Integrated proteomics and network pharmacology reveal autophagy-lysosomal regulation underlying sulforaphane-mediated neuroprotection in Alzheimer's disease.

INTRODUCTION: Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder characterized by amyloid-β (Aβ)-mediated neurotoxicity, impaired autophagy-lysosomal function, and neuronal apoptosis. Although sulforaphane (SFN) has demonstrated neuroprotective properties, its molecular actions against Aβ-related neuronal injury remain incompletely understood. This study investigated the neuroprotective effects of SFN against Aβ-induced neuronal injury and explored the underlying molecular networks associated with its anti-AD activity. MATERIALS AND METHODS: Integrated network pharmacology and proteomics approaches were complemented by experimental validation in retinoic acid-differentiated SH-SY5Y cells exposed to Aβ25-35. Cells were pretreated with SFN, which was withdrawn prior to Aβ exposure. Cell viability, apoptosis-related proteins (BAX/BCL-2), and autophagy-associated markers (LC3B and p62) were evaluated. Data-independent acquisition proteomics was performed to characterize global protein alterations. Target prioritization was conducted through integrated bioinformatics analysis and molecular docking, followed by western blot validation. RESULTS: Network pharmacology identified 331 overlapping targets between SFN and AD, mainly enriched in pathways related to apoptosis, autophagy, and oxidative stress. SFN pretreatment significantly attenuated Aβ-induced cytotoxicity and partially normalized apoptosis- and autophagy-related protein alterations, including reduction of the BAX/BCL-2 ratio, recovery of the LC3B-II/LC3B-I ratio, and decrease in p62 accumulation. Integrated proteomic and network pharmacology analyses identified 28 candidate targets, among which CTSB, CTSD, CASP6, and IGF1R were validated by molecular docking and western blotting. CONCLUSION: SFN pretreatment protected against Aβ-induced neuronal injury in this in vitro model and was associated with modulation of autophagy-lysosomal and apoptosis-related pathways. The integration of network pharmacology, proteomics, and experimental validation provides a systems-level framework for understanding the multitarget neuroprotective actions of SFN.