An Experimental Study To Evaluate The Effect Of “Muziq Balgham Prepared From Uyghur Medicine ” On APP/PS1 A Transgenic AD Mice Model

Objective: Uyghur medicine with it’s vast valuable expertise showcases the treatment of diseases pertaining to temperamental analysis which gives us an opportunity to discuss the effect of Uyghur medicine in management of alzheimer’s disease and we take it forward for its scientific validation with an experimental study to evaluate the effect of abnormal phlegmatic munziq(APMq) on the APP/PS1 transgenic mice alzheimer’s disease model of learning and memory, antioxidant and acetyl cholinesterase activity, immune-histo-chemistry, and the function of the protein expression in the hippocampus, to clarify the mechanism of abnormal phlegmatic munziq and further provide scientific basis for its clinical application. Method: 3 months APP/PS1 transgenic mice were randomly divided into 6 group which model group, Abnormal Phlegmatic Munziq high,-middle,-low dose group and Donepezil group,each group of 18;With 18 same months background C57BL/6J mice as normal control group. six months after the treatment, by Morris water maze test, measure the change of learning and memory ability in mice; with HE staining, Congo red stain, observed the pathological morphological change of the hippocampal CA1 area; with Transmission electron microscope test the hippocampal CA1 area, microtubule, microfilament change; and super oxide dismutase(SOD), glutathione peroxidase(GSH-px) and catalase(CAT), total antioxidant capacity(T-AOC), monoamine oxidase(MAO) and acetylcholinesterase(AchE) activity were determined. using Immunohistochemistry and Western bolt test for APP、Aβ、PS1、LRP1、RAGE、IDE、β-actin、AMBP、GAP43、NGF、Tau. Result:Water maze experiment results show that the APP/PS1 transgenic mice escape latency time was significantly longer than normal mice(P < 0.05), compared with normal group, search space experiment indicate that the cross of the original set position significantly reduced(P < 0.01);Compared with model group, abnormal phlegmatic mature agent groups escape latency time significantly shorten(P < 0.05), abnormal phlegmatic mature agent search space experiment groups mice cross the effective area time increased(P < 0.05).(2) HE staining:Abnormal phlegmatic Munziq groups mice brain tissue hippocampal CAI area, HE staining pyramidal cell layer integrity is a good, the structure of morphology is normal form normal, cell is neat, compared with model group difference was statistically significant.(3) Congo red staining: result showed that normal group not seen obviously neurofibrillary tangles and senile plaque.In model group it showed a large number of scattered red sediments,positive staining showed eosinophilic red or red brick like changing,suggesting the formation of the senile plaques.in the high dose group morphological observation is similar to donepezil group,showed a neuroprotective effect. This study was limited to the CA1 hippocampal region.(4) Transmission electronic microscope :In model group,Ultrastructure observation showed that neurons distribution and nuclear membrane is unclear, mitochondrial structure is disordering,synaptic morphology is abnormal.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)has smooth cytoplasmic membrances marked only infrequently by small irregular in foldings,Rough endoplasmic reticulum weas identified by the presence of ribosomes on its surface,mitochondria appeared as electron-dense oval structures with regular cristae inside and nuclei displayed smooth membrance.different from many cells with apparently normal morphology,in model group mice brian,some neurons and their organelles appeared to have undergo transformations, such as dilated rough endoplasmic reticulum and dilated mitochondria with fewer cristae inside,or even both cytoplasmic and nuclear membrance showing deep infoldings.few of the damaged structures displayed specific chromatin heterocondensation and marginalisation,typical of apoptotic death. In APMq treated brains,many neurons appeared to be with similar morphology to normal ones.except for occasionally deformed mitochondrions recognized by remmants of cristae,rare autophagosomes,and dilated Golgi complex and reticulum,no additional organells could be identified abnormal. APMq can increase the number of synapses,can improve the structure of synaptic before and after membrance, reduce the damage of mitochondria,curative effecrt is than that of donepezil group.(5) biochemistry biomarker:Antioxidant biomarkers were changed in mice AD model group.SOD,GSH-Px and CAT activity were decreased,MAO was increased compared with normal group(p<0.01). there was had significant difference in total antioxidant capacity of AD model group and normal group(p<0.05),indicated that there were severe oxidative damage, oxidative decomposition of monoamine neurotr-ansmitters in the model group. Acetylcholineesterase(AChE) activity was increased in the brain of AD model group compared with the normal group,there was significance difference(p<0.05).after APMq intervention of treatment,activities of antioxidant enzymes have improved in the varying degrees,statistical result showed that high dose of APMq can significantly improve the oxidative stress status of mice,increase the activity of antioxidant enzymes and total antioxidant capacity,high dose of APMq group could significantly inhibit the AChE activity, increased ther CAT activity, there is no statistical significance between donepezil group and high dosage group.