Integrating network pharmacology and experimental validation to decipher the protective effect of icariin against testicular aging

During male reproductive aging, levels of reactive oxygen species (ROS) increase progressively in the testes. Excessive ROS directly reduce testosterone synthesis and impair spermatogenesis in the testes, inducing male reproductive senescence. As a bioactive monomer derived from traditional Chinese medicine, icariin (ICA) exerts protective effects against ROS after exposure to environmental toxicants, including di(2-ethylhexyl) phthalate (DEHP). In the present study, we aimed to explore the therapeutic effects of ICA on male reproductive senescence. In particular, we examined the protective effects of icariin in aged murine models through oral gavage and elucidated its molecular mechanisms via integrated network pharmacology, molecular docking, and molecular dynamics simulation approaches. We identified 125 potential targets of icariin in testicular aging. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses indicated that these targets are associated with ROS scavenging, lipid storage regulation, androgen metabolism, and ferroptosis as well as cellular senescence and PI3K/AKT signaling pathways. The top five hub targets (ESR1, TP53, BCL2, HIF1A, and TNF) demonstrated high binding potential with icariin, as supported by molecular docking and molecular dynamics simulations. Furthermore, in vivo experiments showed that icariin treatment reduces ROS, malondialdehyde, and Fe2 + levels significantly in aging testes. Icariin activated the ESR1/AKT pathway, thereby restoring testosterone synthesis and mitigating cellular senescence in 12-month-old mice. These findings demonstrate the critical role of icariin in ameliorating testicular aging and provide a foundation for detailed analyses of its molecular mechanisms.