RANKL is a critical molecule in osteoclast differentiation and bone resorption. As shown in Fig. 4a, osteoclast formation was clearly detected in medium containing 100 ng/ml of RANKL, whereas TRAP-positive osteoclasts were not detected in the absence of RANKL. In order to observe the effect of DPB-LPS on osteoclast formation, we performed the reaction in media with low levels of RANKL (10 ng/ml), and DPB-LPS was added to cultures of isolated BMMs. The number of TRAP-positive osteoclasts was significantly reduced in response to 10 ng/ml RANKL, as compared with 100 ng/ml RANKL; however, osteoclast formation in 10 ng/ml RANKL condition was significantly stimulated by DPB-LPS treatment. The stimulating effect of DPB-LPS was inhibited by the EETC treatment. The bone resorption activity of mature osteoclasts was measured simultaneously. Consistent with the osteoclast formation studies, the formation of resorption pits was remarkably increased by DPB-LPS treatment and suppressed by the EETC treatment. In addition, the mRNA expression of RANKL was significantly stimulated by DPB-LPS treatment in the hFOB1.19 human osteoblast cells but remained the same for osteopontin (OPG) (Fig. 4b). Treatment with the EETC inhibited RANKL mRNA expression but did not affect OPG mRNA expression in DPB-LPS–treated hFOB1.19 cells (Fig. 4c). Consequently, the increased RANKL/OPG ratio in the DPB-LPS–treated hFOB1.19 cells was remarkably decreased by treatment with EETC. Thus, bone destruction by DPB-LPS was caused by the cooperative stimulation of osteoclast formation in BMMs and of RANKL expression in osteoblasts. EETC could be an important means of preventing bone loss due to oral bacteria.Fig. 4Effect of EETC on DPB-induced osteoclastic bone resorption. a For osteoclast formation, mouse BMMs were treated with M-CSF and RANKL and were stained to detect expression of TRAP. Pit formation was also performed in M-CSF and RANKL on calcium phosphate apatite-coated plates, and light microscopy indicated resorptive pitting. To observe the stimulating effect of DPB-LPS on osteoclast formation and pit formation, both assays were performed at a low concentration of RANKL (10 ng/ml). The graph shows the total number of TRAP-positive multinucleated (≥3 nuclei) osteoclasts (MNC) per well. Data are expressed as mean ± SE of three independent experiments. *P < 0.01 vs. only 10 ng/ml RANKL condition, # P < 0.001 vs. DPB1#-LPS plus 10 ng/ml RANKL condition. b RANKL and OPG mRNA expression was analyzed in hFOB1.19 human osteoblastic cells. Eco-LPS (LPS from Escherichia coli) was the positive control. The data shown are representative of three independent experiments. c Effect of EETC on DPB#1-LPS-induced RANKL and OPG mRNA expression were analyzed by RT-PCR. The ratio of RANKL to OPG was determined after normalization to the intensity of GAPDH. The data shown are representative of three independent experiments. *P < 0.001 vs. control (Ctl), # P < 0.001 vs. DPB#1-LPS condition