Fig 1.
Ononin chemical structure.
Fig 2.
The diagram of experimental design and grouping.
Abbreviation: hematoxylin-eosin (HE), toluidine blue (TB), Safranin O (SF), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), matrix metalloproteinase 13 (MMP-13).
Fig 3.
Safety evaluation of ononin in the treatment of OA rats.
(A) HE staining of liver, spleen and kidney sections (scale bar: 50 μm). (B) Curve of body weight of rats over time, n = 10 (C) The organ coefficients of liver, spleen and kidney were measured after 8 week of post-surgery, n = 3. NS: No significant differences from each group.
Fig 4.
Ononin alleviated cartilage damage in OA rats.
Representative pictures of (A) macroscopic observation and (histological staining of HE, TB and SF (scale bar: 50 μm) and (B) Osteoarthritis Research Society International (OARSI) scores of the femoral, n = 6. All data were presented as mean ± standard deviation. ***P < 0.001 vs. Sham group; ###P < 0.001 vs. OA group; $ $P < 0.01 vs. On-Low group; △△P< 0.01 vs. On-Mid group. ★★★P < 0.001 among the three groups. Red circles represent “superficial” area, yellow circles represent “migratory” area, green circles represent “radial” area, and blue circles represent “cartilaginous matrix calcifications” area.
Fig 5.
Ononin reduced the increase levels of IL-1β, TNF-α and IL-6 in OA rat serums.
The levels of IL-1β (A), TNF-α (B) and IL-6 (C) inflammatory cytokines were accessed by ELISA, n = 10. All data were presented as mean ± standard deviation. ***P < 0.001 vs. Sham group; ###P < 0.001 vs. OA group; $ $ $P < 0.001 vs. On-Low group; △△△P < 0.001 vs. On-Mid group; ★★★P < 0.001 among the three groups.
Fig 6.
Effect of ononin on expression of collagen II and matrix metalloproteinase 13 (MMP-13) in OA cartilage.
(A) Immunohistochemical staining was used to detect collagen II and MMP-13 levels in cartilage tissue (scale bar: 50 μm). Quantitative analysis of relative positive cells collagen II (C) and MMP13 (D) in the cartilage samples, n = 3. (B) Protein levels of collagen II and MMP-13 were determined by western blotting. Relative protein expressions of collagen II (E) and MMP-13 (F) was qualified by Image-J software, n = 4. All data were presented as mean ± standard deviation. ***P < 0.001 vs. Sham group; #P < 0.05 vs. OA group; ##P < 0.01 vs. OA group; ###P < 0.001 vs. OA group; $ $P < 0.01 vs. On-Low group; $ $ $P < 0.001 vs. On-Low group; △P < 0.05 vs. On-Mid group; △△P < 0.01 vs. On-Mid group; △△△P < 0.001 vs. On-Mid group; ★★★P < 0.001 among the three groups. The positive cells with black arrows.
Fig 7.
Ononin repressed the activation of MAPK and NF-κB signaling pathways in OA rats.
(A) The phosphorylation and total proteins were determined by western blotting. Relative phosphorylation levels of proteins were qualified by ImageJ software and was normalized by corresponding total protein content, n = 4. All data were presented as mean ± standard deviation. ***P < 0.001 vs. Sham group; ###P < 0.001 vs. OA group; $ $P < 0.01 vs. On-Low group; $ $ $P < 0.001 vs. On-Low group; △△P < 0.01 vs. On-Mid group; ★★★P < 0.001 among the three groups.
Fig 8.
The results of molecular docking between the key ingredients (ligands) and core targets (receptors).
(A) The binding effect of ononin and ERK1 (affinity: −8.7 Kcal/mol). (B) The binding effect of ononin and ERK2 (affinity: −7.8 Kcal/mol). (C) The binding effect of ononin and JNK1 (affinity: −10.8 Kcal/mol). (D) The binding effect of ononin and JNK2 (affinity: −8.2 Kcal/mol). (E) The binding effect of ononin and p38 (affinity: −9.3 Kcal/mol). (F) The binding effect of ononin and p65 (affinity: −7.7 Kcal/mol).