Increased osteoclast-induced bone resorption is an important factor leading to periprosthetic osteolysis and osteoporosis2,23. As the balance between osteoblast-mediated bone formation and osteoclast‐mediated bone resorption have been reported to be responsible for bone turnover and remodeling, it is important to find out an available strategy on them for the treatment of these diseases5. In our study, we figured out for the first time that mangostin could inhibit RANKL‐induced osteoclastogenesis through inhibition of NF‐κB and MAPK signaling in vitro and hinders LPS-induced osteolytic bone loss in a mouse calvarial model.
Mangostin has been investigated possessing extensive biological activities and pharmacological properties–antioxidant, antineoplastic, anti-proliferation and induces apoptosis14,24,25. Recent report reveals the use of mangostin in treating rheumatoid arthritis and a mangostin-loaded self-micro emulsion (MG-SME) was designed25. Recently, it has been reported that mangostin can block LPS-induced activation in RAW264.7 cells, thereby inhibiting the secretion of IL-1β, IL-6, NO and COX-226. Mangostin had been reported via inhibiting the activation of TAK1-NF-κB to exert anti-inflammatory effects, which makes it a potential choice for treating inflammatory diseases27. Previous studies have shown that mangostin has an inhibitory effect on the osteoclast differentiation of RAW264.7 cells. However, compared with RAW264.7 cells, it is more scientific to select mouse bone marrow-derived macrophages as the research object for in vitro experiments, and the results produced are more credible, which can also explain the effects in vivo. What’s more, we confirmed the role of mangostin in inhibiting osteoclasts at the animal level. Our current research reminds us that mangostin has great potential for treating osteoporosis.
At the beginning, we wandered if mangostin had a toxic effect on BMMs and whether it can inhibit the osteoclast differentiation. The results of CCK-8 reminded us that mangostin had no obvious inhibitory effect on BMMs cells at concentrations lower than 2µmol/L. In order to determine that the effect of mangostin on the formation of osteoclasts is indeed achieved by inhibiting their differentiation rather than promoting cell apoptosis, we also did apoptosis flow cytometry assay and western blotting, and the results were consistent with our previous conclusions. In supplementary figure 1, we clearly found that when the mangostin dose reached 4µM, the expression of apoptosis-related protein was significantly increased. The flow cytometry results suggested that there was apoptosis in BMM cells, and the results of Hoechst staining further confirmed this conclusion. This is the basis for our follow-up study to select the drug dosage. TRAP staining showed that at concentrations below cytotoxic levels, mangostin treatment had a significant protective effect on RANKL-induced osteoclast differentiation. As the concentration of mangostin increased, the number of TRAP-positive cells decreased, and mature osteoclasts were scarcely observed in the high-concentration (2 µmol/L) treatment group. Based on the conclusions of previous studies of natural compounds, the inhibitory effect of berberine on osteoclast formation was used as our positive control28. To further study at which stage of osteoclastogenesis mangostin unleashed its inhibitory effect, we added mangostin (2 µmol/L) at different stages of osteoclastogenesis. The experimental results were in line with our expectations. As shown in figure 1D, the earlier the mangostin intervention, the stronger the effect of obstructing osteoclast formation. Compared with the control group, the osteoclastogenesis inhibition of mangostin was hardly seen in the late-stage group. Based on the above data, we concluded that mangostin could inhibit the formation of osteoclasts, especially in the early stage of osteoclastogenesis.
Under the stimulation of RANKL, the up-regulation of the expression of several specific genes is closely related to the differentiation of osteoclasts8. Therefore, real-time PCR was then utilized to measure the inhibitory effect of mangostin on RANKL-induced mRNA expression of these genes (TRAP, NFATc1, CTSK, V-ATPase d2, CTR and DC-stamp). As expected, results indicated that mangostin blocked RANKL‐stimulated osteoclast-related genes in a dose and time-dependent. This proved from another aspect that mangostin indeed suppressed the osteoclasts differentiation. After verifying the inhibitory effect of mangostin on osteoclast formation and RANKL-induced osteoclastic marker gene expression, it was naturally explored whether mangostin can also function in osteoclast bone resorption. The results that large bone resorption in the control group and less number and area of bone resorption in the mangostin group demonstrated the hinder effect of mangostin on osteoclast function.
