Sudachitin, a Polymethoxyflavone Derived From Citrus Sudachi, Suppresses Lipopolysaccharide-Induced Inflammatory Bone Resorption Because of Inhibiting Osteoclast Formation in Mice

Objective: Inflammatory bone diseases are associated not only with production of inflammatory cytokines but also with local oxidative state. Sudachtin, a polymethoxyflavone derived from Citrus sudachi, possesses antioxidant properties and regulates various functions of mammalian cells.¹ However, the mechanism for osteoclastogenesis and inflammatory bone destruction remains unknown. Extensive studies have indicated that the induction of osteoclast differentiation by RANKL requires the activation of NF-kB and MAPK signaling pathways, which leads to the expression of NFATc1, a critical transcription factor for osteoclast differentiation. NFATc1, in turn, induces osteoclast formation and bone-resorbing activity.

Here, we examined the effect of sudachitin on osteoclast differentiation and lipopolysaccharide (LPS)-induced inflammatory bone resorption, and found that sudachitin suppressed osteoclast formation and function both in vitro and in vivo.

Materials and Methods: In vitro osteoclast formation has been previously described.² Bone marrow cells obtained from 4~8-week-old male C57BL/6 mice were cultured for 3 days in the presence of M-CSF to isolate osteoclast precursors. The osteoclast precursors were then treated with M-CSF and soluble RANKL to form mature osteoclasts. After culturing for the desired time, TRAP-positive multinucleated cells formed in the culture, which were considered to be osteoclastic cells, were counted. Western blotting analysis and quantitative RT-PCR was performed according to the ordinary protocols. To evaluate the effect of sudachitin on in vivo LPS-induced calvarial bone destruction, 8-week-old mice were injected with LPS subperiosteally in the calvarial bone daily for 5 days. After 6 days, μCT scanning of the calvariae was performed. The mean values of the groups were compared by unpaired Student’s t test or by one-way ANOVAs. P < 0.05 was considered significant.

Results: When osteoclast precursors were treated with sudachitin in the presence of sRANKL and M-CSF, the number of TRAP-positive multinucleated osteoclasts formed in the culture was decreased compared with sudachitin-untreated culture in a dose-dependent manner [stachitin (10 µM) group vs. untreated group: 6.8 ± 0.9 cells/well vs. 625.3 ± 18.0 cells/well, p<0.05]. Since sudachitin had no effect on the proliferation and viability of osteoclast lineage cells, the inhibitory effect of sudachitin was attributed to the direct action in osteoclastogenesis. Consistent with the inhibition, sudachitin suppressed the activation of Erk signal in the osteoclast precursors. Furthermore, sudachitin decreased expression of c-fos and NFATc1 in mRNA and protein levels. Followed by the down-regulation of those transcription factors, the expressions of osteoclast differentiation-related molecules such as TRAP, cathepsin K, DC-STAMP and Atp6v0d2 were also decreased. In addition, sudachitin suppressed the production of intracellular reactive oxygen species. Consistent with the in vitro experiments, when inflammatory bone destruction of calvariae was induced by LPS injection, simultaneous administration of sudachitin (50 mM) with LPS reduced the elevated bone resorption as well as the increased TRAP and cathepsin K expression in vivo [stachitin (10 µM) group vs. untreated group: relative amount of TRAP mRNA; 4.69 ± 0.93 vs. 2.66± 0.30 , p<0.05; relative amount of cathepsin K mRNA: 6.0 ± 0.27 vs 2.52 ± 0.37 , p<0.05].

Conclusion: Sudachitin, a polymethoxyflavone, inhibits osteoclast formation and suppresses LPS-induced inflammatory bone resorption in mice. Therefore, sudachitin could be an effective component for the prevention of inflammatory bone disorders.

References

1. Yuasa K, Tada K, Harita G, Fujimoto T, Tsukayama M, Tsuji A: Sudachitin, a polymethoxyflavone from Citrus sudachi, suppresses lipopolysaccharide-induced inflammatory responses in mouse macrophage-like RAW264 cells. Biosci Biotechnol Biochem 76:598-600, 2012

2. Nakayachi M, Ito J, Hayashida C, Ohyama Y, Kakino A, Okayasu M, Sato T, Ogasawara T, Kaneda T, Suda N, Sawamura T, Hakeda Y. Lectin-like oxidized low-density lipoprotein receptor-1 abrogation causes resistance to inflammatory bone destruction in mice, despite promoting osteoclastogenesis in the steady state. Bone 75:170-182, 2015