Fig 1: HucMSCs-EVs alleviate knee osteoarthritis in DMM-induced OA mice model. A. Safranin O and Fast Green staining of knee sections and micro-CT analysis of knee joint in mice with DMM-induced OA. Expression levels of COL2A1, Aggrecan, ADAMTS5, and MMP13 were determined by immunofluorescence staining. Scale bar = 400 µm for Safranin O and Fast Green staining; Scale bar = 1 mm for Micro-CT; Scale bar = 100 µm for immunofluorescence staining. B. OARIS scores of knee sections in each group (n = 10). C. Osteophyte score of each group (n = 10). D. Quantification of mean fluorescence intensity of COL2A1, Aggrecan, ADAMTS5, and MMP13 in each group (n = 10). E. Levels of IL-1ß, IL-18, and TNF-a in mice knee tissue as determined by ELISA (n = 3). Data are presented as mean ± SD. ns, no significance; *p < 0.05; **p < 0.01; ***p < 0.001
Fig 2: The protection effect of knee OA by hucMSCs-EVs was abolished in NLRP3-/- mice. A. Safranin O and Fast Green staining of knee sections and micro-CT analysis of knee joint in NLRP3-/- mice with DMM-induced OA. Expression levels of COL2A1, Aggrecan, ADAMTS5, and MMP13 were determined by immunofluorescence staining. Scale bar = 400 µm for Safranin O and Fast Green staining; Scale bar = 1 mm for Micro-CT; Scale bar = 100 µm for immunofluorescence staining. B. OARIS scores of knee sections in each group (n = 10). C. Osteophyte score of each group (n = 10). D. Quantification of mean fluorescence intensity of COL2A1, Aggrecan, ADAMTS5, and MMP13 in each group (n = 10). E. Levels of IL-1ß, IL-18, and TNF-a in mice knee tissue as determined by ELISA (n = 3). Data are presented as mean ± SD. ns, no significance; *p < 0.05; **p < 0.01; ***p < 0.001
Fig 3: Inhibition of DMT1 restored IL-1ß induced chondrocytes cartilage degeneration. (A) The effect of Dmt1 silence was evaluated using RT-PCR analysis. (B,C) Chondrocytes were transfected with scrambled siRNA control or Dmt1 siRNA, then treated with IL-1ß or TNF-a for 24 h and iron influx into chondrocytes was determined by the quenching of calcein fluorescence. Data are presented as mean ± SD. *P < 0.05 IL-1ß or TNF-a vs. siDmt1 group. (D–G) Chondrocytes were transfected with Dmt1 siRNA, then treated with IL-1ß for 24 h. Protein levels of ADAMTS5, MMP13, COL2, and iNOS were determined by western blot (D,F) and quantification analysis (E,G). Data are presented as mean ± SD. All experiments were repeated three times independently. *P < 0.05; **P < 0.01; ***P < 0.001 vs. scrambled siRNA control (NC); #P < 0.05; ##P < 0.01 vs. IL-1ß treatment group.
Fig 4: Expression of matrix degrading enzymes in knee articular cartilage of 9.5-month-old Vector-Control and HMWTgFGF2 mice treated with BGJ398. Eight-month-old Vector-Control (Vec) and HMWTgFGF2 (HMW) female mice were sq injected with vehicle or BGJ398 5 days/week for 6 weeks. At the end of the experiment (9.5-month- old), knee joints were collected for IHC staining. Representative immunohistochemical staining images for (A) MMP13 and (B) ADAMTS5. Quantitative analysis of percent-positive staining area for (C) MMP13 and (D) ADAMTS5 in articular cartilage. MMP13 and ADAMTS5 expression were increased in knees of HMWTgFGF2 vehicle-treated mice compared with Vector-Control vehicle-treated mice; these were rescued by BGJ398 treatment. Data are shown as mean and individual points. n = 7–9 mice/group. *p < 0.05 by two-way ANOVA.
Fig 5: Vitamin D alleviates knee osteoarthritis in DMM-induced OA mice model. (A) Severity of DMM-induced OA mice model as determined by Safranin-O and Fast Green, and micro-CT analysis. Expression levels of COL2A1, aggrecan, MMP13 and ADAMTS5 as determined by immunofluorescence staining. Scale bar = 400 µm for Safranin-O & Fast Green staining; Scale bar = 100 µm for immunofluorescence staining. (B) OARIS scores in each group (n = 10, one-way ANOVA). (C) Osteophyte score of each group (n = 10, one-way ANOVA). (D) Quantification of mean fluorescence intensity of COL2A1, aggrecan, MMP13 and ADAMTS5 in each group (n = 10, one-way ANOVA). (E) Levels of IL-1ß, IL-18 and TNF-a in mouse knee tissue as determined by ELISA (n = 3, one-way ANOVA). Data are presented as mean ± SD. ns, no significance; *p <0.05; **p <0.01; ***p <0.001.
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