Figure 3

Effect of WFA on osteoclastogenesis indirectly through osteoblasts. (a) WFA increases mRNA levels of OPG but decreases mRNA levels of RANKL in primary osteoblasts. Ratio of RANKL: OPG were determined and quantified with qPCR and normalized with GAPDH. Values represents mean±S.E. ***P<0.001 and **P<0.01 of three independent experiments(n=3) when compared with vehicle-treated cells. (b) WFA treatment to OVx mice reduces osteoclastogenesis and increases osteoprogenitor cells in bone marrow. BMCs from mice of various experimental groups were seeded into 48-well plates and osteoclast differentiation was induced as described. Cells were stained for TRAP activity for osteoclast formation. Figure shows quantitative representation of TRAP +ive mononuclear and multinuclear cells. (c) WFA increases mRNA levels of OPG but decreases mRNA levels of TRAP and RANK in osteoclast culture. BMCs were isolated from 4- to 6-week-old mice. After overnight culture cells were cultured for 6 days in the presence of MCSF and RANKL. mRNA levels of TRAP and RANK related to osteoclastogenesis was determined by qPCR from the total RNA made from BMC’s. Data represent mean±S.E.M.; n=3. **P<0.01 and ***P<0.001 compared with vehicle-treated cells. (d) WFA treatment decreases expression of inflammatory cytokines in osteoblasts. qPCR data show mRNA levels IL-6, MCP-1. Values represents mean±S.E. **P<0.01 and ***P<0.001 of three independent experiments (n=3) when compared with control. (e) WFA inhibits TNFα-induced NF-kB nuclear translocation in osteoblast cells. Representative photomicrograph of sub cellular localization of p65 was determined by immuno fluorescence (magnification × 40) under control and treatment conditions from three independent experiments (n=3). (f) WFA treatment abolished the nuclear translocation of the NF-kB in osteoblasts. Osteoblasts were treated with TNFα and WFA. Cytoplasmic or nuclear extracts were prepared and NF-kB protein levels were detected by immunoblotting. TNFα enhanced the nuclear translocation of NF-kB, WFA abolished it. Histone H3 was used as a loading control for nuclear extract, β-actin was used as loading controls for cytoplasmic fraction. (g) This figure shows the densitometric analysis of NF-kB from three independent blots. Values represents mean±S.E. of three independent experiments (n=3), ***P<0.001, **P<0.01 when compared with control