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

Figure 1 Endothelial cell-exosomes identification and uptake. A: Transmission electron microscopy, orange arrows indicate exosomes; B: Nanoparticle tracking analysis for exosome size distribution; C: Western blotting for detection of exosomes surface markers; D: Exosome phagocytosis experiment. ECs: Endothelial cells; EC-Exos: Endothelial cell-exosomes.

Figure 2 Endothelial cell-exosomes attenuate high glucose-induced oxidative stress and ferroptosis. A-C: Comparison of reactive oxygen species levels, malondialdehyde levels and glutathione levels among the control group, high glucose (HG) group, and HG + exosomes (Exos) group; D: Reactive oxygen species fluorescence images of the control group, HG group, and HG + Exos group; E: Comparison of mRNA expression levels of ferroptosis indicators prostaglandin endoperoxide synthase 2, solute carrier family 3 member 2, solute carrier family 7 member 11, glutathione peroxidase 4 among the control group, HG group, and HG + Exos group. The bars indicate the mean ± SD from three independent experiments (n = 3). ^aP < 0.05 vs control, ^bP < 0.05 vs high glucose. Con: Osteoblasts treated with normal glucose; HG: Osteoblasts treated with high glucose; HG + Exos: High glucose + endothelial cell-exosomes; ROS: Reactive oxygen species; DCF: Dichlorofluorescein; MDA: Malondialdehyde; GSH: Glutathione; PTGS2: Prostaglandin endoperoxide synthase 2; SLC3A2: Solute carrier family 3 member 2; SLC7A11: Solute carrier family 7 member 11; GPX4: Glutathione peroxidase 4.

Figure 3 High glucose + exosomes and high glucose group microRNAs differential expression analysis. A: Heatmap of differentially expressed microRNAs (miRs), red to blue represents expression levels from high to low; B: Volcano plot of differentially expressed miRs. Orange dots represent up regulated miRs, green dots represent down regulated miRs, gray dots represent no difference; C: Validation of the top 10 miRs that were significantly upregulated in high glucose (HG) + exosomes (Exos) compared to HG expression, ^aP < 0.05 vs high glucose; D: Differences in miR-335-3p expression among Exos, HG group, and HG + Exos group; E: Interaction diagram of miR with target proteins; F: Kyoto Encyclopedia of Genes and Genomes pathway analysis diagram. The bars indicate the mean ± SD from three independent experiments (n = 3). ^bP < 0.05 vs high glucose, ^cP < 0.05 vs high glucose + exosomes. Con: Osteoblasts treated with normal glucose; HG: Osteoblasts treated with high glucose; HG + Exos: High glucose + endothelial cell-exosomes; miR: MicroRNA; Exos: Exosomes.

Figure 4 MicroRNA-335-3p targets prostaglandin endoperoxide synthase 2 3’ untranslated region: Prediction and validation. A: MicroRNA binding site database prediction of microRNA-335-3p binding site with prostaglandin endoperoxide synthase 2 3’ untranslated region; B: Dual-luciferase reporter gene assay. ^aP < 0.05. PTGS2: Prostaglandin endoperoxide synthase 2; UTR: Untranslated region; WT: Wild type; miR: MicroRNA; Mut: Mutant; NC: Negative control.

Figure 5 Inhibition of microRNA-335-3p abolishes endothelial cell-exosomes-mediated protection against high glucose-induced oxidative stress in osteoblasts. A: Detection of microRNA-335-3p levels in osteoblasts induced by high glucose after inhibition of microRNA-335-3p by reverse transcription-quantitative polymerase chain reaction; B-D: Results of reactive oxygen species, malondialdehyde and glutathione quantitative analysis; E: Detection of cellular reactive oxygen species levels in each group using dichlorofluorescein fluorescence probe. The bars indicate the mean ± SD from three independent experiments (n = 3). ^aP < 0.05 vs control, ^bP < 0.01 vs control, ^cP < 0.01 vs high glucose, ^dP < 0.01 vs high glucose + exosomes + negative control. Con: Osteoblasts treated with normal glucose; HG: Osteoblasts treated with high glucose; HG + Exos: High glucose + endothelial cell-exosomes; HG + Exos + NC: High glucose + negative control transfection + endothelial cell-exosomes; HG + Exos + inhibitor: High glucose + microRNA-335-3p inhibitor + endothelial cell-exosomes.

Figure 6 Inhibition of microRNA-335-3p abolishes endothelial cell-exosomes-mediated protection against high glucose-induced ferroptosis in osteoblasts. A: The relative expression levels of mRNA for prostaglandin endoperoxide synthase 2 (PTGS2), glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11), and solute carrier family 3 member 2 (SLC3A2) in each group of cells; B: Western blotting to detect the protein expressions of key ferroptosis markers PTGS2, GPX4, SLC7A11, SLC3A2; C: The relative protein expressions of PTGS2, GPX4, SLC7A11, SLC3A2. The bars indicate the mean ± SD from three independent experiments (n = 3). ^aP < 0.01 vs control, ^bP < 0.01 vs high glucose, ^cP < 0.01 vs high glucose + exosomes + negative control. Con: Osteoblasts treated with normal glucose; HG: Osteoblasts treated with high glucose; HG + Exos: High glucose + endothelial cell-exosomes; HG + Exos + NC: High glucose + negative control transfection + endothelial cell-exosomes; HG + Exos + inhibitor: High glucose + microRNA-335-3p inhibitor + endothelial cell-exosomes; PTGS2: Prostaglandin endoperoxide synthase 2; SLC3A2: Solute carrier family 3 member 2; SLC7A11: Solute carrier family 7 member 11; GPX4: Glutathione peroxidase 4; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase.

Figure 7 Altered serum levels and diagnostic significance of prostaglandin endoperoxide synthase 2 and microRNA-335-3p in diabetic osteoporosis. A: Comparison of serum prostaglandin endoperoxide synthase 2 levels between the type 2 diabetes and diabetic osteoporosis groups; B: Comparison of serum microRNA (miR)-335-3p levels between the type 2 diabetes and diabetic osteoporosis groups; C: Correlation analysis of serum prostaglandin endoperoxide synthase 2 with miR-335-3p; D: Receiver operating characteristic curve analysis of miR-335-3p. ^aP < 0.05. DM: Patients with type 2 diabetes; DOP: Patients with type 2 diabetes with osteoporosis; miR: MicroRNA; PTGS2: Prostaglandin endoperoxide synthase 2; AUC: Area under the curve; CI: Confidence interval.