Fig 2: The expression of Rspo2 in RA mouse model and RA patients. A Representative images and statistical analysis of HE staining and Rspo2 immunohistochemical staining in control and RA human synovial tissue (n = 8 per group). Scale bars: 50 μm. B Western blot of Rspo2 expression in human synovial tissue. C Representative images and statistical analysis of Safranin O/Fast Green staining and Rspo2 immunohistochemical staining in control and RA human cartilage tissue (n = 8 per group). Scale bars: 100 μm. D Representative images and statistical analysis of HE staining and Rspo2 immunostaining in the synovium from a RA mouse model and control group (n = 6 per group). Scale bars: 50 μm. E Representative images and statistical analysis of Safranin O/Fast Green staining and Rspo2 immunostaining in knee cartilage from a RA mouse model and control group (n = 6 per group). Scale bars: 50 μm. F Rspo2 concentrations assessed by an ELISA in the serum of sham and RA mice at 4 and 8 weeks after AIA modeling (n = 6 per group). Student’s t-test. *P < 0.05, **P < 0.01. Data are shown as mean ± SEM

Fig 3: Rspo2 disrupt chondrocyte homeostasis in RA. A Immunofluorescence staining and statistical analysis of Col2a1 in the knee cartilage from sham and RA mice administered with a vehicle, rmRspo2, Rspo2-NAb, or rmDKK1 (n = 6 per group). Scale bar: 25 μm. B, C Representative immunohistochemical images and statistical analysis of ACAN and MMP13 in the knee cartilage from sham and RA mice administrated with a vehicle, rmRspo2, Rspo2-NAb, or rmDKK1 (n = 6 per group). Scale bar: 25 μm. D, E Western blot (D) and quantitative PCR analysis (E) of ANAN, Col2a1, Sox9, and MMP13 in rmRspo2-treated chondrocytes with or without Rspo2-NAb or rmDKK1 (n = 3 per group). One-way analysis of variance (ANOVA) and Tukey’s multiple comparison test. *P < 0.05, **P < 0.01. Data are shown as mean ± SEM

Fig 4: Rspo2 enhances FLS aggressive phenotype in RA synovium. A Immunohistochemical staining and statistical analysis of Ki67 in synovial tissues from sham and RA mice treated with vehicle, rmRspo2, Rspo2-NAb, and rmDKK1 for 4 and 8 weeks (n = 6 per group). Scale bars: 50 μm. B, C Immunofluorescence dual staining and statistical analysis of Vimentin and MMP3 in synovial tissues from sham and RA mice treated with vehicle, rmRspo2, Rspo2-NAb, and rmDKK1 for 4 weeks (B) and 8 weeks (C) (n = 6 per group). Scale bars: 12.5 μm. One-way analysis of variance (ANOVA) and Tukey’s multiple comparison test. *P < 0.05, **P < 0.01. Data are shown as mean ± SEM

Fig 5: Rspo2 causes strikingly progressive synovial hyperplasia, production of inflammatory cytokines, and articular cartilage loss in RA mice. A HE staining of synovial tissues from sham and RA mice administrated with vehicle, rmRspo2, Rspo2-NAb, and rmDKK1 for 4 and 8 weeks. Scale bars: 50 μm. B Safranin O/Fast Green staining of knee cartilage from sham and RA mice treated with vehicle, rmRspo2, Rspo2-NAb, and rmDKK1 for 4 and 8 weeks. Scale bars: 50 μm. C Synovitis score for the joints described in A (n = 6 per group). D Osteoarthritis Research Society International grades for the joints described in B (n = 6 per group). E–H TNF-α and IL-1β concentrations assessed by an ELISA in the serum of sham and RA mice treated with vehicle, rmRspo2, Rspo2-NAb, and rmDKK1 for 4 and 8 weeks (n = 6 per group). One-way analysis of variance (ANOVA) and Tukey’s multiple comparison test. *P < 0.05, **P < 0.01. Data are shown as mean ± SEM