【作者】 帕丽丹·吾术尔；

【导师】 哈木拉提·吾甫尔（Halmurat·UPUR）； 努尔买买提·艾买提（Nurmuhammat·AMAT）；

【作者基本信息】 新疆医科大学 ， 内科学， 2013， 博士

【摘要】 目的：以维医体液病理学证候模型拟临床研究概念为指导,在前期疾病模型研究和临床研究的基础上,首次建立老年痴呆异常黑胆质病证结合大鼠模型,从行为学及病理、生化等多层面、多指标揭示异常黑胆质证的本质、揭示病证结合模型发展规律及其内在演化本质,并用异常黑胆质成熟剂对此模型进行干预,评价药物的作用和作用机理,从疾病内在本质构建符合维医药作用特点的药物评价技术及研究平台。方法：运用维吾尔医学异常黑胆质论,从产生异常黑胆质证的病因因素入手,应用干寒环境+慢性电刺激+干寒属性饲料,观察动物体表特征如舌象、毛皮、体重、饮食、活动、反应情况等验证维医体液证侯改变特点,建立异常黑胆质证大鼠模型基础上,再予以聚集态Aβ1-40双海马定向注射制备老年痴呆异常黑胆质病证结合大鼠模型。应用Morris水迷宫及跳台法等行为学试验观察病证结合大鼠模型的行为学改变;采用HE染色,Bielschowsky氏改良染色、刚果红染色等方法观察各组模型大鼠海马形态学的变化：采用电镜技术观察各组大鼠海马神经超微结构观察神经元、神经突触的变化；采用免疫组化法检测海马区Aβ1-40、COX-2、IκB-α、nNOS蛋白的表达；应用紫外分光光度法检测模型大鼠海马组织超氧化物歧化酶(SOD)活性、谷胱甘肽过氧化物酶(GSH-Px)活性、过氧化氢酶(CAT)活性、总抗氧化能力(T-AOC)、单胺氧化酶(MAO)活性、丙二醛(MDA)浓度；采用分光光度法检测各组大鼠胆碱乙酰转移酶(ChAT)、乙酰胆碱酯酶(AChE)活性,应用酶联免疫分析(ELISA)检测乙酰胆碱浓度；代谢组学技术检测各组模型代谢物的改变,在此模型的基础上以异常黑胆质成熟剂高、中、低剂量干预治疗后,应用上述方法检测异常黑胆质成熟剂对变化指标的影响。结果：①生物表征观察结果显示：异常黑胆质证模型组出现了舌质暗红、红紫色黯、欠津,毛色发黄黯淡无光泽,反应迟钝,烦躁争抢食水但体重明显下降,出现消瘦、腹部凹陷松软的表象等生物表征。而正常对照组大鼠发育正常。上述生物表征在造模第14天时明显于第7天。与正常对照组比较异常黑胆质证模型组大鼠每100g体重平均进食饮水量明显增加(P<0.01),而体重明显减轻(P<0.01),异常黑胆质证模型的生物表征发生了明显的改变；痴呆异常黑胆质病证结合模型生物表征的改变较单纯异常黑胆质证模型更加明显；病证结合组与异常黑胆质证组比较争食水减少,反应迟钝,舌质紫黯更明显且舌欠津；不仅掉毛增多,由于活动减少,较少清理毛发故毛中见木屑多,形体日渐消瘦,腹部内凹、弓背以病证结合组更为明显。统计数据也表明与正常对照组相比病证结合组体重减轻(P<0.01),饮食、水量反较异常黑胆质期减少,体重减轻明显,体型瘦弱。②Morris水迷宫定位航行实验中,病证结合组与单纯痴呆组学习记忆能力明显下降,病证结合组与单纯痴呆组相比,潜伏期明显延长；空间探索实验中,病证结合组与单纯痴呆组相比经过有效区域次数明显减少(P<0.01)；跳台测试结果显示：单纯痴呆组、异常黑胆质证组和痴呆异常黑胆质病证结合组学习记忆成绩明显降低,与正常对照组比较(P<0.01)；异常黑胆质证组开始训练时成绩与正常对照组差异不明显,但第3、4天与正常对照组寻找平台的时间差异明显,提示异常黑胆质证对模型的学习行为能力有影响。病证结合组从开始训练到实验结束游泳轨迹都杂乱无章,在每个时间点都与正常对照组有显著差异(P<0.01)。③HE染色结果显示：痴呆异常黑胆质病证结合模型组大鼠海马神经元细胞明显减少,与正常对照组、单纯痴呆组比较数目减少有显著统计学差异(P<0.01),提示神经元坏死数目减少比单纯痴呆组明显：⑤Bielschowsky氏改良染色：正常对照组结果显示阴性,未见明显神经纤维缠结及老年斑。痴呆异常黑胆质病证结合组神经元纤维增粗,排列紊乱,密集成团块状较单纯痴呆组多见,轴突呈编织状遍布细胞质并深入到细胞突起,在胞浆内有形成螺旋扭曲,形成神经纤维缠结。⑤刚果红染色：痴呆异常黑胆质病证结合组可见大量散在分布的橘红色沉积物,阳性染色呈现嗜红色或者砖红色样改变,提示老年斑形成。⑥透射电镜结果显示：痴呆异常黑胆质病证结合模型组核膜不清楚,线粒体结构紊乱,突触形态异常,结构模糊,前、后膜不清晰,数量明显减少,神经损伤最为严重。⑦免疫组织化学结果显示：Aβ1-40阳性细胞在病证结合模型组反应强度明显高于正常对照及单纯痴呆组,在海马区阳性细胞数与前两组比较,差异有显著性意义(P<0.01)。而nNOS的阳性表达明显减弱,COX-2、IκB-a在病证结合模型组的表达强度和阳性细胞数的增加与正常对照组比较(P<0.01)。⑧生化指标检测结果显示：与正常对照组比较,痴呆异常黑胆质病证结合组大鼠海马SOD、GSH-PX、CAT活性及总抗氧化能力均降低,MAO活性增强,氧化产物MDA含量增高(P<0.01)。异常黑胆质证组各项指标较正常对照组都有改变,CAT的减少与正常对照组比较差异有显著统计学意义(P<0.01)。痴呆异常黑胆质病证结合大鼠模型的总抗氧化能力与单纯痴呆组比较有明显差异(P<0.05)。痴呆异常黑胆质病证结合大鼠模型氧化损伤、脂质过氧化损伤、单胺类神经递质被氧化分解最为严重。⑨胆碱能神经指标检测结果显示：异常黑胆质证组大鼠模型的乙酰胆碱含量及胆碱乙酰转移酶活性的改变与正常对照组比较,差异有明显统计学意义(P<0.05),而痴呆异常黑胆质病证结合组与正常对照组比较乙酰胆碱含量、胆碱乙酰转移酶活性及乙酰胆碱酯酶活性均有显著差异(P<0.01),痴呆异常黑胆质病证结合组大鼠模型在中枢胆碱能的损伤最为明显。⑩异常黑胆质证导致模型发生了糖代谢紊乱与能量代谢障碍,代谢物的变化表明不同模型间的代谢产物具有差异性,痴呆异常黑胆质病证结合动物模型体内糖代谢紊乱和能量代谢障碍与单纯痴呆模型及异常黑胆质证模型比较代谢产物及代谢途径在病变过程中发生了演变,痴呆异常黑胆质病证结合动物模型的代谢变化更为复杂。0药物干预治疗结果显示：异常黑胆质成熟剂高剂量组在第二天的平均潜伏期成绩明显优于其他组,与模型组大鼠比较,异常黑胆质成熟剂高、中、低剂量组在第3、4天空间记忆显著提高,潜伏期明显缩短,与多奈哌齐组比较无统计学差异(P>0.05)；与正常对照组及多奈哌齐组比较,各剂量组经过有效区域次数均无明显差异(P>0.05)；与模型组比较跳台实验中个治疗组大鼠潜伏期缩短和错误次数降低(P<0.01),与多奈哌齐组也无明显差异(P>0.05)。HE染色异常黑胆质成熟剂中、低剂量组对组织病理学改变作用不明显,但高剂量干预组HE染色其锥体细胞层较完整,细胞排列较整齐,结构形态正常,染色正常,仅可见少量固缩状的坏死神经元,Bielschowsky氏改良染色正常组未见神经纤维缠结,刚果红染色可见少量散在老年斑,高剂量干预组形态学观察与多奈哌齐组接近,对损伤的神经具有相同的改善作用。电镜超微结构见高剂量的异常黑胆质成熟剂可增加神经突触的数量,改善突触前后膜的结构,保护线粒体的损伤,疗效优于多奈哌齐组；与模型组比较,有高剂量异常黑胆质成熟剂可明显抑制Aβ1-40、COX-2,1κB-α的表达,而促进nNOS表达(P<0.01)；抗氧化酶活性在异常黑胆质成熟剂干预治疗后均有不同程度的改善,其中异常黑胆质成熟剂高剂量显著提高抗氧化酶活性及总体抗氧化能力,减少模型大鼠海马区的脂质过氧化物MDA的含量,有效保护模型大鼠神经细胞的氧化损伤；异常黑胆质成熟剂明显减低MAO酶活性,明显抑制病证结合模型的AchE活性,增强ChAT活性,增加Ach的含量,和多奈哌齐组比较组间差异无统计学意义。