Icariin targets Rho family GEF FARP2 to modulate GTPase signaling for neuroprotection

Background Oxidative stress is a primary driver of neuronal injury in neurodegenerative conditions including ischemia stroke (IS). Icariin (ICA), a natural flavonoid, exhibits neuroprotective activity. However, its direct molecular target remains elusive. Here, we identified the direct protein target of ICA and elucidated the downstream pathways mediating its protection against hydrogen peroxide (H 2 O 2 )-induced neuronal injury. Methods Neuronal B35 cells were treated with H 2 O 2 to induce oxidative stress. Cell viability was evaluated using CCK-8 assays and Calcein-AM/PI double staining. The direct binding target of ICA was screened by drug affinity responsive target stability-mass spectrometry (DARTS-MS) proteomics, followed by validation through molecular docking, molecular dynamics (MD) simulations as well as cellular thermal shift assay coupled with Western blot (CETSA-WB). RNA sequencing (RNA-seq) was performed to identify involved the signaling pathways. Network pharmacology and machine learning approaches including LASSO, random forest, and support vector machine, were applied to identify hub genes associated with IS. Finally, WB, phalloidin staining and siRNA-mediated FARP2 knockdown rescue experiments were performed to confirm the effects and mechanism of icariin in IS. Results ICA dose-dependently restored B35 cell viability following H 2 O 2 exposure. FARP2 was identified as a direct binding target of ICA. Molecular docking and MD stimulations validated stable ICA-FARP2 binding (ΔG_bind = −21.38 ± 2.19 kcal/mol) driven by van der Waals and electrostatic interactions with Thr914 and Gln907. Transcriptomic profiling indicated that H 2 O 2 exposure upregulated RhoGTPase signaling, extracellular matrix organization, and cilium assembly pathways. Integrated analyses further identified caspase-3 as a core regulatory gene and implicated Rap1 and PI3K-Akt signaling in IS. Finally, the functional validation confirms that FARP2 siRNA blocks the protective effect of icariin against H 2 O 2 -induced B35 cell injury. Conclusion: ICA exerts neuroprotective effects against H 2 O 2 -induced neuronal injury by targeting FARP2 and modulating RhoGTPase and PI3K-Akt signaling, suggesting its potential in regulating cytoskeletal dynamics and cellular stress responses.