ObjectiveTo investigate the effect of Staphylococcal peptidoglycan (PGN-sa) on raw264.7 cells differentiating into osteoclasts. MethodsThere were 5 groups in the experiment: 100 ng/mL PGN-sa group, 200 ng/mL PGN-sa group, 400 ng/mL PGN-sa group, positive control group [100 ng/mL receptor activator of nuclear factor κB ligand (RANKL)], and blank control group (PBS). Raw264.7 cells were cultured with different concentrations of PGN-sa, RANKL, or PBS for 5 days, and then tartrate resistant acid phosphatase (TRAP) staining was used to detect the formation of osteoclast-like cells; Image-Pro Plus 6.0 software was used to detect the bone resorption areas of osteoclast-like cells; and MTT assay was used to observe the proliferation activity of raw264.7 cells. ResultsTRAP staining showed that PGN-sa and RANKL can induce raw264.7 cells to differentiate into osteoclast-like cells; different concentrations of PGNsa groups had more osteoclast-like cells formation than blank control group (P < 0.05), and the number of osteoclast-like cells significantly increased with the increase of PGN-sa concentrations (P < 0.05). Bone resorption cavity experiment showed that bone resorption cavities were obvious in different concentrations of PGN-sa groups and in positive control group, and the area of bone absorption cavities was increased with the increasing PGN-sa concentrations, showing significant difference between groups (P < 0.05). MTT assay showed that no significant difference was found in the absorbance (A) value between different concentrations of PGN-sa groups and blank control group, and between different concentrations of PGN-sa groups (P > 0.05). ConclusionPGN-sa can promote raw264.7 cells to differentiate into osteoclasts with bone resorption activity.
ObjectiveTo investigate the molecular mechanism of osteoclast differentiation induced by Staphylococcal peptidoglycan (PGN-sa). MethodsRaw264.7 cells were stimulated with PGN-sa and with PGN-sa+SC75741[a potent inhibitor of nuclear factor κB (NF-κB) activation] in a concentration of 200 ng/mL. The protein expression of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) was tested at 0, 1, 2, and 3 days; the proteins related to osteoclast differentiation of extracellular regulated protein kinases (ERK), p38, c-Jun N-terminal kinase (JNK), NF-κB, inhibitor of NF-κB (IκB-α), Akt, and the phosphorylation forms of p38, ERK, JNK, Akt, NF-κB were measured at 0, 5, 10, 20, 40, and 60 minutes by Western blot. In addition, Raw264.7 cells were stimulated with PGN-sa in the concentrations of 100 ng/mL (group A), 200 ng/mL (group B), 400 ng/mL (group C), and with PBS (group D) for 1, 2, and 3 days; the expression levels of tumor necrosis factor α (TNF-α), interleukin 1α (IL-1α), and IL-6 were detected by ELISA. ResultsThe results of Western blot showed that the expression of NFATc1 increased gradually with time, showing significant difference between different time points (P<0.05). However, after SC75741 was added, the expression of NFATc1 was inhibited at 2 and 3 days, showing significant difference when compared with no addition of SC75741 (P<0.001). After stimulation of PGN-sa, the expression of IkB-α decreased significantly at 5 and 10 minutes when compared with those at the other time points (P<0.001), and returned to normal at 20 minutes. Meanwhile, the expression of p-NF-κB increased significantly at 5 and 10 minutes when compared with those at the other time points (P<0.001), and returned to normal at 20 minutes; and the expression of p-NF-κB at 5 minutes was significantly higher than that at 10 minutes (P<0.001). After the addition of SC75741, there was no change in the expressions of IκB-α and p-NF-κB, showing no significant difference between different time points P>0.05). Moreover, the expressions of ERK, p38, JNK, NF-κB, Akt, p-p38, p-ERK, p-JNK, and p-Akt showed no significant change between different time points P>0.05). ELISA results showed that there were no expressions of TNF-α and IL-1α in groups A-D at different time points. The expression of IL-6 had an increasing trend with time prolonged in each group, showing significant differences between different time points (P<0.05). Moreover, at 1 day after culture, the expression of IL-6 showed no significant difference among groups P>0.05). At 2 and 3 days after culture, the expression of IL-6 in groups A-C showed an increasing trend and was significantly higher than that in group D, showing significant difference among groups (P<0.05). ConclusionPGN-sa can promote osteoclast differentiation through NF-κB signaling pathway, and IL-6 may play a role in this process.
Objective To investigate the effect ofstaphylococcal lipoteichoic acid (LTA-sa) on RAW264.7 cells differentiation into osteoclasts. Methods RAW264.7 cells were cultured with LTA-sa of 100 ng/mL (group A), LTA-sa of 200 ng/mL (group B), LTA-sa of 400 ng/mL (group C), receptor activator of nuclear factor κB (NF-κB) ligand (RANKL) of 100 ng/mL as positive control (group D), and equal volume of PBS as blank control (group E) respectively for 5 days. And then, tartrate resistant acid phosphatase staining (TRAP) was used to detect the formation of osteoclast-like cells, Image-Pro Plus 6.0 software to measure the areas of bone resorption pits in Corning Osteo Assay Surface (COAS) wells, and MTT assay to observe the proliferation activity of RAW264.7 cells in group A, B, C, and E. Results After cultured for 5 days, the formation of osteoclast-like cells and bone resorption pits were observed in all groups. The number of osteoclast-like cells and the area of bone resorption pits in groups A, B, C, and D were more than those in group E. And with the increased concentration of LTA-sa, the indexes in groups A, B, and C increased gradually, but were lower than those in group D, and differences were significant between groups (P<0.05). At 5 days after culture, there was no significant difference in absorbance value among the experimental groups (groups A, B, C, and E) (P>0.05). Conclusion LTA-sa has promoting effect on RAW264.7 cells differentiation into osteoclasts.