Fig 1.
Anti-arthritic effects of vitronectin-derived bioactive peptide (VnP-16) in spondyloarthritis (SpA) mice.
Curdlan (3 mg/kg) was injected intraperitoneally into SKG mice with the ZAP-70W163C mutation to induce SpA. The treatment groups were as follows: 1) vehicle (phosphate-buffered saline) administration; 2) oral celecoxib 10 mg/day; 3) subcutaneous VnP-16 600 μg/week; and 4) oral celecoxib 10 mg/day with subcutaneous VnP-16 600 μg/week. The in vivo experiments were repeated twice and pooled data are presented (n = 10 per group). (A) Weekly mean clinical score for 12 weeks. Black dot, vehicle group; green dot, celecoxib-alone group; blue dot, VnP-16 alone group; red dot, VnP-16+celecoxib group. (B) Hematoxylin and eosin (H&E)/Safranin O-stained images of ankle joints isolated from SpA mice 12 weeks after curdlan injection; bar graphs show the arthritis score. Data are means ± standard error of the mean (SEM) of assessments by three independent experimenters. ns, non-significant; *P < 0.05, ** P < 0.01, **** P < 0.0001.
Fig 2.
VnP-16 reduces the spondylitis score of SpA mice.
In vivo experiments were repeated twice and pooled data are presented (n = 10 per group). Spine tissues were obtained from the vehicle-, celecoxib-, VnP-16 single-, and VnP-16+celecoxib-treated groups 12 weeks after curdlan injection and stained with H&E and Safranin O. Black arrows indicate inflammatory cell infiltration. Bar graphs show the spondylitis score. Ns, non-significant, *P < 0.05.
Fig 3.
VnP-16 reduces inflammatory cytokine expression in the nucleus pulposus of mice with SpA.
IL-1β, IL-6, TNF-α, IL-17A expressing cells were enumerated in the nucleus pulposus by immunohistochemical staining (n = 6 per group). ns, non-significant, *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 4.
VnP-16 reduces inflammatory cytokine expression in the cartilaginous end plate of mice with SpA.
Immunohistochemical staining of IL-1β, IL-6, TNF-α, and IL-17A in the cartilaginous end plate (n = 6 per group). Data are means ± SEM. ns, non-significant, *P < 0.05, ***P < 0.001, ****P < 0.0001.
Fig 5.
VnP-16 regulates type 17 helper T cell (Th17) and regulatory T cell (Treg) populations in the annulus fibrosus of SpA mice.
Spine tissue was stained with CD4–FITC, IL-17–PE, CD25–APC, Foxp3–PE to evaluate (A) Th17, (B) IL-22+ Th17, and (C) Treg populations (n = 6 per group). Double-positive cells are shown in the bar graph. Data are means ± SEM. ns, non-significant, *P< 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Fig 6.
VnP-16 regulates type 17 helper T cell (Th17) and regulatory T cell (Treg) differentiation in the spleens of SpA mice.
Splenocytes were subjected to flow cytometry using antibodies against IL-17A, CD4, CD25, and Foxp3 to determine the Th17 and Treg populations (n = 3 per group). Data are means ± SEM. ns, non-significant, **P < 0.01.
Fig 7.
VnP-16 suppresses pSTAT3 s727 expression of splenocytes.
Splenocytes harvested from BALB/c mice were stimulated with vehicle or VnP-16, and total STAT3, pSTAT3 s727, and GAPDH expression were measured by Western blotting (n = 3 per group). Data are means ± SEM. *P< 0.05. P-values are in comparison with the vehicle group.