Fig 2: TBI causes an imbalanced activation between osteoblasts and osteoclasts, and this is completely prevented by long-term administration of ferulic acid. Levels of (A) RUNX2 and OCN, (B) RANKL and CTSK, and (C) OPG in BM of mice groups were evaluated by IHC assay at 60 days post-TBI. (D) Osteoclasts formed in BM of mice were determined by TRAP staining at the same day after TBI. The IHC images show representative results from four different mice. Scale bars in the images for RUNX2, OCN, and CTSK are 200 μm, while those for RANKL, OPG, and TRAP are 100 μm. All data are presented as the mean ± SD (n = 4). * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with control group; # p < 0.05, ## p < 0.01, and ### p < 0.001 compared with TBI group.

Fig 3: DEIX directly suppresses the migration and osteoclastic activation in LPS-stimulated THP-1 cells. (A) The transwell migration images exhibiting the LPS-stimulated migration of THP-1 cells and its inhibition by the direct addition of DEIX 24 h after the incubation (Bar = 500 μm). (B) The number of migrated THP-1 cells/well in the cells (n = 4). (C) TRAP staining images revealing the LPS-enhanced osteoclast formation in THP-1 cells and its suppression via the DEIX treatment five days after the incubation (Bar = 300 μm). The number of (D) osteoclasts/well and of (E) the nuclei per osteoclast, along with (F) the mean diameter (mm2) of osteoclasts that were formed by the THP-1 cells exposed to LPS, DEIX, or both in the presence of RANKL (n = 4). (G) Western blot images showing the immunoreactive bands of c-Fos, NFATc1, cathepsin K, and MMP-9 in the THP-1 cells exposed to LPS, DEIX, or both for four days. Original blots are presented in Figure S8. (H) Densitometric results show these proteins’ ratio to that of β-actin (n = 4). *p < 0.05 by an unpaired non-parametric test (Kolmogorov-Smirnov test). ns, not significant.

Fig 4: Supplementation with DEIX inhibits osteoclastic activation in the inflamed periodontium of rat groups. (A) TRAP staining images showing the ligature-mediated osteoclast formation and its suppression by DEIX (Bar = 100 μm), in which the tetragonal regions in the upper panels were 4-fold magnified. (B) The TRAP-positive area (%) in the magnified area of rat groups (n = 4). (C) IF assay images indicating the expression levels of RANKL (green) and cathepsin K (red) in the inflamed periodontium of rat groups (Bar = 50 μm). The intensities (a.u.) of (D) RANKL- and (E) cathepsin K-specific immunofluorescences were determined using the ImageJ program (n = 4). *p < 0.05 by unpaired non-parametric test (Kolmogorov-Smirnov test). ns, not significant.