Network pharmacology, bioinformatics and in vitro/in vivo validation elucidate the anti-lung cancer activities and potential targets of Rhoifolin.

Background: Rhoifolin (ROF), a naturally occurring flavonoid, exhibits broad bioactivities, but its therapeutic potential and underlying mechanisms in lung cancer remain largely unknown. This study was designed to systematically investigate the anti-tumor effects of ROF and identify its key molecular targets. Materials and methods: Anti-tumor activities of ROF were assessed using CCK-8, colony formation, flow cytometry, wound healing, and Transwell assays, respectively. An integrated approach combining network pharmacology, transcriptomic analysis with machine learning was employed to identify primary targets. The Kaplan-Meier survival and ROC curve analyses also evaluated the targets' clinical outcomes and tumor microenvironment through the Cancer Genome Atlas (TCGA) data and single-cell RNA sequencing. The confirmed experimentally via RT-qPCR, Western blot, and immunofluorescence. The drug-target interaction was characterized by molecular docking and dynamics simulations. Finally, the Results: ROF potently inhibited lung cancer cell proliferation (IC Conclusion: Rhoifolin exerts potent and selective anti-lung cancer activity by directly targeting and downregulating EPHB2, providing a strong rationale for its further development as a novel therapeutic agent for lung cancer.