Exploring the Role of Gut Microbiome-Derived Urolithin A in Alzheimer's Disease via Novel Target Proteins: An Integrative Study

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by cognitive impairment and hallmark neuropathological changes. Current treatments are inadequate, necessitating novel therapeutic approaches. This study utilized integrated of network pharmacology, bioinformatics, and in vitro to identify key proteins associated with AD and relevant gut microbial metabolites. Initially, we employed the network pharmacology and bioinformatics to identify 19 target proteins, and 17 gut microbial metabolites associated with AD. Therefore, molecular docking, density functional theory (DFT), MM-GBSA, dynamic simulations, principal component analysis (PCA), and dynamic cross-correlation matrix (DCCM) analyses assessed the binding affinity, stability, and interactions of protein-ligand complexes. Among the evaluated gut metabolites Urolithin A (Uro.A) demonstrated the strongest binding energy, stability, and correlation with catalase (CAT), interleukin-4 (IL-4), cathepsin D (CTSD), and insulin receptor substrate-1 (IRS1). Furthermore, the neuroprotective potential of gut metabolite Uro.A was validated through in vitro experiments using human neuroblastoma SH-SY5Y cells. The cytotoxicity assays showed no toxic effects of Uro.A. Therefore, pretreatment with Uro.A significantly improved cell viability in amyloid-beta (Aβ)-exposed cells. Moreover, qRT-PCR results showed that Aβ treatment upregulation of CAT, CTSD, and IL4 genes, while IRS1 expression was downregulated compared to the control group. However, pretreatment with Uro.A significantly decreased the mRNA expression of CAT, CTSD, IL4, and IRS1, while IRS1 expression exhibited a negative correlation in Aβ-induced cells, suggesting a potential neuroprotective effect. Additionally, in silico analysis revealed consistent overexpression of CAT and IRS1 across regions and region-specific patterns for CTSD and IL4 in AD brains. Finally, enrichment analyses identified pathways related to longevity, FoxO, autophagy, PI3K-Akt, and oxidative stress; these play critical roles in regulating AD pathology by targeting these proteins. In conclusion, these preliminary integrated findings suggest gut metabolite Uro.A may have neuroprotective effects against AD to targeting the CAT, CTSD, and IL4. Further in vivo investigation is necessary to validate Uro.A's therapeutic potential in AD via these proteins.