Abstract
In chronic infectious diseases caused by gram-negative bacteria, such as osteomyelitis, septic arthritis, and periodontitis, osteoclastic activity is enhanced with elevated inflammation, which disturbs the bone homeostasis and results in osteolysis. Lipopolysaccharide (LPS), as a bacteria product, plays an important role in this process. Recent evidence shows that an antimalarial drug artesunate attenuates LPS-induced osteolysis independent of RANKL. In this study we evaluated the effects of artesunate on LPS-induced osteoclastogenesis in vitro and femur osteolysis in vivo, and explored the mechanisms underlying the effects of artesunate on LPS-induced osteoclast differentiation independent of RANKL. In preosteoclastic RAW264.7 cells, we found that artesunate (1.56−12.5 μM) dose dependently inhibited LPS-induced osteoclast formation accompanied by suppressing LPS-stimulated osteoclast-related gene expression (Fra-2, TRAP, Cathepsin K, β3-integrin, DC-STAMP, and Atp6v0d2). We showed that artesunate (3.125−12.5 µM) inhibited LPS-stimulated nuclear factor of activated T cells c1 (NFATc1) but not NF-κB transcriptional activity; artesunate (6.25, 12.5 μM) significantly inhibited LPS-stimulated NFATc1 protein expression. Furthermore, artesunate treatment markedly suppressed LPS-induced Ca2+ influx, and decreased the expression of PP2B-Aα (calcineurin) and pPLCγ1 in the cells. In addition, artesunate treatment significantly decreased the expression of upstream signals TLR4 and TRAF6 during LPS-induced osteoclastogenesis. Administration of artesunate (10 mg/kg, ip) for 8 days effectively inhibited serum TNF-α levels and ameliorated LPS (5 mg/kg, ip)-induced inflammatory bone loss in vivo. Taken together, artesunate attenuates LPS-induced inflammatory osteoclastogenesis by inhibiting the expression of TLR4/TRAF6 and the downstream PLCγ1-Ca2+-NFATc1 signaling pathway. Artesunate is a valuable choice to treat bone loss induced by gram-negative bacteria infection or inflammation in RANKL-independent pathway.
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Acknowledgements
This work was supported by the National Natural Science Foundation of China (81773740, 81073119, and 81611130078), the Natural Science Foundation of Guangdong Province, China (2015A030313287), and the Natural Science Foundation of Hainan Province, China (817129).
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XJL, XZZ and SWL designed the research; XZZ, YYZ, QY, SW, BHZ and YHT performed the research; XZZ, SW and MZ analyzed the data; XZZ, QY, XJL and SWL wrote the paper.
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Chemical compounds studied in this article: artesunate (PubChem CID: 16394563); cyclosporin A (CsA) (PubChem CID: 5284373); BAY11-7082 (PubChem CID: 5353431).
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Zeng, Xz., Zhang, Yy., Yang, Q. et al. Artesunate attenuates LPS-induced osteoclastogenesis by suppressing TLR4/TRAF6 and PLCγ1-Ca2+-NFATc1 signaling pathway. Acta Pharmacol Sin 41, 229–236 (2020). https://doi.org/10.1038/s41401-019-0289-6
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DOI: https://doi.org/10.1038/s41401-019-0289-6
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