Article

Artesunate attenuates LPS-induced osteoclastogenesis by suppressing TLR4/TRAF6 and PLCγ1-Ca2+-NFATc1 signaling pathway

Authors: Xiang-zhou Zeng1,2,3, Yue-yang Zhang1,2, Qin Yang1,2, Song Wang4, Bin-hua Zou1,2, Yan-hui Tan1,2, Min Zou2, Shu-wen Liu2, Xiao-juan Li1,2
1 Laboratory of Anti-inflammatory and Immunomodulatory Pharmacology, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China
2 Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China
3 Department of Pharmacology, Hainan Medical College, Haikou 571199, China
4 Department of Surgery, Guangdong Hospital of Traditional Chinese Medicine, Guangzhou 510120, China
Correspondence to: Shu-wen Liu: liusw@smu.edu.cn, Xiao-juan Li: lixiaoj@smu.edu.cn,
DOI: 10.1038/s41401-019-0289-6
Received: 6 May 2019
Accepted: 11 July 2019
Advance online: 20 August 2019

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.
Keywords: artesunate; osteoclast; LPS; PP2B-Aα; Ca2+; NFATc1; TLR4; RAW264.7 cells; chronic infectious diseases

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