Fig 2: Model depicting the mechanisms of miR-17 in maintaining cartilage homeostasis and prevention of OA.MiR-17 is a regulator for HIF-1a in articular chondrocytes, and catabolic and anabolic balance was maintained under physiological HIF-1a expression. The cellular and ECM change of the articular cartilage was shown in mouse models of DMM or miR-17~92 cKO. The faded color in superficial and middle layers indicates ECM destruction or degeneration. In DMM-induced OA, pathological catabolic factors targeted by miR-17 were increased. In miR-17~92 cKO mice, miR-17 deficiency caused pathological increase of HIF-1a, and decreased the physiological catabolism and anabolism in chondrocytes. Supplementation with exogenous miR-17 or inducing endogenous miR-17 by GDF-5 treatment prevented OA and restored cartilage homeostasis.

Fig 3: Osthole delays OA-related degeneration in IL-1ß-treated chondrocytes. (A,B) Flow cytometry results of chondrocytes treated with osthole and IL-1ß were analyzed. (C-E) Western blot and qPCR analysis of COL2A1, ADAMTS5, and MMP13. All data were presented as mean ± SD (n = 3). ** p < 0.01 compared to the control group. ## p < 0.01 compared to the IL-1ß group. ++ p < 0.01 compared to the osthole group. OA, osteoarthritis; IL-1ß, interleukin-1ß; qPCR, quantitative real-time polymerase chain reaction; COL2A1, Collagen type II a1; ADAMTS5, a disintegrin and metalloproteinase with thrombospondin motifs-5; MMP13, matrix metalloprotein-13.

Fig 4: Phgdh expression in an in vivo and in vitro model of OA. (A) DEGs between the 2 week sham and OA groups. (B) DEGs between the 8 week sham and OA groups. (C) Boxplot of Phgdh expression based on microarray analysis of the different groups. (D) Immunofluorescence assay for detection of Col2a1 in chondrocytes harvested from newborn rats. (E) Immunofluorescence analysis for detection of Phgdh in chondrocytes treated with IL-1ß. Scale bar, 200 µm. (F) mRNA and (G) protein expression levels of Phgdh in IL-1ß induced chondrocytes. Data are presented as the mean ± SD (n=3). *P<0.05, **P<0.01, ***P<0.001 vs. Control. Phgdh, 3-phosphoglycerate dehydrogenase; OA, osteoarthritis; DEG, differentially expressed gene; Col2a1, collagen type II a 1 chain.

Fig 5: DIM alleviates OA development in the DMM mice model (A) Assessment of knee OA progression in a mice DMM model was conducted by digital X-ray imaging. Joint space narrowing was evident in the OA group (white arrows) (B) H and E staining of different experimental groups (scale bar, 100 µm) (C) Safranin O/fast green staining was used for the assessment of cartilage destruction (scale bar, 100 µm; partially enlarged image scale bar, 20 µm) (D) Mice articular cartilage OARSI scores (E,F) Immunohistochemical staining of cleaved caspase-3, LC3 II, and p-PI3K expressions in cartilage samples of different experimental groups (scale bar, 50 µm; partially enlarged image scale bar, 20 µm) and AODs were analyzed by the ImageJ software (U.S. National Institutes of Health, Bethesda). The data are presented as mean ± SD values from three independent experiments (n = 10 in each group). Using one-way ANOVA, NS, no statistical difference; # p < 0.05 vs. the Sham group; ## p < 0.01, vs. the Sham group, * p < 0.05 vs. the DMM group; ** p < 0.01 vs. the DMM group.