The Effect Of Dihydromyricetin On Alzheimer’s Disease By Activating Neuronal Autophagy

Objective: Alzheimer’s disease is a neurodegenerative disease closely related to age.Autophagy disorder may be one of the important causes of AD.This article aims to investigate whether dihydromyricetin can protect or treat Alzheimer’s disease mouse models and cell models by affecting autophagy,and explore the potential mechanism.Methods: In animal experiments,7-month-old APP/PS1 transgenic dementia model mice were randomly divided into AD control group and DHM treatment group.7-month-old wild-type C57BL/6J mice were used as WT normal control group,each group was 10 only.The mice in the DHM treatment group were intraperitoneally injected with DHM at a dose of 1 mg/(kg·d),and the mice in the AD control group and the WT normal control group were injected with an equal amount of DMSO solution diluted with normal saline for 28 days.In the cell experiment,human neuroblastoma cell line SH-SY5 Y cells and APP-SH-SY5 Y cells stably transfecting APP gene were selected as cell models,and APP-SH-SY5 Y after treatment with DHM at a concentration of 40 μmol/L for 24 h cells are the DHM-APP group,SH-SY5 Y cells treated with the same concentration of DMSO dilution for 24 h are the CON group,and APP-SH-SY5 Y cells are the APP group.Morris water maze test was used to detect the learning and memory ability of mice in each group;immunohistochemical staining and thioflavin S staining were used to detect the number of senile plaques in the cortex and hippocampus of mice,and Elisa method was used to detect the expression of Aβ in the brain;Detection of autophagy effector protein Beclin-1,autophagy marker LC3,autophagy fusion protein Rab7,autophagy-related protein P62,lysosomal associated membrane protein LAMP1 and cathepsin D in mouse brain and cells.The level of transmission electron microscopy to detect the ultramicroscopic changes of autophagosomes,autophagolysosomes and other autophagy-related structures in the brain and cells of mice.The dual-fluorescence mRFP-eGFP-LC3 virus was used to infect the cells of each group to trace the formation and degradation of autophagosomes,and the inhibitors 3MA,Bafilomycin,CQ group and agonist Rapamycin group were established as controls.Results: The water maze showed that the average escape latency and escape length of the platform found by the DHM group was significantly shortened,the direction of the movement trajectory was clear,and the number of times of crossing the platform was significantly increased.Immunohistochemical staining and thioflavin S staining showed that the number and area of senile plaques in the brain of the DHM group were reduced compared with the AD group.Elisa results showed that the expression of Aβ40 and Aβ42 in the brain of the DHM group was higher than that in the AD group Significantly reduced.WB results showed that in animal models,there was no significant change in Beclin1 in AD group,the ratio of LC3-Ⅱ/LC3-Ⅰ decreased,and the expression of P62,LAMP1,and Cathepsin D protein increased.In the DHM group,the ratio of Beclin1,LC3-Ⅱ/LC3-Ⅰ increased,and the expression of P62,LAMP1,and Cathepsin D protein decreased.In the cell model,the expression of Rab7 protein in the APP group increased,and the expression of Rab7 protein in the DHM+APP group decreased.The results of other protein expressions were basically consistent with the animal models.Transmission electron microscopy results showed that in animal models,AD organs had severe edema in the organelles in the brain,and deep-stained late autophagosomes were found in many places.In the DHM group,organelle edema in the brain was significantly alleviated,and normal autophagolysosomes were found in many places.In the cell model,a large amount of autophagolysosomes accumulated in the APP group,and lipid droplets appeared.Early monolayer autophagosomes and mature autophagosomes were found in the DHM+APP group.The 3-MA group blocked the activity of PI3 K in the early stage of autophagy,and no autophagosomes were observed.The Bafilomycin group inhibited the fusion of autophagosomes and lysosomes in the middle of the autophagy process,and a large number of deep-stained late autophagosomes appeared.The CQ group inhibited the degradation of lysosomes,and the accumulation of late autophagosomes appeared.The Rapamycin group acted as an agonist in the process of autophagy,a large number of autophagosomes appeared in the cells,and even pseudo-inclusion bodies in the nucleus were found.The results of dual fluorescent protein labeling showed that the green and red fluorescent spots of the DHM+APP group increased.After the image fusion,the yellow fluorescent spots(autophagosomes)and red fluorescent spots(autophagolysosomes)increased.No green fluorescent dots were found in the 3-MA group.The Bafilomycin group pictures were mostly yellow fluorescent spots after fusion,and were distributed at the far end of the nucleus.After the CQ group pictures were fused,the yellow and red fluorescent spots were located near the nucleus,and Increased red fluorescent dots.Conclusion: DHM can accelerate the fusion of autophagosomes and lysosomes,promote the degradation of autophagolysosomes,restore their autophagic function,reduce Aβ precipitation in the brain,reduce the pathological characteristics of AD mice,and thus improve their cognitive function,That is,DHM induces autophagic degradation to effectively prevent the progression of AD.