Osteoporosis (OP) is a systemic bone metabolic disease that results from an imbalance between bone formation and bone resorption. The accumulation of iron has been identified as an independent risk factor for osteoporosis. Ferroptosis, a novel form of programmed cell death, is driven by iron-dependent lipid peroxidation. Nevertheless, the precise role of ferroptosis in iron accumulation-induced osteoporosis remains uncertain. We utilized proteomics and ELISA to screen key regulatory molecules related to iron accumulation in osteoporosis populations. HE staining was used to assess osteocyte changes in Hamp knockout (KO) iron accumulation mouse models. Western Blot, qPCR, ALP staining, and Alizarin Red staining were employed to explore the effects of siRNA-mediated gene knockdown on osteogenic differentiation in the MC3T3 cell line. ELISA, micro-CT, von Kossa staining, toluidine blue staining, TRAP staining, and calcein analysis were used to study the bone phenotype of conditional gene knockout mice. RNA-seq, endoplasmic reticulum activity probes, transmission electron microscopy (TEM), Western Blot, co-immunoprecipitation (Co-IP), flow cytometry, and ChIP-seq were employed to investigate the regulatory mechanisms of the target gene in osteogenic differentiation. OVX and Hamp KO mice were used to establish osteoporosis models, and AAV-mediated overexpression was employed to explore the intervention effects of the target gene on osteoporosis. The experiments demonstrate that iron accumulation can lead to changes in COPB1 expression levels in bone tissue. Cellular and animal experiments revealed that COPB1 deficiency reduces the osteogenic ability of osteoblasts. Transcriptome analysis and phenotypic experiments revealed that COPB1 deficiency induces ferroptosis and endoplasmic reticulum stress in cells. Further investigation confirmed that COPB1 plays a key role in endoplasmic reticulum stress by inhibits SLC7A11 transcription via ATF6. This reduces cystine uptake, ultimately inducing ferroptosis. Overexpression of COPB1 can restore osteogenic function in both cells and mice. This study elucidated the essential role of COPB1 in maintaining bone homeostasis and highlights it as a potential therapeutic target for treating iron accumulation-related osteoporosis. Our data elucidate the critical role of COPB1 in maintaining bone homeostasis and demonstrate that COPB1 can directly promote bone formation, making it a potential therapeutic target for the future treatment of osteoporosis.

Background. The purpose of this study was to demonstrate the pharmacodynamic material basis and molecular mechanism of pilose antler (PA) in the prevention and treatment of osteoporosis (OP) by the method of network pharmacology. Methods. First, the active components of PA were screened by BATMAN-TCM database, and the component targets were obtained from the SwissTargetPrediction online tool. Moreover, the relevant target genes of OP were obtained by searching the DisGeNET database. Second, the Venn diagram was drawn to obtain the PA-OP common targets, and the protein-protein interaction (PPI) network and drug-component-target (D-C-T) network were constructed by Cytoscape software. Finally, the GO functional annotation and KEGG pathway enrichment analysis of common targets were performed using the Metascape online tool. Results. 82 common targets were identified by generating a Venn diagram. The PPI network of 82 common targets indicated that the top 5 nodal targets, including PIK3CA, MAPK1, ESR1, AKT1, and SRC, were strongly associated with other proteins. The D-C-T network suggested that the active components with high degree of connectivity include Prostaglandin E1, 17-Beta-Estradiol, Alpha-Estradiol, and Estrone. Furthermore, the GO enrichment analysis revealed that the biological process categories were dominated by response to peptide, cellular response to lipid, regulation of MAPK cascade, and so on. Additionally, the KEGG pathway analysis indicated the estrogen signaling pathway, osteoclast differentiation, and HIF-1 signaling pathway might have critical effects on the development of OP. Conclusion. The study shows that PA has the characteristics of multi-component, multi-target, and multi-pathway in treating osteoporosis.