Study On Establishment Of Alzheimer’s Disease Rat Model With Abnormal Savda Syndrome And Intervention Of Abnormal Savda Munziq

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.