Network pharmacological analysis and experimental validation of Anemarrhenae Rhizoma in treating Alzheimer 's disease / 浙江大学学报·医学版

ABSTRACT

OBJECTIVES@#To explore the mechanism of action of Anemarrhenae Rhizoma in treatment of Alzheimer's Disease (AD) through network pharmacology analysis and experimental validation.@*METHODS@#The active ingredients and targets of Anemarrhenae Rhizoma for treatment of AD were screened with network pharmacological methods, the protein-protein interaction (PPI) network was constructed and the core targets were analyzed, enriching Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis was performed. The peripheral blood lymphocytes were extracted and lymphoblast like cell lines (LCL) were constructed and an in vitro cell model of LCL-SKNMC was established. MTT/CCK-8 method was used to detect the activity of SKNMC/LCL cells, 2 ´, 7 ´-dichlorodihydrofluorescein diacetate (DCFH-DA) probe was used to detect reactive oxygen species (ROS) generated, and immunofluorescence staining was used to detect the generation of Aβ1-42 in the co-cultured model; Western blotting was used to detect protein expression in the co-culture model. The lifespan of N2 nematodes was observed under oxidative stress, normal state, and heat stress; ROS generated by N2 nematodes was detected by DCFH-DA probes. The paralysis time of CL4176 N2 nematodes was evaluated by paralysis assay, and Aβ deposition in the pharynx was detected by Thioflavin S (Th S) staining.@*RESULTS@#Through network pharmacology, 15 potential active ingredients and 103 drug-disease targets were screened out. PPI analysis showed that the Anemarrhenae Rhizoma might play anti-AD roles through albumin, Akt1, tumor necrosis factor, epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGFA), mammalian target of rapamycin (mTOR), amyloid precursor protein (APP), glycogen synthase kinas (GSK) 3β and other related targets. KEGG analysis showed that the pharmacological effects of Anemarrhenae Rhizoma might involve the biological processes of Alzheimer's disease, endocrine resistance, insulin resistance; and neuroactive ligand-receptor interaction, phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway, calcium signaling pathway, AGE-RAGE signaling pathway in diabetes complications, neurotrophic factor signaling pathway and others. The in vitro cell experiments showed that Anemarrhenae Rhizoma was able to reduce the production of ROS and Aβ1-42 (all P<0.01), inhibit the expression of β-secretase 1 (BACE1), APP and Aβ1-42 proteins (all P<0.05), up-regulate the expression of p-PI3K/PI3K, p-AKT/AKT, p-GSK3β/GSK3β in SKNMC cells (all P<0.05). Studies further confirmed that Anemarrhenae Rhizoma prolonged the lifespan of C. elegans under stress and normal conditions, reduced the accumulation of ROS and the toxicity of Aβ deposition.@*CONCLUSIONS@#Anemarrhenae Rhizoma may reduce the production of Aβ in AD and inhibit its induced oxidative stress, which may be achieved by regulating the PI3K/AKT/GSK-3β pathway.