Fig 2: Effects of Mume Fructus (MF) on IL-1β-induced signaling in primary chondrocytes.The expression of matrix metalloproteinase (MMP) 3 (A), a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) (B), and collagen type II alpha 1 chain (COL2A1) (C) was evaluated using immunofluorescence staining (n = 5). Scale bar = 25 μm. (D) Fluorescence intensity was quantified using Image J. Data are expressed as mean ± standard deviation (SD). One-way ANOVA was performed, followed by Dunnett’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001 vs. IL-1β-treated group. #P < 0.05, ##P < 0.01, ###P < 0.001 vs. control group.

Fig 3: RORα inhibition reversed IL-6 mediated cartilage degradation in human articular chondrocytes.A IHC staining and positive cell percentage for STAT3 and phosphorylated STAT3 (Y705) of normal and degenerated OA cartilage (n = 6). Scale bars, 50 μm. B IHC staining for phosphorylated STAT3 of articular cartilage after 6 weeks of indicated treatment of mice. Scale bars, 50 μm. C Cells were infected with sh-RORA or control virus (sh-NC) lentivirus and then treated with 50 ng/ml IL-6 for 48 h. The expressions of MMP13, ADAMTS4, ADAMTS5, ACAN, and COL2A1 in human articular chondrocytes were detected by real-time PCR analysis. D Western blot analysis and quantification of ACAN, COL2A1, ADAMTS4, ADAMTS5, and MMP13 protein level in human chondrocytes. Chondrocytes were infected with sh-RORA sh-NC lentivirus and then treated with 50 ng/ml IL-6 for 48 h as indicated. E The expression of RORα in human articular chondrocytes after different durations of IL-6 treatment was detected by western blot. F The expressions of MMP13, aggrecan, and COL2A1 in human chondrocytes were detected by western blot. Cells were treated with either vehicle, IL-6, SR3335, Stattic, or in combination as indicated above. The statistical data in A were analyzed with Student’s t test, and data in C and D were analyzed with one-way ANOVA followed by Dunnett’s test. *P < 0.05, **P < 0.01, ns = no significance. All data shown above are presented as mean ± SD.

Fig 4: The protein amounts of ADAMTS5 and MMP13 are increased in osteoarthritic cartilage from mice with reduced EGFR activity. (a, b) Immunostaining of ADAMTS5 and MMP13 in sham- (left panels) and DMM-operated (right panels) knee joints from WT and EgfrWa5/+ mice (a) and vehicle- and gefitinib-treated mice (b). For ADAMTS5, we observed increased staining in both sham and DMM knees from mice with reduced EGFR activity. However, for MMP13, we observed decreased staining in sham knees but increased staining in DMM knees from mice with reduced EGFR activity. The percentages of ADAMTS5- and MMP13-positive chondrocytes were quantified. * P<0.05; *** P<0.001. (c) qRT-PCR shows that EGFR signaling inhibits chondrogenic adamts5 expression. Primary chondrocytes were serum starved overnight, treated with vehicle (control) or TGF-α (50 ng⋅mL−1) for 1 day, and harvested for mRNA quantification. ** P<0.01.

Fig 5: Pathologic changes in endplate chondrocytes induced by estrogen deficiency. (A) CCK-8 assay of endplate chondrocytes at 72 h. (B) The mRNA expression of Aggrecan, Col2a1, Adamts5 and Mmp13 in endplate chondrocytes. (C) The protein expression of Aggrecan, Collagen II, ADAMTS5 and MMP13 in endplate chondrocytes. Refer to the Supplementary Fig. 1 for full scans of blots. Error bars represent the mean ± SD, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001.