代谢组学分析不同剂量的异常黑胆质成熟剂对与糖代谢相关代谢产物及与氨基酸合成代谢相关代谢产物有不同程度的调节作用,从而改善神经细胞的糖代谢紊乱和能量代谢障碍。结论：(1)首次在异常黑胆质证模型的基础上双海马Aβ1-40注射术建立痴呆异常黑胆质病证结合模型,此模型将“病”和“证”的因素集于一体,与单纯痴呆模型、异常黑胆质证模型比较,病证结合模型大鼠在体征和行为学方面的变化上不仅具有维医体液证候特点,而且具有AD学习记忆受损的西医疾病的特点；学习记忆受损程度,神经损伤从神经元的数目、神经纤维缠结的程度及老年斑的形成以及从加速这种病变的因子的表达最为明显；神经细胞氧化应激损伤、脂质过氧化损伤、单胺类神经递质被氧化分解最为严重,中枢胆碱能神经损伤最为突出,代谢产物差异性更复杂。发现AD与异常黑胆质证具有相对一致的生物学基础紊乱,异常黑胆质证所造成的病理改变对Aβ的神经毒性造成的神经损伤不仅有协同的作用,部分改变还发生了叠加,进一步加重了模型的生物学基础的改变,此模型能更好的成为维医体液论和老年痴呆的基础研究的动物模型,为今后异常黑胆质型痴呆的发病机制及防治研究奠定动物实验基础。(2)异常黑胆质成熟剂对痴呆异常黑胆质病证结合模型大鼠的学习记忆有显著改善作用；可改善受损神经元细胞的组织结构,减少神经细胞损伤,减轻神经纤维缠结的程度及老年斑的形成,抑制Aβ1-40、COX-2、IκB-α表达,增加nNOS表达水平,加速NO合成,减轻氧自由基对模型大鼠神经细胞的氧化损伤,能显著改善模型大鼠神经细胞氧化应激损伤,抑制海马AchE活性,增强ChAT活性,增加ACh的含量,对模型组大鼠的胆碱能系统的损伤有修复作用,不同剂量的异常黑胆质成熟剂对与糖代谢相关代谢产物及与氨基酸合成代谢相关代谢产物有不同程度的调节作用。异常黑胆质成熟剂可通过直接或间接地改善炎症反应、氧化应激、胆碱能系统的损伤,调节代谢障碍。异常黑胆质成熟疗法减轻Aβ神经毒性作用造成大脑海马神经元损伤而提高学习记忆能力、认知水平,改善“痴呆”症状。本实验也证明异常黑胆质成熟疗法具有多靶点、多途径、多层次、整体治疗的特点。更多还原

【Abstract】 Objective:In this study, under the guidance of Hilit phathology theory of Uighur medicine, as well as on the basis of research on disease model and clinical research established Alzheimer disease rat model with Abnormal Savda Syndrome. From more perspectives of behavioral science, the pathological and biochemical indicators reveal the nature of the Abnormal Savda Syndrome, reveal the regulation of development and its inner nature of evolution,comprehensively and accurately revealed the nature of syndrome with diseases. I addition, we used the Abnormal Savda Munziq to treat Alzheimer disease with Abnormal Savda Syndrome rat model, and clarified the mechanism of Abnormal Savda Munziq on Alzheimer’s disease. Method:We applied the Uighur medicine Savda syndrome theory, and proceeded with the etiological factors of Abnormal Savda such as dry-cold environment,(psychological)chronic electronic shock and dry-cold feed to establish Abnormal Savda Syndrome rat model to observe biological character such tongue, for, weight, diet, acitivity, response to validate the biological characteristics of Savda syndrome. Then the incubated Aβ1-40was injected into both hippocampus of each rat, enabling it to obtain the characteristics of both the western medicine of disease, and the characteristics of Savda syndrome. The behavioral abnormalities were investigated by Morris water maze test and step-down phases test. Rat brain sections were detected by HE staining, transmission electron microscopy and immunohistochemistry for Aβ1-40、COX-2、IκB-α、nNOS expression. RT-PCR for COX-2、IκB-α、nNOS expression of mRNA. Acetylcholine (ACh) levels, acetylcholineesterase (AChE) activity, Choline acetyltransferase (ChAT) activity, monoamine oxidase (MOA), oxidative stress markers (SOD, GSHPx, T-AOC, CAT and MDA) were assessed in the hippocampus. Metabonomics technology to detect changes in metabolites. After model stage, ASMq and donepezil were orally administered to AD rats for14days after7days Aβ1-40injected to hippocampus. Invesitigate the therapeutic potential mechanism of ASMq to ameliorate AD-like pathology in the brain of AD rats with Savda syndrome. Results:①The change of biological characterization of the Abnormal savda syndrome model:Abnormal savda syndrome rat model group appeared dark red tongue, red and purple, the hair color is