| Alzheimer's disease(AD),a degenerative disorder of central nervous system,is an important disease in an aging society.A? plaques and neurofibrillary tangles in the brain,decreased selective cholinergic neurons and the loss of neurotransmitters characterize the lesions of AD.Among these,it is believed that formation and deposition of A? plaques is the most important regarding initiation and progression of AD,while the main cause of nerve fiber entanglement and neurotransmitter deficiency is inferred to be oxidative stress.Though being banked on all the time,medicines targeting A? failed in their phase III clinical trials,invariably.This may due to the complexity of AD pathogenesis and lack of insight of the accurate mechanisms of AD.Latest researches indicate that increase of brain blood flow could be a potential AD therapy,after all,brain processes the most abundant bloodstream among the whole body.As known for its effect in improving cerebral blood flow and brain nourishment,Yangxue Qingnao extraxt(YXQN)is a patent medicine,composed of 11 kinds of herbs,aimed at alleviating headache and dizziness for more than 20 years.Four of them come from Siwu Tang,Four Herbs Decoction,one of the most famous prescriptions for activating blood circulation,which is recorded in the Chinese first national pharmacopeia,Prescriptions Peoples of the Welfare Pharmacy.The other seven herbs additionally activate blood circulation,and play the role of anti-oxidation,protection of neurons and regulation of the enzymes targeting the nervous system.YXQN is currently an important clinical medicine to improve cerebral ischemia.However,its potential role to prevent and/or cure AD has not been explored.Therefore,we systematically studied the cognitive impairment and the improvement of brain pathology in AD-model rats,and analyzed the effects of the key signaling pathway and differentially expressed genes and the associated mechanism of oxidative damage in this study.APPswe/PS1dE9 mouse(APP/PS1)is wildly used as one of the animal models for AD research.Therefore,8 months APP/PS1 mice were used as a model for moderate AD.APP/PS1 mice were divided into 5 groups with vehicle,3 different YXQN concentrations(low(0.69 g/kg),medium(2.08 g/kg)and high(6.24 g/kg)),and a drug control,Donepezil(n=16-18),which is currently widely used to improve AD symptom(cholinesterase inhibitors).The drugs were given via continuous gavage within 2 months,until the mice were 10 months-old,during which behavioral,pathological,gene-chip and biochemical tests were performed to detect the pharmacodynamics of YXQN and analyze its mechanism.Besides,even-aged normal wild type mice(WT,n=16)were also tested as healthy control group to further clarify the effect of the medicine.First,we studied the effects of YXQN on learning and memory and behavioral cognitive function in AD model mice.APP/PS1 showed significant decrease in spatial memory and cognition,compared with wild mouse,when they were tested with Morris Water Maze place navigation and spatial probe.During the navigation tests,as the concentration of YXQN elevated,mice performed better,almost got comparable results to wild mice.While in spatial probe experiments,medicine intake restored mice's performance,compared with vehicle group,and yield a close result with wild type mice.Therefore,we are confident that YXQN could significantly improve the long-term memory and spatial cognitive ability of 10 months-old APP/PS1 mice.Further,we proved that both drug administration for 1 month and 2 months,could improve mice performance in Y-Maze,overwhelming saline control group and Donepezil group.Thus,we conclude that YXQN could significantly improve the cognitive ability of APP/PS1 mice,in a concentration dependent manner,resembling the wild-type mice with a better effect than Donepezil.As brain A? deposition characterize AD lesion,we next tested the role of YXQN on brain pathology via thioflavin-S fluorescence staining,Congo red staining and A? specific antibody immunohistochemically staining.We found that,in the hippocampus and cortex of APP/PS1 mice,amyloid plaques of YXQN groups decrease 47% to 72%,compared with vehicle group.In three other pathological examinations of brain plaques,high YXQN concentration group yielded a decrease in deposition by at least 60% to saline control group.The results showed that YXQN could significantly relieve the AD pathology.Further analysis with ELISA proved that levels of soluble A?40,A?42 and insoluble A?40,A?42 in the brain of AD mice were significantly reduced by YXQN intaking.A? was formed by the precursor protein APP,and we found that YXQN increased the CTF-?/CTF-? ratio,which inhibited the pathologic process of APP and promoted the process of APP physiology process.Through Western blot detection and immunohistochemically analysis of the key secretases,which involved in the APP process,we found that YXQN can reduce the level of ?-secretase(BACE1)and ?-secretase(mainly presenilin 1(PS1)),thus reducing the production of A? in the brain.At the same time,YXQN promoted the physiological process of APP by up-regulating the expression of ?-secretase(ADAM10),and produced more sAPP? with neuroprotective effects,and antagonistic to A? formation.These results suggest that YXQN has the function of promoting the physiological process of APP and the antagonistic effect of pathologic process.In an attempt to find key target genes of YXQN's action of AD pathology,we analyzed the expression differences of hippocampus between WT,APP/PS1 and YXQN-treated APP/PS1 mice with microarray.After Gene Ontology(GO)analysis and KEGG pathway database screening,182 AD pathology-related genes in total were confirmed and their cluster analysis was carried out.We also found some dominant genes regulated by YXQN in AD pathology signaling pathway,such as the downregulation of PS1 mRNA expression,in accordance with its protein level in the last part.In particular,the data indicate that YXQN repaired oxidative stress associated pathway and cholinergic systems,so in the next section we focus on the validation.A?-mediated oxidative stress injury is the precondition of the change of AD neuron structure and function and the results of expression microarray suggest that YXQN improves the stress-induced injury caused by A?.To further confirm this preliminary result,we analyzed the regulative effect of YXQN on the multiple oxidative stress indexes in the brain and the effect on the cholinergic system.The results showed that YXQN significantly inhibited the process of peroxidation in the brain,i.e.,significantly reduced MDA content,NO and NOS level,eliminated the brain peroxide products,i.e.,significantly enhanced SOD.Besides,the activity of degradation of GPx and CAT of the peroxide product is stronger than that of Donepezil group.Thus,the effect of YXQN significantly improves the pathology of AD oxidative damage in brain tissue.In particular,YXQN can significantly restore Ach to the physiological level,which is closely related to the correction of oxidative damage and the increase of ChAT.These results indicated that YXQN could protect brain tissue against oxidative damage and cholinergic system imbalance in AD mice,thus improving its cognitive function.In summary,we have confirmed the level of pharmacodynamics of YXQN to improve the memory cognitive impairment caused by AD,relieve the AD pathological changes of amyloid plaques in the brain,antagonistic to oxidative stress in the brain,and thus achieved the level of Ach.In addition,YXQN regulates the AD-related gene signaling pathway,reversing the pathological process of APP.Therefore,this study provides the potential antiviral therapeutic value of YXQN,which lays a good foundation for the further study of the anti-AD drugs based on YXQN.Taken together,our results strongly suggest that YXQN could be a potential safety medicine for AD,and provide new evidence for further AD drug research and development. |
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