祸首。由于脑内特定区域的淀粉样斑块与认知损害的程度相关,且Aβ的蓄积是阿尔茨海默病的早期事件。有人认为当Aβ的产生与清除之间出现不平衡,加上肽段间的自我聚集作用,导致Aβ开始蓄积。这种现象超过一定限度,可能是AD的启动因子。支持这种观点者称之为淀粉样蛋白级联假说("amyloid cascade hypothesis")。不断积累的证据也显示,Aβ具有神经毒性。利用Aβ脑室内或者海马内注射可以复制阿尔茨海默病动物模型。可致大鼠学习记忆障碍,海马胆碱乙酰转移酶(ChAT)活性降低等。该类模型适用于研究Aβ聚集或沉积在AD发病中的作用机制,并可作为评价药物对AD治疗作用的模型。
目前已经用于临床治疗阿尔茨海默病的药物主要是胆碱酯酶抑制剂,正在进行研究的还有抗Ap的单克隆抗体、抵抗tau氧化和聚集作用的小分子试剂以及脑源性神经营养因子(brain-derived neurotrophic factor, BDNF)替代疗法等。同时因为中药的成分天然,作用靶点多,对解决病因复杂的疾病有独特优势,相关实验研究及临床应用逐渐受到重视。
中医传统理论认为,情志所伤、所愿不遂、肝气郁滞是本病的重要病因和基本病机。现代临床医学研究表明,抑郁发生在老年性痴呆的各个阶段,AD患者最常见的行为异常是表情淡漠、情感低落、焦虑不安、易激惹等情绪相关症状。因此,AD的中医病理属性当属肝气郁滞。同时,中医现代研究表明,肝郁证的主要病理基础在大脑高级中枢。
内质网是重要的细胞器之一,蛋白质合成和修饰的场所。为保持内环境的稳态,内质网会将错误折叠的分子跨膜转运到细胞浆中降解。这种机制称为内质网相关性蛋白降解机制。体内外研究证明,内质网应激是AD发病机制之一。
基于内质网是细胞内精密的质量控制系统,在调控和维持细胞内环境稳定中发挥极其重要的作用,其形态结构、生理作用与功能状态均具有疏通的特点,且以调和畅达为至关紧要,与中医肝藏疏泄、调达的生理特性十分吻合,因此我们认为,内质网的结构和功能是肝主疏泄的亚细胞生理基础。基于此,导师吴正治教授结合传统理论与现代分子机制研究进展,提出老年性痴呆“肝气郁滞-内质网应激关联”中西医结合病理假说,该假说的核心观点是：肝气郁滞是老年性痴呆的基本病机,内质网应激为本病肝气郁滞的亚细胞病理基础,疏肝解郁可以缓解和拮抗老年性痴呆内质网应激。
建立Aβ25-35与D-半乳糖复合老年性痴呆模型；观察疏肝解郁代表方剂柴胡疏肝散、补肝益髓中药楮实子对Aβ25-35与D-半乳糖复合老年性痴呆模型大鼠空间学习记忆能力及病理学改变的干预作用;探讨AD内质网应激及细胞凋亡的相关发病机制,验证肝气郁滞-内质网应激关联新假说。
本研究通过双侧海马内脑立体定向注射Aβ25-35联合长期皮下注射D-半乳糖的方法建立AD大鼠模型。将大鼠随机分为5组,即正常对照组、AD模型组、假手术组、中药1组(柴胡疏肝散)组、中药2组(楮实子提取物)组(每组8只,共40只),见表1。通过Morris水迷宫实验检测各组大鼠空间探索实验逃避潜伏期及定位航行试验相关指标的变化,比较不同处理组别间大鼠空间学习记忆能力。观察各组大鼠脑组织病理学改变：HE染色用以观察病理学改变、甲苯胺蓝染色用以计数大鼠海马区神经元数目及刚果红染色法用以观察各组大鼠脑组织淀粉样蛋白沉积。采用免疫组织化学(immunohistochemical, IHC)法检测β淀粉样肽(Aβ)、磷酸化tau蛋白表达情况,以及内质网应激标志蛋白PERK、BiP、GADD153/CHOP的表达情况。原位末端凋亡法(TUNEL)用以检测各组海马区细胞平均凋亡率。