yellow, dim and dull, lags in response, dull copetition for water and feeding, but the weight loss evidently, appeared weight was lossed, representation symptoms of abdominal deboss loosing While normal control group’s rats can growth normally, the above symptoms were clear in model rats in fourteenth days than seventh days. The results show that the amount of eating and drinking consumption of the abnormal savda syndrome rat model group for each100g rat’s weight increased significantly in compared with the normal control group (P<0.01), and the weight loss significantly (P<0.01). Thus it can be seen that, under the condition of three factors the biological characterization of Alzheimer’s disease (AD) with Abnormal Savda Syndrome rat model, combination AD with Abnormal Savda Syndrome rat model, and its biological characterization of the change were obviously than the Abnormal Savda Syndrome. The response of combination disease with syndrome group’s fighting was reduced than the abnormal savda group, dark purple tongue also more obviously than abnormal savda group. Not only hair loss were increased, rat activity were decreased, body weight were decreased, showed abdominal concave were much more obvious of combination disease with syndrome group. Statistics show that weight losing (P<0.01), food, water consumption were much more obvious when compared with the normal control group.②Spatial learning and memory ability:Morris water maze test results showed that decrease ability of learning and memory of the combination AD with Abnormal Savda Syndrome and AD model group’s than another group. The potential latency of the combination AD with Abnormal Savda Syndrome rat model group was decreased much more obviously when compared with AD group, in the space exploration number of through the effective area was significantly reduced (P<0.01). The abnormal savda syndrome group began training the marks were not significantly different than the normal control group, but the third,4th days the searching platform time has differences between groups, indicated that that abnormal savda damaged learning behavior ability of model. The experiment track of combination AD with Abnormal Savda syndrome group were disorder from in the beginning to the end, also at each time point has significant difference (P<0.01).Results of the step-down test also show that learning and memory performance of simple AD group, the abnormal savda syndrome group and Alzheimer disease with Abnormal Savda Syndrome rat model were decreased significantly (P<0.01).③HE staining:Combination AD with Abnormal Savda Syndrome rat model group, the rat’s hippocampal neurons number were significantly reduced than the normal control group and simple AD group, and it also has significant statistical diffeences (P<0.01), necrosis of neurons, decreased in number than the AD group.④Modified Bielschowsky’s staining:results showed that in combination AD with Abnormal savda syndrome thickening neuronal fiber, arranged in disorder, densely integrated mass can be seen more than simply AD group rat, axons was woven throughout the cytoplasm and deep into the cell processes, have formed spirally twisted in the cytoplasm, and formed neurofibrillary tangle.