Fig 2: 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.

Fig 3: Expression trends for IL-6/P-ERK/BMAL-1/MMP3/MMP13/ADAMTS5/COL2 in the CON, CRD, and REC groups. (A) Western blot results for IL-6/P-ERK/BMAL-1/MMP3/MMP13/ADAMTS5/COL2. (B–E) RT-qPCR analysis of MMP3/MMP13/ADAMTS5/COL2. All experiments were performed in triplicate, and the results are expressed as the mean ± SD. *P < 0.05; **P < 0.01, ***P < 0.005. NS, not significant.

Fig 4: The relationship between interleukin 6 (IL-6), extracellular signal–regulated kinase (ERK), estrogen-related receptor ? (ERR?) and temporomandibular joint osteoarthritis (TMJOA). (A) and (B) Representative images of Western blot assays demonstrate the relationship between IL-6, phospho-ERK (P-ERK), ERR?, matrix metalloproteinase 9 (MMP9), MMP13, HIF-1a, vascular endothelial growth factor A (VEGFA), and TMJOA, while the expression levels of total ERK and ß-actin were the same in the cartilage tissues of the control group and the UAC group (n = 12 rats per group). (C) and (D) RT-qPCR analysis of IL-6, ERR?, MMP9, MMP13 VEGFA, COL2, AGG, HIF-1a, and ERRa in cartilage tissues of the control group and the UAC group (n = 12 rats per group). (E) The CCK-8 assay was used to examine the cell proliferation of each group; the absorbance was measured at 450 nm (n = 3). All experiments were performed in triplicate, and the results are expressed as the mean ± SD. *P < 0.05; **P < 0.01. NS not significant. Scale bar: 50 µm.

Fig 5: Increasing IL-33 levels exacerbates OA in vivo. (a) protein and (b) mRNA expression of cartilage-degrading proteases (MMP-13, ADAMTS-5 and MMP-3) and chondrogenic markers (COL2A1, SOX-9 and Aggrecan) in isolated human chondrocytes from Non-OA (n = 20) and OA patients (n = 20) treated with either PBS (vehicle control) or rIL-33 (30 ng mL-1, 24 h). (c) MMP-13 and (d) MMP-3 protein level in cell media supernatants obtained from isolated human chondrocytes from Non-OA (n = 12) and OA patients (n = 12) treated with either PBS (vehicle control) or rIL-33 (30 ng mL-1; 24 h). (e, f) OARSI scoring of cartilage tissue, (g) synovitis scoring and (h) osteophyte maturity scoring of sham-operated (n = 20) or DMM-operated (n = 20) WT mice (12 weeks post-surgery end timepoint) treated intraperitoneally with either PBS (vehicle control) or rIL-33 (33 µg kg-1; daily for 12 weeks post-surgery). (i) von Frey pain assessment of sham-operated (n = 20) or DMM-operated (n = 20) WT mice (12 weeks post-surgery end timepoint) treated intraperitoneally with either PBS (vehicle control) or rIL-33 (33 µg kg-1; daily for 12 weeks post-surgery). (j) mRNA expression of cartilage-degrading proteases (MMP-13, ADAMTS-5 and MMP-3) and chondrogenic markers (COL2A1, SOX-9 and Aggrecan) in whole knee joints of DMM-operated (n = 20) WT mice (12 weeks post-surgery end timepoint) treated intraperitoneally with either PBS (vehicle control) or rIL-33 (33 µg kg-1; daily for 12 weeks post-surgery). All RT-qPCR gene expressions were normalised to the endogenous level of 18 s in respective groups. Data are expressed as mean ± S.E.M. with unpaired 2-tailed Student’s t-tests (c, d), two-way analysis of variance followed by the Tukey-Kramer test (f, g, h), or repeated measures 2-way ANOVA with Bonferroni’s post hoc tests was used to compare groups at each time point (i; DMM PBS vs DMM rIL-33). n indicates the number of human specimens or mice per group. NS = non-significant. P < 0.01, P < 0.001 or P < 0.0001 are represented as **, *** or ****, respectively.