实验结果用SPSS 13.0 for windows统计软件分析,均用均数±标准差(X±S)表示,P<0.05为差异有统计学意义。对实验数据大鼠逃避潜伏期、游泳距离、搜索时间百分比、搜索距离百分比、Aβ、tau平均阳性细胞数、BiP、PERK、GADD153/CHOP蛋白免疫组化平均灰度值、DG区平均神经元数目、平均凋亡率,分别进行各组间多个样本均数比较。若方差齐性,采用单因素方差分析方法(one-way ANOVA),用LSD法进行多重比较；若方差不齐,用Welch近似方差分析法进行检验,用Dunnett's T3法进行多重比较。
1、Aβ与D-半乳糖复合老年性痴呆模型的行为变化：Aβ25-35与D-半乳糖复合老年性痴呆模型大鼠较正常对照组大鼠活动及打斗减少,对抓触逃避反应明显减弱,游泳速度较其余组别大鼠减低。
2、药物对老年性痴呆模型大鼠定位航行实验的影响：实验结果经统计学分析表明,大鼠搜索并找到安全平台的逃避潜伏期及游泳距离各组间有显著差异(分别为F=5.004,P=0.007；F=3.256,P=0.037),结果见表2。AD模型组逃避潜伏期较正常对照组及假手术组均延长(分别为P=0.017,P=0.041),中药1组组、中药2组组较AD模型组逃避潜伏期均缩短(分别为P=0.013,P=0.010),中药2组组游泳距离较AD模型组缩短(P=0.046)。
3、药物对老年性痴呆模型大鼠空间探索实验的影响：实验结果经统计学分析表明,大鼠在原平台所在象限搜索时间及搜索距离所占总时间和轨迹长度的百分比各组间有显著差异(分别为F=44.487,P<0.001；F=37.828,P<0.001),结果见表3。AD模型组原平台象限搜索时间百分比较正常对照组及假手术组均缩短(分别为P<0.001,P<0.001),中药1组组、中药2组组较AD模型组原平台象限搜索时间百分比均延长(分别为P<0.001,P<0.001),AD模型组原平台象限搜索距离百分比较正常对照组及假手术组均缩短(分别为P<0.001,P<0.001),中药1组组、中药2组组较AD模型组原平台象限搜索距离百分比均延长(分别为P<0.001,P<0.001)。
4、常规HE染色显示正常对照组大鼠海马神经细胞呈锥形,有长的突起,胞核明显,为圆形或椭圆形,着色均匀,为浅蓝紫色,核浆比值大,可见深染的核仁。在CA1区锥体细胞排列为3-4层。模型组大鼠海马CA各区及齿状回(DG区)神经元数量较其余各组明显减少,排列不规则,可见神经细胞胞体缩小,胞浆浓缩,核固缩,CA1区细胞排列层数减少。
5、药物对海马大鼠DG区平均神经元数目的影响：实验结果经统计学分析表明,DG区平均神经元数目各组间有显著差异(F=10.560,P<0.001),结果见表6。AD模型组DG区平均神经元数目比正常对照组及假手术组均降低(分别为P=0.002,P=0.012),中药1组和中药2组组DG区平均神经元数比AD模型组均升高(分别为P=0.001,P<0.001)。
6、淀粉样蛋白刚果红染色显示：AD模型组大鼠海马区可见明显嗜刚果红淀粉样斑块着色,正常组及假手术组大鼠海马区未见明显嗜刚果红淀粉样斑块着色,中药干预组大鼠海马区嗜刚果红淀粉样斑块着色较少。
7、药物对老年性痴呆模型大鼠海马Aβ和磷酸化tau蛋白阳性表达细胞数目的影响：实验结果经统计学分析表明,Aβ、tau平均阳性细胞数各组间有显著差异(分别为F=46.824,P<0.001；F=30.553,P<0.001),结果见表4。AD模型组Aβ、tau平均阳性细胞数比正常对照组及假手术组高(分别为P<0.001,P<0.001,P<0.001,P<0.001),中药1组和中药2组组Aβ、tau平均阳性细胞数均比AD模型组低(分别为P<0.001,P<0.001,P<0.001,P<0.001)。
