Study On The In Vitro And In Vivo Mechanism Of Action Of Schisandra Chinensis(Turcz.) Baill-Acorus Tatarinowii Schott In The Treatment Of Alzheimer’s Disease

Objective:Alzheimer’s disease（Alzhermer’s disease（AD））is an underlying,progressive neurodegenerative disorder.Schisandra chinensis（Turcz.）Baill and Acorus tatarinowii Schott（Sc-At）are often used to treat neurological disorders,but their mechanisms for treating AD are unclear.In this study,the mechanism of action of Sc-At in vitro was investigated using metabolomics and bioinformatics.The mechanism of action of Sc-At in AD rats was also investigated and validated using untargeted metabolomics in combination with targeted metabolomics.Methods:（1）In this study,an in vitro model of AD was constructed using Aβ_25-35damaged SH-SY5Y cells,and the apoptosis rate was detected by Annexin V-FITC/PI fluorescent double-staining apoptosis kit.（2）The metabolic interference of Sc-At on Aβ_25-35damaged SH-SY5Y cells was analyzed by UPLC-QTOF/MS untargeted metabolomics method and screened for differential metabolites.（3）The pathways of action and potential targets of Sc-At exerting anti-AD effects were analyzed by network pharmacology.（4）AD pathology correlation analysis of potential targets using bioinformatics and simulation prediction of drug active ingredients to potential targets using molecular docking.（5）Pathway-based association analysis of differential metabolites with potential targets to speculate on possible mechanisms of action.（6）The AD rat model was constructed and the model was evaluated by H&E staining and related inflammatory indicators.（7）The metabolic interference of Sc-At on AD rat brain tissue was analyzed by UPLC-QTOF/MS untargeted metabolomics.（8）Quantification of testosterone and 17β-estradiol in brain tissue using UPLC-MS/MS targeted metabolomics.（9）ELISA for brain aromatase activity.Results:（1）10μM Aβ_25-35 acting on SH-SY5Y cells for 24h could construct an in vitro cell model of AD.（2）Sc-At can decrease the apoptosis rate of Aβ_25-35 injured SH-SY5Y cells in a concentration-dependent manner.（3）Sc-At can improve the metabolic disorder caused by Aβ_25-35 on SH-SY5Y cells.There were 68 differential metabolites between the model and blank groups,mainly involving the metabolism of sphingomyelin lipids,glycerophospholipid metabolism,and linoleic acid metabolic pathways.（4）Bioinformatics revealed that Sc-At may exert anti-AD effects through 54 targets,14 of which were associated with Aβand 7 with tau protein,and some targets were demonstrated to be differentialy expressed between the control and AD groups by the GEO dataset.Molecular docking predicted the best results for aromatase with the active ingredient.（5）Rats in the AD model group showed disordered neuronal arrangement and reduced cell numbers in the CA1 region of the hippocampus in H&E staining compared to the blank group.Also the expression of inflammatory factor TNF-αwas increased in the model group.There were 30 differential metabolites between the model group and the blank group,mainly involving linoleic acid metabolism,unsaturated fatty acid biosynthesis,glycerophospholipid metabolism,andα-linolenic acid metabolic pathway.（6）Compared with the blank group,the brain aromatase activity and 17β-estradiol content were reduced in the AD model group,while the aromatase activity and 17β-estradiol content were increased after the Sc-At intervention.Conclusion:The present study,in vivo,in vitro,combined with metabolomics and bioinformatics techniques,it was shown that Sc-At can improve the metabolic disorders caused by Aβ_25-35,and it was found that the anti-AD effect may be exerted by increasing the activity of brain aromatase in rats,which in turn increases the content of 17β-estradiol in rat brain tissue.