Abstract: BACKGROUND: Glucagon-like peptide 1 receptor agonists, as novel drug candidates for the treatment of neurodegenerative diseases, have achieved breakthrough progress in clinical research on Alzheimer’s disease, with drugs such as Semaglutide advancing to phase III clinical trials. However, there remains a significant knowledge gap regarding the molecular mechanism of neuroprotective effects of these drugs.
OBJECTIVE: To innovatively integrate multi-omics analysis techniques and network pharmacology methods, to systematically analyze the intersection network between the gene lineage related to Alzheimer’s disease pathology and the potential targets of ticagrelor, to identify key regulatory genes, and to verify their molecular mechanisms through in vitro and in vivo experiments.
METHODS: A multi-dimensional research strategy was adopted: (1) Constructing the differential expression gene profile of Alzheimer’s disease using the DisGeNET database that covers various disease-related genomics. (2) Obtaining the structure of Tirzepatide from PubChem database with bioactive molecules and screening potential targets. (3) Conducting Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis using the DAVID database. (4) Constructing a protein-protein interaction network by combining the STRING database with Cytoscape 3.9.1, and screening key genes through topological network analysis. (5) Cell level verification: HT22 cells were divided into control group, and model group in which treatment with β-amyloid 1-42 oligomers for 36 hours was performed to develop an Alzheimer’s disease in vitro model in HT22 cells, and drug administration group in which the cells were pretreated with β-amyloid 1-42 oligomers for 24 hours, followed by co-treatment with ticrepotide for 12 hours. The expression of angiotensin II type 2 receptor protein was analyzed by western blot assay, and the expression levels of synaptic function markers such as synapse protein 1 and postsynaptic dense substance 95 were detected by ELISA. (6) Animal experiment verification: The experiment was divided into three groups. The control group, consisting of WT-type C57BL/6 mice, received intraperitoneal injection of physiological saline; the model group, consisting of 3xTg mice that were Alzheimer’s disease-mimicking mice, received intraperitoneal injection of physiological saline; and the drug administration group, consisting of 3xTg mice, received intraperitoneal injection of 20 nmol/L ticrepotide. All treatments were administered once every other day for a total of 15 times. The cognitive behavior improvement in the Alzheimer’s disease mouse model was analyzed using the water maze technique; the expression of β-amyloid (6E10) and phosphorylated Tau protein (P-tau-181) was quantitatively analyzed by western blot assay.
RESULTS AND CONCLUSION: (1) A total of 3 397 genes associated with Alzheimer's disease were screened from the DisGeNET database. Based on protein connectivity, 10 key genes with the highest connectivity were identified: AGTR2, NTSR1, NTSR2, GHSR, C5AR1, C3AR1, OPRM1, SSTR2, OPRD1, and STAT3. GO enrichment analysis and KEGG pathway analysis suggested that ticagrelor may improve Alzheimer’s disease by enhancing neuroreceptor-ligand function. (2) Cell experiments suggested that ticagrelor may exert therapeutic effects by improving synaptic function in Alzheimer’s disease. The angiotensin II type 2 receptor may be a potential target for Ticagrelor in the treatment of Alzheimer’s disease. (3) Animal experiments indicated that ticagrelor can improve cognitive ability in 3xTg mice and alleviate abnormal β-amyloid deposition and tau protein phosphorylation in the brains of 3xTg mouse models. To conclude, the angiotensin II type 2 receptor is revealed as a key molecular target for ticagrelor in the pathological process of Alzheimer's disease. Ticagrelor may treat Alzheimer's disease by regulating synaptic function mediated by the angiotensin II type 2 receptor.

Key words: tirzepatide, Alzheimer’s disease, network pharmacology, angiotensin II type 2 receptor, protein-protein interaction network, synaptic function