Fig 2: miR-200a-3p regulates MMP-3 and MMP-13 expression in EfS-treated BMSCs. a mRNA expressions of MMP-3 and MMP-13 were increased on stimulation of EfS in a dose-dependent manner with 24 h incubation. β-actin was used as internal control. b Protein expressions of MMP-3 were upregulated with increasing concentration of EfS after 48 h incubation. c Protein expressions of MMP-13 were upregulated with increasing concentration of EfS after 48 h incubation. In b and c, the levels of β-actin were used as internal control. d MMP-3 protein levels were detected by western blot in BMSCs treated with EfS (10%) together with miR-200a-3p mimics (100 nM) or inhibitors (100 nM). e MMP-13 protein levels were detected as in d. *p < 0.05; NS, not significant

Fig 3: Immunohistochemistry of MMP3, MMP9 and MMP13 Antibody-Positive C28/I2 Chondrocytes in Response to IL-6 and the Effect of Tofacitinib (50nM) on MMP Antibody-Positive Chondrocytes. The scale is 50μm (A–J). Confluent C28/I2 chondrocytes were pre-treated with Tofacitinib (50nM) for 30 min followed by incubation with rhIL-6 (10ng/mL) or rhIL-6 plus Tofacitinib (50nM) for 1 hr. The “no-additions” group served as the control for rhIL-6-treated chondrocytes. The arrows in each panel indicate an MMP3, MMP9 or MMP13-positive C28/I2 chondrocyte. Panel J is a “blank” which shows the appearance of individual chondrocytes stained with hematoxylin. MMP3 (A–C); MMP9 (D–F); MMP13 (G–I); Appearance of confluent C28/I2 chondrocytes stained with DAB and hematoxylin (J).

Fig 4: Inhibition of miRNA-495 expression during in vitro osteoarthritis progression protects chondrocytes from IL-1β-mediated disruption of SOX9 and COL2A1. (A) Protein levels of SOX9 were analyzed in TC28a2 cells transfected with anti-miR negative control (NC, 100 nM), anti-miR-495 (50 or 100 nM) by Western blotting to determine the effective dose of anti-miR-495. (B) TC28a2 cells transfected with anti-miR NC (50 nM) or anti-miR-495 (50 nM) were seeded at 1×105 cells per well in 24-well culture plates. The cells were treated with 10 ng/mL of IL-1β or with no IL-1β as a control. Safranin O staining was performed to detect glycosaminoglycans (GAGs). Stained cells were destained with 10% cetylpyridinium for quantitative analysis. Absorbance was measured at 490 nm. *p<0.05 compared to Group 2 (n=3 experimental replicates). (C) GAG content was measured using the Blyscan sulfate GAG assay. The sulfated GAGs were quantified in the supernatant and standardized using the chondroitin 4-sulfate standard solution. *p<0.05 compared to group 2 (n=3 experimental replicates). (D) Protein levels of SOX9, COL2A1, COX-2, ADAMTS5, and MMP13 were analyzed in 10 ng/mL of IL-1β-treated TC28a2 cells transfected with anti-miR NC (50 nM) or anti-miR-495 (50 nM) using Western blotting. The graphs represent the average values of the band intensity for each blot, and each blot was normalized to the band intensity value of β-actin (n=3 experimental replicates). The data are presented as means±SD. *p<0.05 vs. Group 2. IL-1β, interleukin-1β; SFO, safranin O; CV, crystal violet.

Fig 5: The effects of suppressed GAS5 expression on mechanical stimulation-induced MMP-13 expression were investigated. (A) The expression of GAS5 was detected by qRT-PCR in sh-GAS5-transfected chondrocytes. (B) The cell viability was determined by CCK-8 assays. (C) The expression of miR-27a was determined by qRT-PCR. The mRNA (D) expression of MMP-13 was determined by qRT-PCR, and the protein expression of MMP-13 (E-F) and Col2a1 (E and G) were detected bywestern blotting assays. (H) The immunofluorescence intensity of MMP-13 was detected. (I) The calculation of fluorescence activity of MMP-13. * p < 0.05 and **p < 0.01.