Endothelial cell-derived exosomes inhibit high glucose-induced osteoblast ferroptosis by activating microRNA-335-3p/prostaglandin endoperoxide synthase 2.

BACKGROUND: Diabetic osteoporosis (DOP), a serious complication of type 2 diabetes mellitus (T2DM), involves ferroptosis-mediated disruption of bone metabolism. While endothelial cell-derived exosomes (EC-Exos) demonstrate inherent bone-targeting properties, their role in counteracting high glucose (HG)-induced osteoblast ferroptosis remains unexplored. AIM: To investigate whether EC-Exos protect against HG-induced osteoblast ferroptosis through microRNA (miR)-335-3p-mediated regulation of prostaglandin endoperoxide synthase 2 (PTGS2) and evaluate clinical relevance in DOP. METHODS: Mouse vascular endothelial cells (bEND.3) and osteoblasts (MC3T3E1) were used. Exosomes were isolated and subsequently characterized by transmission electron microscopy, nanoparticle tracking analysis, and western blotting for CD63 and CD81. miR expression profiles were compared between HG-treated osteoblasts and exosome-cocultured groups using high-throughput sequencing and quantitative reverse transcription polymerase chain reaction. Targeting of PTGS2 mRNA by miR-335-3p was validated by dual-luciferase reporter assay. Ferroptosis markers, reactive oxygen species, malondialdehyde, glutathione (GSH), PTGS2, GSH peroxidase 4, solute carrier family 7 member 11, and solute carrier family 3 member 2, were quantified following miR-335-3p inhibition. Serum samples from 30 T2DM patients and 32 DOP patients were analyzed. miR-335-3p levels were measured by quantitative reverse transcription polymerase chain reaction, and PTGS2 concentrations were determined via enzyme-linked immunosorbent assay. Diagnostic performance was assessed using receiver operating characteristic curves and logistic regression. RESULTS: EC-Exos significantly reduced reactive oxygen species levels and malondialdehyde, while increasing GSH in HG-treated osteoblasts. miR-335-3p expression increased 3.7-fold in exosome-treated cells vs HG controls. miR-335-3p directly bound the PTGS2 3' untranslated region. Inhibition of miR-335-3p abolished exosomal protection against ferroptosis, as demonstrated by increased PTGS2 expression and reduced levels of GSH peroxidase 4, solute carrier family 7 member 11, and solute carrier family 3 member 2. DOP patients exhibited lower serum miR-335-3p and higher PTGS2 compared with T2DM controls, showing a strong inverse correlation. miR-335-3p demonstrated diagnostic potential for DOP. CONCLUSION: EC-Exos affect ferroptosis in osteoblasts induced by HG by activating miR-335-3p/PTGS2. Serum miR-335-3p may be a novel diagnostic biomarker.