摘要Icariin is a natural product that possesses numerous pharmaceutical properties.Thus,this study aimed to investigate the molecular mechanism by which icariin prevents ferroptosis in aged-related osteoporosis.Firstly,mRNA transcriptomics was used to analyze differentially expressed genes and ferroptosis markers.Next,a weighted correlation network analysis was conducted on these genes.Then,common genes among ferroptosis,Yinyanghuo,and differentially expressed genes were identified as target genes.Single-cell and spatial transcriptomics were analyzed to evaluate expression changes of target genes in bone marrow.Furthermore,to validate the results of bioinformatics analysis,MC3T3-E1 cells were used to model the preventive effects of icariin in vitro,and ferroptosis markers and mitochondrial function were both examined.In vivo,4-month-old mice were fed a diet containing icariin for 14 months,following which bone proteomics was assessed to identify essential proteins.Hematoxylin-eosin staining and immunohistochemistry assays were performed on mouse femurs.Finally,Western blot and polymerase chain reaction(PCR)analyses were performed to analyze the effects of icariin on ferroptosis target biomarkers.Ptgs2 and Hmox1 were identified as target genes related to both icariin and ferroptosis.The expression of ferroptosis-related genes was up-regulated with age.Moreover,single-cell and spatial transcriptomics analyses revealed that up-regulation of these two genes inhibited osteogenic capability.The results of the in vitro experiments indicated that icariin mitigated the accumulation of reactive oxygen species and β-galactosidase.Similarly,icariin prevented aberrant changes in the levels of ferroptosis-related proteins,consistent with the results of the in vivo experiments.Specifically,10 mg/(kg·day)of icariin inhibited ferroptosis and osteoporosis in aged mice.Overall,this study revealed that icariin could serve as a dietary supplement to prevent age-induced osteoporosis.Furthermore,Ptgs2 and Hmox1 were identified as ferroptosis-related targets through which icariin exerts its protective effects.