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Iron accumulation is an independent risk factor for postmenopausal osteoporosis, but mechanistic studies of this phenomenon are still focusing on molecular and genetic researches in model animal. Osteoporosis with iron accumulation is a distinct endocrine disease with complicated pathogenesis regulated by several proteins. However, the comprehensive proteome-wide analysis of human bone is lacking. Using multiplex quantitative tandem mass tag-based proteomics, we detected 2900 and quantified 1150 proteins from bone of 10 postmenopausal patients undergoing hip replacement. Comparing with non-osteoporosis patients, a total of 75 differentially expressed proteins were identified, comprising 53 downregulated proteins and 22 upregulated proteins. These proteins primarily affect oxidoreductase activity, GTPase activity, GTP binding, and neural nucleus development, were mainly enriched in neural, angiogenesis and energy-related pathways, and formed complex regulatory networks with strong interconnections. We ultimately identified 4 core proteins (GSTP1, LAMP2, COPB1, RAB5B) that were significantly differentially expressed in the bone of osteoporosis patients with iron accumulation, and validated the changed protein level in the serum of the medical examination population. Our systemic analysis uncovers molecular insights for revealing underlying mechanism and clinical therapeutics in osteoporosis with iron accumulation.

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Objective Iron plays a significant role in multiple biological processes. The purpose of this study was to measure whether iron mediated osteoclast differentiation through regulation of triggering receptor expressed in myeloid cells‐2 (Trem‐2) expression and the PI3K/Akt signaling pathway. Methods The effects of six different concentrations of ferric ammonium citrate (FAC) (100, 80, 40, 20, 10 and 0 μmol/L) on RAW 264.7 cells proliferation were assessed by Cell Counting Kit‐8 (CCK‐8) gassay. Tartrate resistant acid phosphatase (TRAP) assay was performed to detect the effects of FAC on osteoclast formation. The expression of osteoclast differentiation‐related (TRAP, NFATc‐1, and c‐Fos) and Trem‐2 mRNA and proteins was analyzed by reverse transcription‐polymerase chain reaction and western blot, respectively. Si‐Trem‐2 was constructed and transfected to RAW264.7 to measure the effects of Trem‐2 on FAC‐mediated osteoclast formation. TRAP assay and osteoclast differentiation‐related gene analyses were further performed to identify the role of Trem‐2 in osteoclastogenesis. The Search Tool for the Retrieval of Interacting Genes (STRING) was used to explore the target genes of Trem‐2. Trem‐2‐related gene ontology and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway were used for further in‐depth analysis. PI3K/Akt pathway‐related proteins were detected by immunofluorescence and western blot. Results In groups with FAC concentration of 10 (102.5 ± 3.1), 20 (100.5 ± 1.5), and 40 μmol/L (98.7 ± 3.1), compared with the control group (100.1 ± 2.2), cell viability was not significantly different from the control (P > 0.05). When the concentration of FAC exceeded 80 μmol/L, cell viability was significantly decreased (87.5 ± 2.8 vs 100.1 ± 2.2, P < 0.05). FAC promotes Trem‐2 expression and osteoclast differentiation in a dose‐response manner (P < 0.05). The number of osteoclast‐like cells was found to be reduced following transfection with the siRNA of Trem‐2 (42 ± 3 vs 30 ± 5, P < 0.05). We observed that most of Trem‐2 target genes are primarily involved in response to organic substance, regulation of reactive oxygen species metabolic process, and regulation of protein phosphorylation. The STRING database revealed that Trem‐2 directly target two gene nodes (Pik3ca and Pik3r1), which are key transcriptional cofactors of the PI3K/Akt signaling pathway. KEGG pathways include the “PI3K‐Akt signaling pathway,” the “thyroid hormone signaling pathway”, “prostate cancer,” the “longevity regulating pathway,” and “insulin resistance.” Expression of p‐PI3K and p‐Akt protein, measured by immunofluorescence and western blotting, was markedly increased in the FAC groups. Trem‐2 siRNA caused partial reduction of these two proteins (p‐PI3K and p‐Akt) compared to the FAC alone group. Conclusion The FAC promoted osteoclast differentiation through the Trem‐2‐mediated PI3K/Akt signaling pathway. However, its regulation osteoclastogenesis should be verified through further in vivo studies.

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.