Effects Of Yangxue-Peiben-Jiannao Method On Learning And Memory And Apoptosis-related Proteins In AD Rats Induced By A?_25-35

Obj ectiveTo collect and sort out relevant literatures ahout the prevention and treatment of Alzheimer's disease(AD)and explore the theoretical basis for the prevention and treatment of AD with traditional Chinese medicine.Combined with the ADanimal model,the effects of Yangxue-Peiben-Jiannao method on the learning and memory ability and apoptosis-related proteins in AD rats were determined with the use of animal behavior,histomorphology and molecular biology experiments,laying a foundation for the development of new drugs for AD and the promotion and inheritance of Chinese medicinetheory.Methods1 Theoretical research:This study collected and summarized the literatures related to AD prevention and treatment,combined the theory of"blood and spirit"in TCM with neuron apoptosis in modern medicine,and therefore explained the connotation of Yangxue-Peiben-Jiannao Recipe on AD prevention and treatment.2 Behavioral research:84 male SPF SD rats were divided into 7 groups randomly as normal group,sham operation group model group,three Yangxue-Peiben-Jiannao Recipe groups with high,middle and low dose(Yanggao group,Yangzhong group,and Yangdi group,respectively),and Aricept as positive group,with 12 rats in each group.The model group and every treatment group were injected A?25-35preparation into CAl region of bilateral hippocampal as AD models,and the normal and sham operation group was administrated with same quantity of normal saline.After 5 days,rats in each dose group were given intragastrically with Yangxue-Peiben-Jiannao Recipe,the positive control group was given the equal dose of Aricept,and the sham operation and normal group were given the same amount of normal saline,for continuous intragastric administration 4 weeks.At the end of 3rd week,Morris water maze was used to detect the spatial learning and memory ability of rats in each group.All rats were sacrificed to take serum and hippocampal tissue for follow-up experiments after the final intervention.3 Morphological research:Hematoxylin-eosin staining(HE)was used to observe the morphology of hippocampal tissue and the neuron number ofrats in each group after treatment intervention.4 Mechanisms research:(1)The levels of serum AP25-35 in each group after intervention was determined by Enzymelinked immunosorbent assay(ELISA).(2)The apoptosisrelated proteins expression of Cleaved Caspase-3 and Bcl-2 in hippocampal CAl region were tested using Immunohistochemical staining(IHC).(3)The apoptosisrelated proteins expression levels of Cleaved Casepase-3,Bax and Bcl-2 in the hippocampus of rats in each group after the intervention was detected using(WB).Results1 Theoretical research:Yangxue-Peiben-Jiannao Recipe could supplement the innate basis to prevent dementia caused by blood deficiency and stasis.2 Behavioral research:Morris water maze results showed that compared with normal group and sham operation group,learning and memory ability of rats in the model group was significantly impaired,which showed that escape latency was prolonged in directional navigation experiments,the times of crossing platform area,percentage of time of target quadrant and track length of target quadrant were reduced in space exploration experiments,and time of first arrival at platform area was increased,implying that Jiannao intervention could significantly improve the learning and memory ability of AD rats.3 Morphological research:HE staining showed that compared with the sham operation group,there were a number of neurons which had unclear outline and severe atrophy,together with pyknotic nucleus,decreased cytoplasm,atrophicnucleus in disorder and scattered distribution,followed by proliferative glial cells in the hippocampal CA1 area of rats in the model group.There were clearer outline,more neurons,more abundant cytoplasm,less nucleus atrophy,more orderly arrangement and less glial cells in the western medicine and Yanggao groups than those in the model group.But there was no obvious changes among the Yangdi group,Yangzhong group and model group,4 Mechanisms research:(1)ELISA analysis showed that compared with sham-operated group,the A?25-35 content in serum in model group increased significantly(P<0.01).And the AP25-35 content in Yanggao group,Yangzhong group and western drug group decreased compared with the model group(P<0.01).(2)IHC results showed that compared with sham-operated group,the expression of Bcl-2 in CA1 area of hippocampus in model groupsignificantly decreased(P<0.01),and the expression of Cleaved Caspase-3 significantly increased(P<0.01);compared with model group,the expression of Bcl-2 in Yangzhong group,Yanggao group and western drug group increased((P<0.01),while the expression of Cleaved Caspase-3 in each treatment group decreased(P<0.01).(3)WB analysis results illustrated that,compared with the sham-operated group,Bax and Cleaved Caspase-3 protein expressions significantly increased(P<0.01),while Bcl-2 expression significantly reduced(P<0.01)in the model group.In comparison with the model group,the expression levels of Bcl-2 protein increased in each intervention group(P<0.05,P<0.01),and the expression levels of Bax and Cleaved Caspase-3 decreased in the Yanggao group and the western drug group(P<0.05,P<0.01).ConclusionYangxue-Peiben-Jiannao Recipe has a protective effect on the learning and memory disorders induced by A?25-35 in AD rats,and the mechanism might be related to the up-regulation of Bcl-2 expression,and the down-regulation of Bax,Cleaved Caspase-3 in the hippocampus and the A?25-35 content in serum of AD rats,implying that Yangxue-Peiben-Jiannao Recipe could suppress cell apoptosis in the hippocampus of AD rats.