Quantitative LFQ-DIA proteomics reveals FTH1-MCM5/WNT axis mediated osteoblastic dysfunction via ferroptosis drives diabetic osteoporosis.

In diabetes mellitus osteoporosis (DMOP), a common and severe chronic complication of diabetes mellitus (DM), long-term challenges are posed to public health. Recent evidence has implicated ferroptosis-a form of regulated cell death driven by iron-dependent lipid peroxidation-in the pathogenesis of DMOP. Ferritin heavy chain 1 (FTH1) plays a critical role in regulating iron metabolism during ferroptosis. To elucidate the regulatory mechanisms by which FTH1 modulates osteoblast (OB) ferroptosis and aberrant bone metabolism under high glucose and high fat (HGHF) conditions, we performed quantitative LFQ-DIA proteomics combined with bioinformatic analysis. MC3T3-E1 cells cultured under high glucose and high palmitic acid (HGPA) conditions were subjected to lentiviral-mediated FTH1 knock down (KD) or over expression (OE), and their protein expression profiles were systematically compared. We identified 857 differentially expressed proteins (DEPs) in the FTH1(KD)/HGPA group and 129 DEPs in the FTH1(OE)/HGPA group. Gene Ontology (GO) analysis revealed that, relative to HGPA/NC controls, DEPs in the KD/HGPA group were predominantly enriched in pyruvate biosynthesis and ADP metabolic processes, whereas DEPs in the OE/HGPA group were mainly associated with retinoid-like and glucuronate metabolic processes; in the HGPA/NC group, DEPs were enriched in cell-cell adhesion and regulation of inflammatory response. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that these DEPs are involved in glycolysis, glutathione metabolism, and ferroptosis pathways. Protein-protein interaction (PPI) network analysis further identified minichromosome maintenance complex component 5(MCM5) and glucose 6 phosphate isomerase (GPI) as top hub proteins in the KD group. Functional validation of MCM5-the highest-scoring node by MCODE (score = 18.0)-demonstrated that FTH1(KD) significantly upregulated MCM5 expression, whereas MCM5(KD) reduced OBs ferroptosis, enhanced osteogenic differentiation, and activated WNT signaling. These results suggest that MCM5 is a key mediator of OB differentiation under HGHF conditions. Collectively, our findings reveal that altered FTH1 expression under HGHF conditions reshapes the OB protein interaction network and identify MCM5 as a potential therapeutic target for DMOP.