Fig 1: The inhibitor of miR-690 partially reversed the up-regulation osteogenesis and down-regulation adipogenesis induced by M2D-Exos. (A) The level of miR-690 in BMSCs after treated with M2D-Exos or PBS. *p < 0.05 compared to the BMSC group. (B) The level of miR-690 in M2 macrophages after treated with lipo3000, miR-690 inhibitor, or inhibitor control measured by qRT-PCR analysis. Control group was M2 macrophages treated with lipo3000. *p < 0.05 compared to the inhibitor control group. (C) The level of miR-690 in BMSCs after treated with M2D-Exos, miR-690 inhibited M2D-Exos, or inhibitor control M2D-Exos before cultured in osteogenic inducer were measured by qRT-PCR analysis. *p < 0.05 compared to the M2D-Exos NC inhibitor group. (D) The level of miR-690 in M2 macrophages after treated with lipo3000, miR-690 inhibitor, or inhibitor control before cultured in adipogenic inducer were measured by RT-PCR analysis. *p < 0.05 compared to the M2D-Exos NC inhibitor group. (E) IRS-1, TAZ, OCN, RUNX2 mRNA level of BMSCs after treated with M2D-Exos, miR-690 inhibited M2D-Exos, or inhibitor control M2D-Exos during osteogenesis were measured by qRT-PCR analysis. *p < 0.05 compared to the M2D-Exos NC inhibitor group. (F) IRS-1, TAZ, C/EBPβ, and PPARγ mRNA level of BMSCs after treated with M2D-Exos, miR-690 inhibited M2D-Exos, or inhibitor control M2D-Exos during adipogenesis were measured by qRT-PCR analysis. *p < 0.05 compared to the M2D-Exos NC inhibitor group. (G) A-R staining was used to measure the formation of bone nodules in BMSCs following treated by M2D-Exos, miR-690 inhibited M2D-Exos, or inhibitor control M2D-Exos for 14 days. Scale bar = 50 µm, (H) The statistical data of Alizarin red-mediated calcium staining. (I) Oil red O staining was used to measure the formation of lipid droplets in BMSCs following treated by M2D-Exos, miR-690 inhibited M2D-Exos, or inhibitor control M2D-Exos for 14 days. Scale bar = 50µm. (J) The statistical data of Oil red O staining. *p < 0.05 compared to the M2D-Exos NC inhibitor group.
Fig 2: Exosomes derived from M2 macrophages enhanced osteogenic differentiation of BMSCs. (A, B) Osteogenic proteins, OCN and RUNX2, were measured by western blotting analysis and quantified, Control group was BMSCs treated in osteogenic inducer. (C) Osteogenic genes, OCN and RUNX2, were measured by RT-PCR analysis, Control group was BMSCs treated in osteogenic inducer. (D) Alizarin red staining was used to measure the formation of bone nodules in BMSCs after treated by PBS (control group) or M2D-Exos for 14 days. Scale bar = 200 µm. (E) The statistical data of Alizarin red-mediated calcium staining. *p < 0.05 compared to the control group. Control group was BMSCs treated in osteogenic inducer.
Fig 3: Secreted ucOCN limited OA development in DMM mice model. (A) Experimental design schematic for testing ucOCN as a therapeutic regimen with the OA mouse model. 12-week-old mice had their right knees subjected to DMM surgeries, intra-articularly injected with mouse IgG, recombinant ucOCN or OCN antibody respectively twice a week at the 7th day after surgeries and the animals were sacrificed at 20 weeks old. (B) Articular cartilage morphology shown by Safranin O and fast green staining of representative paraffin sections, with statistical analysis of OARSI score on the right. (C) IHC staining of representative paraffin sections for MMP13 and COL10a1 expression of articular cartilage, with statistical analysis on the right. (D) Synovitis is shown by H&E staining, with statistical analysis of synovitis score on the right. n = 6 per group. Scale bar, 100 μm. One-way ANOVA. *p < 0.05. **p < 0.01. ***p < 0.001.
Fig 4: HIF-1α is positively regulated by ucOCN in mice OA model. (A-D) IHC staining of representative paraffin sections of HIF-1α and TIMP3 expression in articular cartilage of human LFC (CON) and MFC (OA) with OA (n=3 per group) and mice subject to sham and DMM surgery (n=6 mice per group). (E) IF analysis of representative paraffin sections of HIF-1α expression of articular cartilage treated with mouse IgG, recombinant ucOCN or OCN antibody respectively 2 months after DMM surgery, statistical analysis on the right. (F, G) IHC staining of representative paraffin sections of TIMP3 and p-mTOR expression of articular cartilage treated with mouse IgG, recombinant ucOCN or OCN antibody respectively 2 months after DMM surgery, statistical analysis on the right. (H) Schematic diagram of ucOCN regulation in the maintenance of cartilage integrity. ucOCN activates HIF-1α by interacting with GPRC6A. HIF-1α reduces hypertrophic markers by increasing the expression of TIMP3 and increases autophagy by down regulate AKT/mTOR pathway, thus inhibits chondrocyte hypertrophy to protect cartilage from degradation. One-way ANOVA *p < 0.05; **p < 0.01; ***p < 0.001. Scale bar, 100 μm. n = 6 per group.
Fig 5: Secreted ucOCN protects chondrocyte from hypertrophy in vitro. (A) ELISA analysis of total OCN, ucOCN, cOCN of supernatant harvested from WT chondrocytes treated with control (CON) or OCN-overexpressing lentivirus (LV-OCN). (B) Gene expression analysis of hypertrophic markers of WT chondrocytes treated with supernatant harvested from WT chondrocytes treated with control (CONs) or OCN-overexpressing lentivirus (LV-OCNs) for 24 hours. (C-E) Gene expression and western blotting analysis of MMP13 and COL10a1 of WT primary chondrocytes treated with LV-control (CON), LV-OCN, LV-OCN + OCN antibody respectively for 24 hours. (F, G) Flow cytometric analysis of apoptosis of WT chondrocytes treated with mouse IgG (CON), IL-1β, IL-1β + recombinant ucOCN, IL-1β + OCN antibody respectively for 24 hours. (H, I) Gene and IF analysis of MMP13 of WT chondrocytes treated with mouse IgG (CON), IL-1β, IL-1β + recombinant ucOCN, IL-1β + OCN antibody respectively for 24 hours. (J) Western blotting analysis of MMP13 and COL10a1 of WT chondrocytes treated with ucOCN at different time point (1-6 day). (K, L) Gene expression and western blotting analysis of MMP13 and COL10a1 of OCN-/- primary chondrocytes treated with WT chondrocytes supernatant (WTs), OCN-/- chondrocytes supernatant (OCN-/- s), WTs with OCN antibody, OCN-/-s with 5 ng/mL recombinant ucOCN respectively for 24 hours. (M) Western blotting analysis of COL10a1 and MMP13 of WT primary chondrocytes treated with mouse IgG, IL-1β, IL-1β + recombinant ucOCN, IL-1β + recombinant cOCN respectively for 24 hours. (N, O) GAG and CTX II release detect from cartilage explants treat IgG, IL-1β+TNFα, IL-1β+TNFα+ recombinant ucOCN, IL-1β+TNFα+ recombinant cOCN respectively. Cells were maintained under monolayer culture conditions. Student's t-test for two groups, one-way ANOVA for three or more. *p < 0. 05. **p < 0. 01. ***p < 0. 001. WTs, wild type chondrocytes supernatant. OCN-/-s, OCN-/- chondrocytes supernatant. OCN Ab, OCN antibody.
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