Osteoarthritis
Figure 1 Excessive mechanical loading induces chondrocyte senescence in vitro and in mice. (A,B) Quantitative PCR analysis of Col2a1 and Mmp13 in primary chondrocytes treated with different elongation strain loading (5%,10% or 20%) for 0, 6, 12 and 24 hours. n=5 per time point. (C,D) Representative images and quantification of SA- β Gal staining in primary chondrocytes treated with different elongation strain loading (5%, 10% or 20%) for 0, 6, 12 and 24 hours. n=5 per time point. Scale bar: 50 µm. (E,F) Representative images and quantification of F-actin (phalloidin) staining in chondrocytes described in (C) . n=10 per time point. Scale bar: 50 µm. (G) Representative images of safranin O/Fast green staining (top) and immunofluorescence of p16 INK4a (middle) and p21 (bottom) in articular cartilage of mice treated with multiple loading episodes at peak loads of 9.0 and 13.5 N. Scale bars: 100 µm (first row) and 50 µm (second and third rows). (H) Quantitative analysis of the OARSI scale. n=5 per group. (I) Quantification of p16 INK4a -positive and p21-positive chondrocytes as a proportion of the total chondrocytes in mice cartilage described in (G). n=5 per group. Data are shown as mean±SD. Statistical analyses were conducted using one-way analysis of variance followed by Dunnett’s multiple comparison test (A,B,I), two-way analysis of variance followed by Dunnett’s multiple comparisons test (D,F) or Kruskal-Wallis test followed by Dunn’s multiple comparisons test (OARSI score) (H). Boxed area is enlarged in the bottom right corner. *P<0.05, **P<0.01. DAPI, 4',6-diamidino-2- phenylindole; NS, not significant; OARSI, Osteoarthritis Research Society International.
as previously reported. 30 These results indicate that the loss of FBXW7 protein at 24 hours under 20% elongation strain loading might be caused by the initial drop in the mRNA level at 6 hours (online supplemental figure S3G–J). Moreover, IF staining showed that FBXW7 was decreased in association with cartilage damage in patients with OA, which was further confirmed by western blotting analysis (figure 2B–E and online supplemental figure S4A–C). Inter- estingly, a loss of FBXW7-expressing chondrocytes was observed in aged (24-month-old) mice compared with young (4-month-old) mice (figure 2F–H and online supplemental figure S4D,E). Additionally, FBXW7-positive articular chon- drocytes were progressively reduced in a mechanical load- induced (destabilisation of the medial meniscus (DMM))
cycloheximide (CHX) (50 µ M) to block new protein synthesis under 20% elongation strain loading for 24 hours. When treated with CHX alone, the expression level of FBXW7 was reduced significantly. However, there was no significant difference in the levels of FBXW7 protein under mechanical overloading whether treated with CHX or not (online supplemental figure S3E). Furthermore, inhibition of proteolysis by MG132 increased FBXW7 protein level in control cells but could not prevent the marked decrease of FBXW7 induced by 20% elongation strain loading (online supplemental figure S3F). The results suggested that the loss of FBXW7 in response to mechanical overloading is mainly caused by a decrease in FBXW7 mRNA synthesis. In addition, when protein synthesis was blocked by CHX, we found that the level of FBXW7 decreased time-dependently
Zhang H, et al . Ann Rheum Dis 2022; 81 :676–686. doi:10.1136/annrheumdis-2021-221513
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