Astragalin Regulates Autophagic Flux By Inhibiting PI3K/Akt-mTOR Pathway In Hippocampal Neurons Of AD Mice

Objective:To investigate the neuroprotective effect of astragalin（AST）on mouse hippocampal neurons of Alzheimer’s disease（AD）and the regulatory effect and mechanism of proteins involved in autophagic flux,and to provide theoretical support and experimental basis for the application of AST in the treatment of AD.Methods:In vivo experiments:（1）Animal grouping and administration:28 8-month-old APP/PS1 male mice were randomly divided into four groups,including model group（APP/PS1）,low-dose AST treatment group（10mg/kg AST+APP/PS1）,medium-dose AST treatment group（20 mg/kg AST+APP/PS1）,and high-dose AST treatment group（40 mg/kg AST+APP/PS1）,and C57BL/6 male mice were set as control group（WT）,there were 7 mice in each group.The animals in the AST-treated group were injected intraperitoneally with different doses of AST working solution,and the mice in the WT and APP/PS1 groups were given equal amount of PBS solution containing 1%DMSO intraperitoneally.（2）Behavioral test:Before and after administration,the spatial learning and memory capacity of mice was estimated by the Morris water maze test,and the learning and memory function of mice was assessed by the step-down passive avoidance test.（3）Hematoxylin and eosin staining（HE）staining and Nissl staining:Morphological and structural changes of hippocampal neurons in each group of mice were observed.（4）Thioflavin S（Th-S）and immunofluorescence double staining and ELISA assay:The amyloid-β（Aβ）plaques in mice brain and the levels of Aβand Aβ₄₂ in mice serum were detected.（5）Multiplex immunofluorescence staining and Western Blot:multiplex immunofluorescence staining was performed to observe the co-expression of autophagic flux-related proteins LC3B,p62,Beclin-1,ATG5,ATG12,LAMP-1 and Neu N in the hippocampus of mice,and Western Blot was used to detect specific changes in the mice hippocampus of LC3B,p62,Beclin-1,ATG5,ATG12 and LAMP-1 proteins involved in autophagic flux-related proteins.（6）HE staining of liver and kidney tissues:The morphological structure of liver and kidney tissues in each group of mice were observed.In vitro experiments:（1）The mouse hippocampal neuronal cell line（HT22）cells were treated with 0μM～160μM Aβ_25-35/AST,and the effect of AST on cell proliferation after Aβ_25-35 damage on HT22 cells was detected by CCK8 assay.（2）Immunofluorescence staining and Western Blot:The changes in the expression levels of autophagic flux-related proteins LC3B,p62,Beclin-1 and LAMP-1 involved in HT22 cells were detected.（3）Western Blot:The protein levels of PI3K,p-PI3K,Akt,p-Akt,m TOR and p-m TOR in HT22 cells were detected.Results:In vivo experiments:（1）Behavioral tests:the Morris water maze test:The escape latency of APP/PS1 mice was actually increased（P<0.001）,the time to reach the original target platform quadrant was virtually prolonged（P<0.001）and the number of times to cross the target platform quadrant was virtually increased（P<0.001）within 1 min.After AST treatment,APP/PS1 mice showed a decrease in escape latency（P<0.01）,a decrease in the time to reach the original target platform quadrant（P<0.05）and a decline in the number of times to cross the target platform quadrant（P<0.01）.The step-down passive avoidance test:APP/PS1 mice had a shorter step-down latency（P<0.001）and made more errors（P<0.001）.After AST treatment,the latency of step-down in APP/PS1 mice was actually increased（P<0.01）and the number of errors made by APP/PS1 was virtually decreased（P<0.01）.（2）HE staining and Nissl staining:the morphological structure of hippocampal neurons in WT group was intact,while the hippocampal neurons were sparsely arranged and the Nissl staining percentage was reduced（P<0.01）in APP/PS1 mice.After AST treatment,the hippocampal neurons were neatly arranged and clearly structured,and the Nissl staining percentage was increased（P<0.05）in APP/PS1 mice.（3）Th-S and immunofluorescence double staining and ELISA assay:The deposition of Aβplaques in the brain of APP/PS1 group was virtually expanded（P<0.001）,and after AST treatment,the deposition of Aβplaques was virtually decreased（P<0.01）.Compared with WT group,the levels of Aβand Aβ₄₂ in the serum were largely increased（P<0.001）,while the serum levels of Aβand Aβ₄₂ were reduced（P<0.05）after AST treatment.（4）Multiplex immunofluorescence staining and Western Blot:The co-expression of LC3B,p62,Beclin-1,ATG5,ATG12,LAMP-1 and Neu N was present in the hippocampus of all groups of mice.The expression levels of LC3B,Beclin-1,ATG5,ATG12 and LAMP-1 were significantly decreased in the hippocampus of APP/PS1 mice（all P<0.01）,and the level of p62 was virtually increased（P<0.01）.After AST treatment,the protein levels of LC3B,Beclin-1,ATG5,ATG12 and LAMP-1 were increased（all P<0.05）,and p62 level was decreased（P<0.05）.（5）HE staining of liver and kidney tissues:The hepatocytes in each group were neatly arranged,and the liver lobules were complete and clear.The glomerular epithelial cells in each group of mice had normal morphology,and the glomeruli and tubules were structurally intact.In vitro experiments:（1）The cell viability of HT22 cells gradually decreased,and the IC₅₀ was 20.44μM in Aβ_25-35-injured HT22 cells.After treated with AST for 24 h,the viability of HT22 cells was maintained at about 90%.The viability of HT22 cells damaged by Aβ_25-35 pretreated with40μM AST was（97.17±2.50）%.（2）Immunofluorescence staining and Western Blot:LC3B,Beclin-1and LAMP-1 levels were significantly decreased（all P<0.01）and p62level were virtually increased（P<0.01）in Aβ_25-35-injured HT22 cells,and the levels of LC3B,Beclin-1 and LAMP-1 were significantly increased（all P<0.01）and p62 level were actually decreased（P<0.01）in HT22 cells treated with Donepezil（DNP）+Aβ_25-35 and AST+Aβ_25-35.The levels of LC3B,Beclin-1 and LAMP-1 were significantly decreased（all P<0.01）,and the level of p62 was significantly increased（P<0.01）in 3-MA+AST+Aβ_25-35 group.（3）Western Blot:The ratios of p-PI3K/PI3K,p-Akt/Akt,and p-m TOR/m TOR were significantly increased in Aβ_25-35-injured HT22 cells（all P<0.01）.The ratios of p-PI3K/PI3K,p-Akt/Akt,and p-m TOR/m TOR were significantly decreased in DNP+Aβ_25-35 and AST+Aβ_25-35 groups（all P<0.01）,and the ratios of p-PI3K/PI3K,p-Akt/Akt,and p-m TOR/m TOR were significantly increased in HT22 cells of the 3-MA+AST+Aβ_25-35 group（all P<0.01）.Conclusion:AST could exert effective neuroprotective effect on cognitive impairment in APP/PS1 mice,and enhance autophagic flux in hippocampal neurons of APP/PS1 mice,and this mechanism might be related to the inhibition of PI3K/Akt-m TOR pathway.