⑤Congo red staining:results showed that staining in normal group and abnormal savda syndrome group showed negative, not seen obviously neurofibrillary tangles and senile plaques. Congo red staining in combination AD with Abnormal savda syndrome showed a large number of scattered red sediments, positive staining showed eosinophilic red or red brick like changing, suggesting the formation of senile plaques.⑥Transmission electron microscope:Ultrastructure observation showed that neurons distribution and nuclear membrane unclear in combined AD with Abnormal savda syndrome group, we found that disordering structure of mitochondrial, synaptic’s abnormal morphology, fuzzy structure, before and after the film is not clear, the number decreased evidently, nerve injury is the most severe.⑦Immunohistochemical:Aβ1-40positive cells in the combination AD with Abnormal savda syndrome group in the reaction intensity was significantly higher than that of normal control and the AD group, positive cells number in the hippocampus compared with the two other groups, they have significance diffirences in their cells number (P<0.01). While the expression of nNOS was significantly decreased, COX-2, IκB-a expression were increased in the combination of AD with Abnormal savda syndrome group compared with normal control group (P<0.01);⑧Biochemistry biomarker:Antioxidant biomarkers were changed in rats combination AD with Abnormal savda syndrome to compare with the normal contract group. SOD, GSH-Px and CAT activity were decreased, MAO was increased compared with normal control group (P<0.01). There was had significant difference in total antioxidant capacity of combination AD with Abnormal Savda Syndrome rat model’s comparered with simple AD group (P<0.05), indicated that there were severe oxidative damage, lipid peroxidation damage, oxidative decomposition of monoamine neurotransmitters in the combination AD with abnormal savda syndrome’s.⑨Study on the damage of cholinergic neurons:there was acetylcholinesterase (AChE) activity in brain increased obviously in the combination AD with Abnormal Savda Syndrome model’s, but choline acetyltransferase activity significantly decreased, as well as acetylcholine content was decreased when compare with the normal control group (P<0.01). Acetylcholine content was decreased, and acetylcholineesterase (AChE) activity was increased in the brain of Abnormal savda syndrome compared with the normal control group, there was significant difference (P<0.05), while content of acetylcholine, acetylcholineesterase (AchE) activity, choline acetyltransferase activity of the combination of disease with syndrome group have significant difference with the normal control group (P<0.01). damage of central cholinergic of combination AD with Abnormal Savda Syndrome model’s is the most obvious.