β-Ecd inhibited LPS-induced osteoclast formation
To eliminate the inhibitory effects of β-Ecd on osteoclast formation could have been due to its cytotoxicity on RAW264.7 cells, CCK-8 assay were performed. The data showed that the cell viability of RAW264.7 cells was not distinctly reduced by β-Ecd with concentrations of 5, 10, 20 and 40μM, indicating that β-Ecd under 40μM was not nontoxic to RAW264.7 cells (Fig.1).
To observe the effect of β-Ecd on LPS-induced osteoclastogenesis, RAW264.7 cells were treated with different concentrations of β-Ecd as mentioned above for 72 h in the presence or absence of LPS, and then stained with TRAP, an osteoclast-specific protein. Data showed that LPS can exactly stimulate RAW264.7 cells differentiation into osteoclast. β-Ecd treatment caused osteoclast less rounded and few nuclei than that of LPS-treated group (Fig.2A), and indeed inhibited LPS-induced osteoclast formation in a dose-dependently manner (Fig.2B). Notably, 40 μM of β-Ecd nearly inhibited LPS-induced osteoclast formation, the osteoclast number was counted with three different microscope fields and the average number of osteoclasts was only 12.5 % of the LPS-treated group. Besides, β-Ecd at 5 μM inhibited osteoclastogenesis less severely than the other β-Ecd-treated groups.
β-Ecd inhibits LPS-induced osteoclast-related gene expression
To further understand the inhibitory function of β-Ecd on osteoclast formation, RAW264.7 cells were treated with different concentrations of β-Ecd in the presence or absence of LPS, and expression of osteoclast differentiation-related genes including RANK, TRAF6, MMP-9, CK and CAⅡ were analyzed quantitatively using WB. The WB bands containing all the former proteins were shown in Fig.3A. Notably, the data indicated that LPS at 100 ng/mL significantly up-regulated proteins expression of RANK (Fig.3B), TRAF6 (Fig.3C), MMP-9 (Fig.3D), CK (Fig.3E) and CAⅡ (Fig.3F) during osteoclast formation. β-Ecd treatment groups obviously inhibited expression of these relevant genes, and the inhibitory effects were also in a dose-dependent manner. Particularly, 40 μM of β-Ecd exhibited the inhibition on these genes stronger than those of the other three concentrations.
β-Ecd suppressed the release of pro-inflammatory cytokines
It is reported that pro-inflammatory cytokines are considered as mediators of bone-loss . Common inflammatory markers include TNF-α, IL-1β, PGE2 and cyclooxygenase-2 (COX-2). These cytokines enhance osteoclast formation and migration in bone destructive diseases such as rheumatoid arthritis and periodontitis. Consistent with this, RAW264.7 cells incubated with LPS for 24 h resulted in insignificant increases in TNF-α, IL-1β, PGE2 and COX-2 (Fig.4), and their levels were increased to 1.4-, 1.2-, 1.5- and 1.4-fold of untreated control group, respectively. Clearly, β-Ecd treatment for 24 h suppressed LPS-induced the secretion of these cytokines with a dose-dependent manner. Importantly, β-Ecd at 40 μM caused a more obvious reduction of IL-β, PGE2 and COX-2 compared with the remaining concentrations (Fig.4).
β-Ecd inhibited LPS-triggered activation of NF-κB Pathway
NF-κB signaling pathway plays an important role in activating inflammatory response and inducing apoptosis of chondrocytes, which is mediated by the degradation of Iκbα [16, 30]. β-Ecd suppressed LPS-induced osteoclast formation as well as the expression of osteoclast-related genes. Therefore, to illuminate the possible molecular mechanisms underlying the inhibitory effect of β-Ecd on LPS-induced osteoclast formation, we explored whether β-Ecd affects the NF-κB pathway which LPS-induced. Given this, the proteins expression of IκBα and p-IκBα were analysed by WB. The WB bands were presented in Fig.5A. Stimulation of RAW264.7 cells with LPS can up-regulate the ratio of p-IκBα/IκBα, which presented the activation of NF-κB pathway (Fig.5B). When treated with different concentrations of β-Ecd, it can efficiently inhibited LPS-increased NF-κB-related protein levels as assessed by western blot assay. Notably, the level of p-IκBα/IκBα was decreased as the concentration increased. Taken together, these findings illustrated that β-Ecd inhibited LPS-induced osteoclastogenesis through suppressing IκBα phosphorylated activation in NF-κB pathway.
Inhibition NF-κB pathway restrain LPS-induced osteoclast formation and inflammation
The above results preliminary indicated that β-Ecd inhibited the osteoclast formation and inflammation. Next, the effects of inhibition NF-κB pathway on LPS-induced osteoclast formation and inflammation need to be elucidated. For this purpose, TRAP-staining and ELISA assays were performed as previous description. In TRAP-staining assay, RAW264.7 cells were treated with 40μM β-Ecd or sulfasalazine (NF-κB inhibitor) in the presence of LPS. LPS and control group were involved. After interaction for 72 h, RAW264.7 cells were stained with TRAP. As shown in Fig.6A, β-Ecd and sulfasalazine treatment caused osteoclast less rounded and few nuclei than that of LPS-treated group and indeed inhibited LPS-induced osteoclast formation (Fig.6B). Notably, 40 μM β-Ecd can inhibit LPS-induced osteoclast formation, and the number of osteoclast was 64% of the LPS-treated group. Besides, osteoclastogenesis was also inhibited in sulfasalazine group compared with LPS-treated group.
Next, the contents of TNF-α, IL-1β, PGE2, and COX-2 in the culture supernatants were measured by ELISA. RAW264.7 cells incubated with 40 μM β-Ecd or sulfasalazine in the presence of LPS for 24 h. LPS-treated group caused the release of these pro-inflammatory cytokines. When treated with 40 μM β-Ecd and sulfasalazine, the expressions of LPS-induced TNF-α (Fig.6C), IL-1β (Fig.6D), PGE2 (Fig.6E), and COX-2 (Fig.6F) represented a downward trend. Importantly, there was no significant difference in the levels of these cytokines between β-Ecd and sulfasalazine group. These results indicated that suppression of the NF-κB pathway indeed decreased the LPS-induced osteoclast formation and inflammation.