NF-κB signalling is a very classic pathway in those studies of osteoclastogenesis7. When RANKL activates downstream signals, the phosphorylation of NF-κBp65 and IκBα and the degradation of IκBα promote the activation of NF-κB p65 and nuclear translocation, which will increase the expression of s osteoclast‐specific gene and promote the formation and function of osteoclasts29,30. Based on this status quo, many researchers currently focus their research on pharmacological intervention for NF‐κB signalling related checkpoints. In our current study, we found that mangostin restricted the degradation of IκBα and phosphorylation of NF‐κB p65 and IκBα induced by RANKL stimulation. Furthermore, many previous studies have shown that RANKL-induced osteoclastogenesis usually involves the activation of both NF-κB and MAPK signaling pathways. So, in this study, we explored whether mangostin could also inhibit the activation of the MAPK pathway. Not surprisingly, the phosphorylation of all three MAPK pathways (ERK, JNK, and p38) in BMMs stimulated with RANKL was blocked by mangostin at non‐cytotoxicity concentrations.
Previous studies reported that JNK1 regulates RANKL-induced osteoclastogenesis by activating the Bcl-2-Beclin1-autophagy pathway, and our findings suggest that the JNK pathway is regulated by mangostin31. So, we chose anisomycin, a JNK activator, to explore whether the mangostin-mediated inhibition of osteoclastogenesis can be reversed. The results confirmed that anisomycin could indeed reverse the effect of mangostin on the JNK pathway, but the effect of mangostin on inhibiting the formation and function of osteoclasts will not be affected. This may be due to the fact that mangostin works through the three MAPK pathways, so simply changing one of them does not make a significant difference. When the LPS pathway is activated, the TRAF-TBK1-IRF3 pathway will also change32. We used western blot assay to detect the expression of TBK1 and IRF3 proteins, and found that the presence of mangostin did not affect the pathway stimulation of RANKL. Therefore, we concluded that the activation of MAPK and NF-KB pathways was inhibited by mangostin, except for IRF3 (figure S2D).
Since previous results indicated that mangostin obstructed RANKL-induced osteoclast formation and decreased the expression of those osteoclastic related gene via blocking the NF-κB and MAPK signaling cascades in vitro, we investigated whether mangostin could inhibit pathological osteolysis in an LPS-induced murine calvarial osteolysis model33. Analysis of Micro-CT scan results and histological examinations, we concluded that mangostin did have the effect of reducing LPS-induced osteolysis in vivo. Regarding the choice of in vivo drug dosage and administration method, we refer to the method of previous literature34,35. At first, we chose 20mg/kg as the in vivo dose, and we found that there was inflammatory hyperplasia under the skin of the mouse skull. So, we choose to reduce the body dose to 10mg/kg, which is consistent with the effective dose of another research36. In our study, we reported for the first time that mangostin could be used as a potential treatment for these osteoclast-related diseases.
Nevertheless, there are several limitations of the current study. First, since this balance consists of osteoclastic bone resorption and osteoblastic bone formation, further research on the effect of mangostin on osteoblasts are needed to be addressed. Then, the present study proved that mangostin markly suppressed the RANKL-induced osteoclast formation by inhibiting the pathway in vitro. We still need to study whether mangostin also exerts its anti-osteoclastogenesis effect through this pathway in vivo. Unfortunately, we did not explore the exact binding target of mangostin. This target should explain why the presence of mangostin affects the blockade of NF-κB and MAPK pathways caused by RANKL. In the early stage of the RANKL pathway, after RANKL binds to RANK, tumor necrosis factor receptor-related factor 6 (TRAF6) will be recruited to form a complex, and then further activate the MAPK and NF-κB pathways37,38. Since the MAPK and NF-κB pathways share the same upstream promoter TRAF6, it is reasonable to speculate that M plays an interfering role in the binding process of TRAF6 and RANK.