Fig 1: AAV-Runx1 overexpression alleviates the pathological changes in growth plate cartilage. a Histological staining, including H&E, Masson, and safranin O, showing the effects of AAV-Runx1 overexpression on growth plate cartilage at 24 weeks after ACLT surgery. b μ-CT results showing changes in the thickness of growth plate cartilage in AAV-Runx1-overexpressing articular cartilage at 24 weeks after ACLT surgery. c μ-CT results showing the changes in subchondral cancellous bone in AAV-Runx1-overexpressing articular cartilage at 24 weeks after ACLT surgery. d Quantitative analysis of the thickness (width) in b in growth plate cartilage in AAV-Runx1-overexpressing mice. e Quantitative analysis of BV/TV in c in AAV-Runx1-overexpressing mice at 24 weeks after ACLT surgery. f IHC staining showing the changes in Col2a1 in the growth plate in AAV-Runx1-overexpressing articular cartilage at 24 weeks after ACLT surgery. g Immunofluorescence staining showing the changes in SOX9 in the growth plate in AAV-Runx1-overexpressing articular cartilage at 24 weeks after ACLT surgery. h Immunofluorescence staining showing the changes in ColX in the growth plate in AAV-Runx1-overexpressing articular cartilage at 24 weeks after ACLT surgery. i Immunofluorescence staining showing the changes in MMP13 in the growth plate in AAV-Runx1-overexpressing articular cartilage at 24 weeks after ACLT surgery. These results are based on at least three independent experiments (n = 3). All significance data presented in d and e are based on two-tailed Student’s t-tests
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 γ (ERRγ) and temporomandibular joint osteoarthritis (TMJOA). (A) and (B) Representative images of Western blot assays demonstrate the relationship between IL-6, phospho-ERK (P-ERK), ERRγ, matrix metalloproteinase 9 (MMP9), MMP13, HIF-1α, vascular endothelial growth factor A (VEGFA), and TMJOA, while the expression levels of total ERK and β-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, ERRγ, MMP9, MMP13 VEGFA, COL2, AGG, HIF-1α, and ERRα 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.
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