(6)Effect of Abnormal Phlegmatic Munziq on APP/PS1 transgenic mice model of AD amyloid-β formation and aggregation pathway.Comppared with Model group, the average number of APP positive cells in the hippocampal CA1 area, APMq middle and high dose group decreased significantly(P < 0.01);The average number of amyloid beta protein positive cells expression in CA1 area of hippocampus of mice, abnormal phlegmatic munziq middle, high dose group compared with model group,the differences was statistically significant(P < 0.01), and abnormal phlegmatic munziq small dose group compared with the model group decreased significantly.Average number of PS-1 positive cells expression in CA1 area of hippocampus of mice, abnormal phlegmatic munziq high dose and middle dose group compared with the model group decreased significantly(P<0.05/P<0.01); expression in hippocampal CA1 region of Tau positive cell number and abnormal phlegmatic munziq in each dose group compared with the model group decreased significantly(P<0.05).(7) Abnormal Phlegmatic Munziq effect on APP/PS1 transgenic mice model of AD amyloid-β peptide degradation pathway,Average number of IDE positive cells expression in CA1 area of hippocampus of mice, APMq all dose group compared with model group positive cell number increased significantly, there was statistically significant difference(P < 0.05);The protein content of IDE, the Abnormal Phlegmatic Munziq of different dose group compared with model group, showed increased expression of protein.(8) The influence of APMq on APP/PS1 transgenic mice model of AD amyloid-β peptide clearance pathway, Immunohistochemistry and Western blot results showed that, Compared with normal group, in model group, expression of LRP1 in brain tissue was decreased, and the RAGE was increased(P < 0.05),After Abnormal Phlegmatic Munziq treatment,Compared with the model group, in treatment group the expression of LRP1 in brain tissue was enhanced, and the expression of RAGE was decreased(P<0.05).(9) The effect of Abnormal phlegmatic munziq on APP/PS1 transgenic mice to repair nerve damage.Hippocampal CA1 area of mice with beta actin expression in the APMq group compared with model group increased significantly(P < 0.01); expression in CA1 area of hippocampus of mice AMBP, the APMq group compared with model group increased significantly(P < 0.01); mice in CA1 area of hippocampus GAP43 expression, APMq group compared with model group significantly increased(P<0.05).The expression of NGF in hippocampal CA1 area of mice, APMq group compared with the model group, the number of positive cells increased, the difference was statistically significant(P<0.05);(10) Effects of Abnormal phlegmatic munziq on APP/SP1 transgenic mice model of AD on hippocampus neuronal apoptosis and its related gene expression.The number of apoptotic cells in hippocampal CA1 region in mice,treatment, there was significant difference(P<0.05,P<0.01). APMq groups can upregulate the expression of Bcl-2, down regulate the expression of Bax, increase the ratio of Bcl-2/Bax, after statistical analysis, there were significant differences(P < 0.05, P < 0.01). Conclusion: Abnormal Phlegmatic Munziq(APMq) can improve spatial learning and memory in AD mice. it can improve the organizational structure of the damaged neurons cells,reduce the cell damaged,reduce the formation of neurofibrillary tangles and senile plaque,can significantly improve the oxidative stress in mice,reducing the oxygen free radical on nerve cells in a mice model.inhibition of AchE activity,increased CAT activity. APMq treatment to reduce Aβ generation and Aβ neurotoxicity, improve synaptic plasticity, and protect the learning and memory of mice; APMq group significantly increased the IDE content and expression, suggesting that APMq can enhance the IDE activity, so as to improve the ability of degradation of Aβ beta, reduce Aβ levels in the brain, preventing the formation of AD pathology; APMq can reduce the content of RAGE in the mice brain and expression. Enhanced Aβ clearance, and increase the expression of LRP1 protein; APMq intervention microtubule arrangement, visible structure of normal axons in hippocampal neurons,can obviously increase the β-actin, GAP43,NGF and MBP, to reach a normal level, APMq effect Bcl-2, Bax expression in mice in hippocampal neurons, inhibit the pro apoptotic protein Bax and anti apoptotic protein Bcl-2 promotes, protects the nerve Cell injury;Different dosage of APMq on amino acid and sugar metabolism have different degree of regulation. it can also improve the inflation of by direct or indirect way,oxidative stress, cholinergic system damaged, increased Aβ clearance,relieve Aβ metabolic disturbance, relieve hippocampal neurones caused by Aβ neurotoxicity,while improve the learning ability,memory,cognitive level.