The Gut-Brain Axis in Alzheimer’s: From Microbiota Genetics to Stigmasterol’s Neuroprotection Mechanism

Tunan Ding,1,2,* Junlei Chen,1,3,* Yunsheng Xiang,2 Xiaojie Zhou,3 Hongbo Zheng,3 Yihan Bai,1 Weihao Wang,3 Qiang Fu,1 Yan Chen,4 Yin Fu1 1School of Basic Medical Sciences, Heilongjiang University of Chinese Medicine, Harbin, Heilongjiang, 150040, People’s Republic of China; 2School of Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing, 102401, People’s Republic of China; 3First Clinical Medical College of Heilongjiang University of Chinese Medicine, Harbin, Heilongjiang, 150006, People’s Republic of China; 4Beijing Hospital of Integrated Traditional Chinese and Western Medicine, Beijing, 100089, People’s Republic of China*These authors contributed equally to this workCorrespondence: Qiang Fu, School of Basic Medical Sciences, Heilongjiang University of Chinese Medicine, Harbin, 150006, People’s Republic of China, Email [email protected] Yin Fu, School of Basic Medical Sciences, Heilongjiang University of Chinese Medicine, Harbin, 150006, People’s Republic of China, Email [email protected]: 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 Aβ1-42 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 Aβ1-42 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.Keywords: intestinal flora, Alzheimer’s disease, mendelian randomization, bioinformatic analysis, network pharmacology, stigmasterol