Fig 2: Differentiation of ASCs. (a, b) Adipogenic differentiation of ASCs, Oil Red stain, (15 days of cultivation; objective lens 20×, eyepiece 10×; phase contrast). (a) control — culture of third passage ASCs without using a differentiating medium, the subconfluent monolayer is formed by typical fibroblast-like cells. In the cells of the control culture, the formation of fat vacuoles is not fixed. (b) the culture of ASCs after cultivation in a differentiating medium. Fat culture vacuoles are clearly defined in culture cells stained with a specific Red Oil dye. The presence of fat vacuoles in the cells indicates the ability of ASC to differentiate in the adipogenic direction; (c, d) Osteogenic differentiation of ASCs. Osteogenic differentiation of ASCs, stained with alizarin red (15 days of cultivation; objective lens 10×, eyepiece 10×, light microscopy). (c) The control culture of ASCs is presented as a subconfluent monolayer formed by morphologically identical spindle-shaped cells, with no calcium deposits. (d) The culture of ASCs after cultivation in a special differentiating medium. The culture is a subconfluent monolayer. Calcium deposits are clearly visualized, which are stained with a differentiating dye, alizarin red. Calcium deposits indicate the ability of ASC to differentiate in the osteogenic direction; (f) Chondrogenic differentiation of ASCs. Staining of type II collagen deposits in the pellet with polyclonal antibodies (Abcam, ab34712; cell nuclei stained with hematoxylin and eosin (Sigma-Aldrich, Germany); objective lens 20×, eyepiece 10×). Ball formation and deposition of type II collagen in them indicates chondrogenic differentiation of ASCs.

Fig 3: Disorganization of GP chondrocytes during experimental knee osteoarthritis (OA). Destabilization of medial meniscus (DMM) was induced in 12 weeks old mice, that were sacrificed 8 weeks after surgery. Post-traumatic damage accounted for the articular cartilage (AC) lesions, regarding proteoglycan loss, cartilage fibrillation and vertical clefts observed in the AC. (a) Representative mice joint sections stained with Safranin-O Fast Green at × 5 and × 20 magnification from non-operated wild type (NO-WT) and DMM-WT mice (bars = 250 and 100 µm respectively). (b) Histopathological OARSI Score represented as the sum of tibia and femur scores. (c) Representative Haematoxilin-Eosin stained tibia growth plate (GP) sections at × 40 magnification from NO-WT and DMM-WT mice (bars = 50 µm). (d) Percentage of cells in columns and column index (CI) in the GP in NO-WT and DMM-WT mice. Data are shown as median ± interquartile range (IQR). (e) Association between the percentage of cells in columns and OARSI score, and between CI and OARSI score in WT mice. NO-WT, n = 10–16; DMM-WT, n = 11–12.

Fig 4: 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 5: The expression of TNF signaling pathway is up‐regulated in IVD in IVDD humans and mice. (A) KEGG ontology enrichment results of up‐regulated and down‐regulated gene sets of NPCs. (B) Violin plots illustrating the expression levels of key differentially expressed genes (TNFRSF1A, TNFRSF1B, ADAMTS5, NFKB1, ACAN, and COL2A1) in NPCs between normal, mild and severe groups. (C) Violin plots illustrating the expression levels of key differentially expressed genes (TNFRSF1A, TNFRSF1B, ADAMTS5, NFKB1, ACAN, and COL2A1) in AFCs between normal, mild and severe groups. (D) Typical gene expression profiles in each cell cluster from EP (TNFRSF1A, TNFRSF1B, ADAMTS5, and ACAN), comparing non‐degenerated and degenerated groups. (E) Safranin O and Fast Green (SO&FG) staining and immunofluorescence (IF) staining of TNF‐α, Tnfr1, and Tnfr2 of lumbar IVD sections from the 5‐ and 21‐month‐old male C57BL/6 mice. Images of high magnification views of AF, NP, and EP were detailed on the right panels. White dashed lines showed the boundary between NP and AF. Scale bar, 100 μm. (F) Total histological scores of lumbar IVDs of (E). N = 6 mice per group. (G)–(I) Quantitative analysis of the positive areas or cells for TNF‐α (G), Tnfr1(H), and Tnfr2(I) in lumbar IVDs. N = 6 for each group. Results were expressed as mean ± standard deviation (s.d.). ns, no significant difference, *p <0.05, **p <0.01, ***p <0.001, ****p <0.0001.