8、药物对大鼠海马DG区平均凋亡率的影响：实验结果经统计学分析表明,平均凋亡率各组间有显著差异(F=240.155,P<0.001),结果见表7。AD模型组平均凋亡率正常对照组及假手术组均升高(分别为P<0.001,P<0.001),中药1组和中药2组组平均凋亡率比AD模型组均降低(分别为P<0.001,P<0.001)。
9、药物对老年性痴呆模型大鼠脑组织BiP蛋白表达的影响：实验结果经统计学分析表明,BiP蛋白免疫组化平均灰度值各组间有显著差异(F=174.042,P<0.001),结果见表5。AD模型组BiP蛋白免疫组化平均灰度值比正常对照组及假手术组均降低(分别为P<0.001,P=0.001),中药1组和中药2组组BiP蛋白免疫组化平均灰度值比AD模型组均降低(分别为P<0.001,P<0.001),中药1组和中药2组组BiP蛋白免疫组化平均灰度值比正常对照组均降低(分别为P<0.001,P<0.001),中药1组和中药2组组BiP蛋白免疫组化平均灰度值比假手术组均降低(分别为P<0.001,P<0.001)。
10、药物对老年性痴呆模型大鼠脑组织PERK蛋白表达的影响：实验结果经统计学分析表明,PERK蛋白免疫组化平均灰度值各组间有显著差异(F=144.556,P<0.001),结果见表5。AD模型组PERK蛋白免疫组化平均灰度值比正常对照组及假手术组均降低(分别为P<0.001,P<0.001),中药1组和中药2组组PERK蛋白免疫组化平均灰度值比AD模型组均升高(分别为P<0.001,P<0.001),中药1组和中药2组组PERK蛋白免疫组化平均灰度值比正常对照组均降低(分别为P<0.001,P<0.001),中药1组和中药2组组PERK蛋白免疫组化平均灰度值比假手术组均降低(分别为P=0.001,P<0.001)。
11、药物对老年性痴呆模型大鼠脑组织GADD153/CHOP蛋白表达的影响：实验结果经统计学分析表明,GADD153/CHOP蛋白免疫组化平均灰度值各组间有显著差异(F=48.703,P<0.001),结果见表5。AD模型组GADD153/CHOP蛋白免疫组化平均灰度值比正常对照组及假手术组均降低(分别为P<0.001,P<0.001),中药1组和中药2组组GADD153/CHOP蛋白免疫组化平均灰度值比AD模型组均升高(分别为P<0.001,P<0.001),中药1组和中药2组组GADD153/CHOP蛋白免疫组化平均灰度值比正常对照组均降低(分别为P<0.001,P<0.001),中药1组和中药2组组GADD153/CHOP蛋白免疫组化平均灰度值比假手术组均降低(分别为P<0.001,P<0.001)。
Aβ25-35联合D-半乳糖所致AD模型大鼠出现明显的空间学习记忆能力障碍,脑内出现嗜刚果红淀粉样斑块,磷酸化tau与Aβ免疫组化阳性细胞数目显著增多,海马神经元凋亡显著增多,内质网应激标志蛋白表达显著增多；在柴胡疏肝散浓缩液与楮实子浓缩液的干预下,海马神经元凋亡减少,大鼠空间学习记忆能力改善。柴胡疏肝散浓缩液与楮实子浓缩液通过上调保护性内质网分子伴侣GRP78/BiP蛋白,下调PERK蛋白,阻断细胞内质网应激所致凋亡信号通路,减少神经元凋亡,有效改善AD大鼠症状及病理学改变。通过疏肝解郁、补肝益髓,能够有效改善AD模型大鼠行为学改变,肝气郁滞-内质网应激关联假说得到了实验支持。
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.
引文
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