Exploring the protective effect and molecular mechanism of betulin in Alzheimer's disease based on network pharmacology, molecular docking and experimental validation

Alzheimer's disease (AD) is a neurodegenerative disorder that impairs learning and memory, with high rates of mortality. Birch bark has been traditionally used in the treatment of various skin ailments. Betulin (BT) is a key compound of birch bark that exhibits diverse pharmacological benefits and therapeutic potential in AD. However, the therapeutic effects and molecular mechanisms of BT in AD remain unclear. The present study aimed to predict the potential therapeutic targets of BT in the treatment of AD, and to determine the specific underlying molecular mechanisms through network pharmacology analysis and experimental validation. PharmMapper was used to predict the target genes of BT, and four disease databases were searched to screen for AD targets. The intersection targets were identified using the jveen website. Drug-disease target protein-protein interaction networks and hub genes were obtained and visualized using the Search Tool for the Retrieval of Interacting Genes/Proteins database and Cytoscape. The Database for Annotation, Visualization and Integrated Discovery was used for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, and AutoDock was used for molecular docking analysis of BT and hub genes. Subsequently, the network-predicted mechanisms of BT in AD were verified in vitro. A total of 495 BT and 1,386 AD targets were identified, and 120 were identified as potential targets of BT in the treatment of AD. The results of the molecular docking analysis revealed a strong binding affinity between BT and the hub genes. In addition, enrichment analyses of GO and KEGG pathways indicated that the neuroprotective effects of BT mainly involved the 'PI3K-Akt signaling pathway'. The results of in vitro experiments demonstrated that pretreatment with BT for 2 h may ameliorate formaldehyde (FA)-induced cytotoxicity and morphological changes in HT22 cells, and decrease FA-induced Tau hyperphosphorylation and reactive oxygen species levels. Furthermore, the PI3K/AKT signaling pathway was activated and the expression levels of downstream proteins, namely GSK3 beta, Bcl-2 and Bax, were modified following pre-treatment with BT. Overall, the results of network pharmacology and in vitro analyses revealed that BT may reduce FA-induced AD-like pathology by modulating the PI3K/AKT signaling pathway, highlighting it as a potential multi-target drug for the treatment of AD.