Objective: This study aimed to identify novel therapeutic targets for Alzheimer’s disease (AD) by investigating the role of the intestinal flora (IF) via the gut-brain axis, and to predict a potential natural compound for AD treatment and elucidate its underlying mechanism. Methods: Following a primary analytical axis, we first employed Mendelian randomization (MR) to infer causal relationships between gut microbiota and AD. To pinpoint molecular targets, we integrated Summary-data-based MR (SMR) with single-cell and spatial transcriptomics. Subsequently, network pharmacology and molecular docking were used to identify stigmasterol as a candidate compound targeting the causal pathway. Finally, the neuroprotective effects and the STIM1/Orai1-mediated mechanism were experimentally validated in vitro using Aβ_1-42 exposed SH-SY5Y cells. Results: MR-based causal inference identified Desulfovibrio as a risk factor for AD, while Slackia and the Lachnospiraceae NK4A136 group were protective factors. Seven key AD-related genes were identified by combining MR results with databases, which were highly druggable. SMR analysis and multi-omics integration pinpointed STIM1-mediated calcium signaling as the core causal pathway. Following the identification of stigmasterol via network pharmacology and molecular docking, in vitro experimental validation confirmed that stigmasterol significantly inhibited Aβ_1-42 induced neuronal apoptosis and calcium overload by specifically modulating the STIM1/Orai1 pathway and the Bcl-2/Bax ratio. Conclusion: This study decodes the gut-brain axis by establishing the specific causal pathway. We demonstrate that Stigmasterol exerts neuroprotective effects by inhibiting apoptosis through a IF-associated mechanism involving the STIM1/Orai1 pathway, provideing novel insights into AD pathogenesis and offering a promising therapeutic strategy based on natural compounds.

This study aimed to identify biomarkers associated with MIRI and elucidate their potential mechanisms through integrated bioinformatics and experimental va