Fig 2: CB-1 interfered with RANKL-induced activation of NF-?B and NFATc1 pathways. (A, B) Raw264.7 cells were pretreated with CB-1 (10 µM) for 4 h and then treated with RANKL for 15 to 45 min before lysed in RIPA Buffer. Phosphorylated and total I?Ba and NF-?B p65 proteins were detected by specific antibodies. RANKL induced the phosphorylation of I?Ba and NF-?B p65, which was significantly inhibited by CB-1. (C) After treated with or without CB-1 (10 µM), RAW264.7 cells were stimulated by 50 ng/ml RANKL for 1 h and then stained for NF-?B p65 antibody and secondary antibody with FITC. Immunofluorescence demonstrated that NF-?B p65 protein was translocated into nucleus upon RANKL treatment, and this nuclear translocation was inhibited by CB-1. (D, E) NF-?B and NFATc1-binding site-specific luciferase reporter assay showed that RANKL promoted NF-?B and NFATc1 transcription activity, which were suppressed by CB-1. (F) After pretreatment with or without CB-1 (10 µM) for 4 h, the cells were cultured in induction medium for 0, 1, or 3 days. Cells were then collected and lysed for western blot analysis. (G) The relative gray levels of corresponding to c-Fos and NFATc1 were quantified and normalized to ß-actin using ImageJ software. Data are presented as mean ± SD; *P < 0.05 and **P < 0.01 compared with the control group. Data are representative of at least three independent experiments.

Fig 3: Reduced osteoclast differentiation- and autophagy-related proteins and induced apoptosis-related proteins after the addition of polyphenolic compounds. (A). Western blotting of osteoclast differentiation-related molecules such as NFATc1, TRAP, and Cathepsin K protein levels, with GAPDH used as an internal control, was shown at day 6 in the absence or presence of tannic acid (TA), gallic acid (GA), or ellagic acid (EA) during osteoclast differentiation. (B). Western blotting of autophagy-related molecules such as Beclin1, Atg7, Atg5, and LC3B protein levels, with GAPDH used as an internal control, was shown on day 6 in the absence or presence of tannic acid (TA), gallic acid (GA), or ellagic acid (EA) during osteoclast differentiation. (C). Western blotting of apoptosis and cell survival-related molecules such as PARP, BAX2, and Bcl2 protein levels, keeping GAPDH as an internal control, was shown on day 6 in the absence or presence of tannic acid (TA), gallic acid (GA), and ellagic acid (EA) during osteoclast differentiation. (D). Western blotting of down-stream signaling activation-related molecules such as pAkt (T308 and T473), pP38, total Akt, total p38, and total Erk1/2 protein levels, with GAPDH as an internal control, were shown at day 6 in the absence or presence of tannic acid (TA), gallic acid (GA), or ellagic acid (EA) during osteoclast differentiation.

Fig 4: Schematic diagram demonstrating that ephB4-Fc suppresses osteoclast formation and bone resorption induced by Ti particles. Ti particles activated the NF?B pathway in the presence of RANKL. A total of 3 days later, ephrinB2 expression increased significantly. By adding ephB4-Fc, ephrinB2 was activated, which inhibited the C-FOS/NFATc1 signaling pathway and inhibited the gene expression of cathepsin-K, tartrate-resistant acid phosphatase and matrix metalloproteinase 9, by RANKL and Ti particles. Finally, ephB4-Fc inhibits osteoclasts differentiation and bone resorption by Ti particles. Ti, titanium; ephB4, erythropoietin-producing hepatocellular receptor 4; OC, osteoclast; NF-?B, nuclear factor ?B; RANK, receptor activator of NF-?B; RANKL, RANK ligand; TRAP, tartrate-resistant acid phosphatase; C-FOS, Fos proto-oncogene, AP-1 transcription factor subunit; NFATc1, nuclear factor of activated T-cells 1.

Fig 5: OMT inhibits RANKL-induced osteoclast-specific gene expression in vitro. (A–F) BMMs were treated with M-CSF, RANKL and indicated concentrations of OMT for 5 days, the expression of c-fos, Nfatc1, Ctsk, Trap, Atp6v02d, and Mmp9 were analyzed by real-time PCR and normalized relative to Gapdh. (G–L) BMMs were treated with M-CSF, RANKL, and 200 µM OMT for 0, 1, 3, 5 days, the expression of osteoclast-specific genes was tested. Data were presented as means ± SD of 5 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.