Study On The Protective Effect Of Verbascoside Against Alzheimer’s Disease

Alzheimer’s disease（AD）is a latent onset and progressive neurodegenerative disease which is related to aging.It is clinically incurable,and it will eventually manifest itself as dementia.The pathological features of AD in the brain are the insoluble fibrils and plaques which are formed by the soluble oligomers that are spontaneously aggregated by monomer Aβ,neurofibrillary tangles（NFTs）deposits which are formed by phosphorylation tau protein,malnutrition neuritis and nerve fiber filaments.Although the exact pathogenesis of the disease is unknown,it has been shown that Aβcan mediate the cytotoxicity of neurons,eventually leading to the loss and degeneration of a large number of neurons.Endoplasmic reticulum（ER）,one of the largest organelles,is responsible for signal transduction,protein modification and processing,and the transport of substances from the nucleus to cytoplasm,cell membrane,and extracellular.Therefore,ER homeostasis is essential for good health.Aβaggregation causes neuronal endoplasmic reticulum stress（ERS）,which in turn triggers neuronal apoptosis.ERS also up-regulates the expression and secretion of mesencephalic astrocyte-derived neurotrophic factor（MANF）,which can regulate ERS and reduce the toxicity of Aβto neurons.Verbascoside（VB）,also known as Acteoside,is a phenylpropanoid glycoside extracted from the dried scaly succulent stems of Cistanche deserticola Y.C.Ma.It has been reported that VB has anti-apoptotic,antioxidant and neuroprotective effects,but the specific mechanism of VB against AD is still unclear.In this study,the purity of VB is98.38%,purchasing from Chengdu Herbpurify CO.,LTD..It was used as the raw material to clarify the anti-AD effect and pharmacological mechanism through cell experiments and animal experiments.The main experimental methods and results are as follows:In this paper,the effects of VB on U251 cells under ERS state which were induced by Aβ_1-42 were investigated.The MTT method and flow cytometry（FCM）were used to investigate the effect of VB on the viability and apoptosis of U251 cells.DCFH-DA and Fluo-4 AM fluorescent probe staining were used to detect the levels of ROS and Ca²⁺in U251 cells.The morphological changes of ER and mitochondria in U251 cells were observed by transmission electron microscopy（TEM）.At the same time,the levels of ERS-related proteins in U251 cells were determined by Western blot.The experimental results showed that VB can effectively increase the viability of U251 cells while not producing cytotoxicity.VB could control the overload of ROS and Ca²⁺,reduce the damage of ER and mitochondrial,and reduce the expression of ERS-related proteins（such as p-IRE1,p-PERK,ATF6,etc.）,thereby acting on U251 cells protection.APPswe/PSEN1d E9（APP/PS1）mice were selected as research objects to investigate the effect of VB on the behavioral,physiological and biochemical indexes.After 30 days gavage administration of VB,mice were tested by open field test（OFT）and Morris water maze（MWM）.After 42 days gavage administration,the tissue sections of mice were prepared,the proteomic assays of hippocampus were measured,the levels of ERS and mitochondrial apoptosis-related protein in mice brain tissue homogenate and serum were detected by ELISA kits,and the levels of ERS-related protein in brain tissue were measured by Western blot.The results showed that VB was nontoxic to the liver,spleen and kidney of mice.It could significantly improve the memory and cognitive functions,inhibit neuronal apoptosis,reduce Aβdeposition and NFTs which are formed by highly phosphorylated tau protein,and low the expressions of 4-HNE and MANF in the brain of APP/PS1 mice.Moreover,proteomics results suggested that the anti-AD effect of VB may be associated with ERS.The results of ELISA and Western blot showed that VB could down regulate ERS-related factors in the brain and serum of APP/PS1mice,including MANF,BiP,ATF6,p-IRE1/IRE1,XBP1s,p-PERK/PERK,p-eIF2α/eIF2α,ATF4,CHOP,and caspase12.It also showed that VB could down-regulate the expression of mitochondrial apoptosis-related factors.The results of ELISA also showed that VB could down-regulate the expression of mitochondrial apoptosis-related factors.In conclusion,cell and animal experiments showed that VB has significant neuroprotective effects,which may be achieved by reducing ERS.The findings of this study provide new ideas and potential effective therapeutic targets for the clinical treatment of AD.