Figure 6.

Signaling pathway through which MPMBP inhibits the production of inflammatory mediators. (A) Fluorescent staining of cultured neonatal mouse calvaria for alkaline phosphatase activity and immunofluorescent staining for NF-κB/p65. Parietal bones were incubated with the vehicle (a,d,g,j,m), 25 μM MPMBP (b,e,h,k,n) or 2.5 μM zoledronate (c,f,i,l,o) for 48 h and then stimulated with LPS for 4 h. Scale bars = 20 μm. Arrows indicate the nuclear translocation of NF-κB/p65 (solid line circles). Arrowheads indicate the lack of NF-κB/p65 staining in the nuclei (dotted line circles). Figures in the 3rd (g–i) and 5th (m–o) columns correspond to the squares in the 2nd (d–f) and 4th (j–l) columns, respectively. (B) Percentage of NF-κB/p65-positive nuclei in the selected region of interest (ROI)s that contained a relevant number of cells (ranged from 25 to 68 cells) of the parietal bones stained for NF-κB/p65 (mean ± SD, sample size of n = 7 per group, *: p < 0.05, ¶: p < 0.001 versus LPS-alone). (C) Effects of non-NBPs (left panel) and NBPs (right panel) on O₂⁻ generation in the xanthine-xanthine oxidase system. (mean ± SD, n = 5. *: p < 0.05, ¶: p < 0.001 versus control). (D) RT-PCR analysis of the expression of COX-2 in calvaria cultured with 10 μM NOC18, an NO donor, or 10 μM SIN-1, an ONOO⁻ donor, for 72 h. (E) Representative confocal microscopy images of cultured neonatal mouse calvaria immunofluorescent stained for nitrotyrosine (a–d). Parietal bones were treated with the vehicle (a,b), 25 μM MPMBP (c), or 2.5 μM zoledronate (d) for 48 h, and then incubated for a further 4 h in the absence (a) or presence of LPS (b,c,d). Scale bars = 20 μm.