⑩Abnormal black bile leads to the sugar metabolic disorders and energy metabolism dysfunction, metabolite changes showed that the metabolites are different between different models, the metabolic changes of combining AD with syndrom groups are more complex.11Intervention of Abnormal Savda Munziq(ASMq):Results show that Abnormal Savda munziq high dose group significantly improve the average latency performance in the second compared with model group, in the third,4days all the ASMq three dosage groups significantly improved spatial memory and escape latency period, there was no significantly statistical difference compare with donepezil group(p>0.05). There were no significant difference in the navigation test, number of pass the effective area of all treatment group compare with normal control group and donepezil group(p>0.05), there are no significant differences between donepezil group and step-down test(p>0.05). Histological results showed that HE staining, there are not evident curative effect of low dosage treatment with ASMq, But the high dose group, HE staining of the pyramidal cell layer of relatively complete, cells were arranged in order, the structure of morphology is normal, normally staining, only a small amount of pyknotic neuron necrosis can be seen. Modified Bielschowsky’s staining results showed no neurofibrillary tangles, found a altered in a small amount of senile plaques in Congo red staining, in the high dose treatment group morphological observation is similar to donepezil group, showed a neuroprotective effect. This study was limited to the CA1hippocampal region. In normal group brains, no observe indications of neurodegeneration were observed. Most of the neurons with normal morphology (large, round, or oval nuclei, synaptic contacts on the soma, well-developed rough endoplasmic reticulum, and Golgi complex) had smooth cytoplasmic membranes marked only infrequently by small irregular in foldings. Rough endoplasmic reticulum was identified by the presence of ribosomes on its surface, mitochondria appeared as electron-dense oval structures with regular cristae inside and nuclei displayed smooth membranes. Different from many cells with apparently normal morphology, in model rat brains, some neurons and their organelles appeared to have undergone transformations, such as dilated rough endoplasmic reticulum and dilated mitochondria with fewer cristae inside, or even both cytoplasmic and nuclear membranes showing deep infoldings. Few of the damaged structures displayed specific chromatin heterocondensation and marginalisation, typical of apoptotic death. In ASMq treated brains, many neurons appeared to be with similar morphology to normal ones. Except for occasionally deformed mitochondrions recognized by remnants of cristae, rare autophagosomes, and dilated Golgi complex and reticulum, no additional organelles could be identified abnormal. ASMq can increase the number of synapses, can improve the structure of synaptic’s before and after membranes, reduce the damage of mitochondria, curative effect is better than that of donepezil group. Immunohistochemistry research results show that high doses of ASMq could significantly inhibit the number of Aβ1-40positive cells, and increase the expression of nNOS. As to the number of COX-2cells, there is obvious inhibitory effect between high dose group and model group. High dose ASMq reduced the IκB-α immunoreactive expression in the hippocampus, the reaction intensity decreased, the number of positive cells decreased, reaching the level of the normal control grop. The expression of mRNA; After ASMq intervention on treatment, Activities of antioxidant enzymes have improved in the varying