Fig 2: Immunohistochemical analysis of RANKL and OPG in bone tissue. Immunohistochemical staining images showing plenty of RANKL immunostained osteoblasts and few OPG immunostained osteoblasts in OVX bone tissue (red arrows) (a); scale bar, 10 µm. Osteoblasts in PMVS-treated bone specimens revealed weak RANKL and mild OPG immunoreactivity (b). Difference of investigations (mean ± S.E.; n = 4–5) were analyzed using Dunn’s nonparametric comparison test, where asterisks * and ** resemble p < 0.05 and p < 0.01, respectively.

Fig 3: RANKL expressions in the oral mucosa between the groups.Arrows indicate positive cells: streptavidin-biotin peroxidase method. Scale bars = 50 µm.Favi: favipiravir; Vit C: vitamin C

Fig 4: Active osteoclasts and RANKL expression. (A) The expression of RANKL was revealed by immunofluorescence. The control without periodontitis induction (left side) is shown on the individual upper panel, the assays with ligature with or without P. gingivalis (right side) are shown on the 4 other panels (CT = connective tissue, AB = alveolar bone). (B) RANKL mean of fluorescence intensity (MFI) was measured in a 0.082 mm2 area (ROI) surrounding the alveolar bone crest. (C) Active osteoclasts were revealed by immunohistochemistry targeting the active osteoclasts marker Cathepsin K (Ctsk). Red arrowheads show Ctsk+ osteoclasts. Only the right sides of treated mice are shown (CT = connective tissue, AB = alveolar bone). (D) The number of Cathepsin K-positive cells was counted in a 0.082 mm2 area (ROI) surrounding the alveolar bone crest. Scale bars = 50μm. (B, D) Each dot represents a single mouse. Horizontal bars represent the mean +/- SEM. Ordinary one-way ANOVA followed by uncorrected Fisher’s least significant difference (LSD) with a single pooled variance multiple comparison test. **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s.P ≥ 0.05, non-significant.

Fig 5: RANKL expressions in the nasal mucosa between the groups.Arrows indicate positive cells: streptavidin-biotin peroxidase method. Scale bars = 50 µm.Favi: favipiravir; Vit C: vitamin C