Integrated Multi‐Omics and Single‐Cell Analyses Implicate NOX4 as an Icariin‐Associated Oxidative Stress Regulatory Node in Gastric Cancer

ABSTRACT Icariin, a prenylated flavonoid glycoside from Epimedium species, is a representative dietary polyphenol with antioxidant, anti‐inflammatory, and antitumor properties. Gastric cancer (GC) is a major non‐communicable chronic disease characterized by complex molecular alterations and dysregulated redox homeostasis, yet the molecular targets underlying the potential anti‐GC activity of icariin remain poorly defined. In this study, we integrated network pharmacology, bulk transcriptomic analysis (TCGA‐STAD; GSE15497, GSE84437, GSE84433, and GSE26253), and single‐cell RNA sequencing (GSE183904; 142,053 cells) to identify candidate icariin‐associated genes in GC. NOX4 expression, prognostic value, and tumor microenvironment characteristics were systematically evaluated. A NOX4‐positive endothelial cell (NOX4pos_Endo)‐associated molecular subtyping strategy and a LASSO‐Cox prognostic model were constructed and validated. Molecular docking and molecular dynamics simulation were performed to explore a potential interaction between icariin and NOX4, and AGS cells were used for in vitro validation. Network pharmacology identified 24 overlapping icariin–GC targets, among which NOX4 showed prominent prognostic relevance. NOX4 was upregulated in GC and associated with unfavorable prognosis across multiple cohorts. At the single‐cell level, NOX4 was predominantly expressed in an endothelial subpopulation characterized by angiogenesis‐, adhesion‐, and extracellular matrix remodeling‐related transcriptional features. Consensus clustering identified two molecular subtypes with distinct clinical outcomes and biological characteristics. A 5‐gene prognostic model comprising SPIRE1, PLCH1, KCNS3, SLC27A2, and GRP showed moderate predictive performance across independent cohorts. Molecular docking suggested a potential icariin–NOX4 interaction involving Ser576 and Trp377, while molecular dynamics simulation supported the stability of the predicted binding pose. In AGS cells, icariin treatment was associated with reduced NOX4 expression and intracellular ROS levels. Overall, these findings identify NOX4 as a candidate icariin‐associated regulatory node in GC and suggest that NOX4/ROS‐related signaling may contribute to the cellular response to icariin. Further experimental studies are required to determine whether NOX4 is a direct molecular target of icariin and to clarify its therapeutic relevance.