degrees, Statistical results showed high dose of ASMq can significantly improve the oxidative stress statues of rats, increase the activities of antioxidant enzymes and total antioxidant capacity, can significantly reduce the content of lipid peroxide MDA in model rat’s hippocampus, evidently decrese the enzyme activity of MAO, High doses of ASMq could significantly inhibit the combination disease and syndrome model’s AchE activity, Increased ChAT activity, increased Ach contents, and there is no statistical significance between donepezil group and high dosage group. Metabonomics analysis of different doses of ASMq on amino acid and sugar metabolism have different degree of regulation, can improve the sugar metabolic disorders and energy metabolism of nerve cells disorder.Conclusion:(1) We firstly establish combination Alzheimer disease rat model with Abnormal Savda Syndrome according to the basis of abnormal savda syndrome model and bilateral hippocampal injection of Aβ1-40, the model will be combine "disease" and "syndrome" in one time, the learning and memory impairment obviously of the combination AD with Abnormal savda syndrome model than the simple model of AD induced bilateral hippocampal injection of Aβ1-40and Abnormal savda syndrome model, Histopathological changes such as neurone damage, neurofibrillary tangles and senile plaques were was significantly combination AD with Abnormal savda syndrome model than the Aβ1-40induced AD model. Oxidative damage, lipid peroxidation, monoamine neurotransmitters oxidative decomposition are the most serious in the AD with Abnormal savda syndrome model brain. The central cholinergic nervous system damaging was much more obvious, This is would be not only animal model for disease differentiation but also syndrome differentiation on the basis of the Uighur humor theory and AD, should accord with clinical practice of AD, The rat model of combined disease with syndrome can comprehensively reflect the character of AD aspects from both western medicine and Uighur Medicine as well as from etiology and pathology. It will become the ideal animal model study AD, also a ideal model research Uighur medicine effect and its treatment target.(2) ASMq significantly improved learning and memory of combined AD with savda syndrome in rats. It can improve the organizational structure of the damaged neurons cells, reduce the cell damaged, reduce the formation of neurofibrillary tangles and senile plaque, inhibit the positive expression Aβ1-40, COX-2, IκB-α, increased nNOS levels, accelerate the synthesis of NO, can significantly improve the oxidative stress in rats, reducing the oxygen free radical on nerve cells in a rat model. Inhibition of AchE activity, increased ChAT activity, increasing the content of Ach, and agitating M receptor activation of cholinergic neurons. Different doses of ASMq on amino acid and sugar metabolism have different degree of regulation.It can also improve the inflammation by direct or indirect way, oxidative stress, cholinergic system damaged, increased Aβ clearance, relieve Aβ metabolic disturbance, relieve damaging of hippocampal neurons caused by Aβ neurotoxicity, while improve the learning ability, memory, cognitive level, improve AD symptoms.更多还原