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

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

Fig. 1

Artesunate inhibits LPS-induced osteoclastogenesis. a The chemical structure of artesunate. b Artesunate inhibits LPS-induced osteoclast formation as assessed by a TRAP assay in preosteoclastic RAW264.7 cells. c The osteoclast number was counted per well of each group. Bars show the mean ± SEM from three independent experiments. ***P < 0.001, compared with the LPS-treated group

Fig. 2

Artesunate suppresses LPS-induced osteoclast-related gene expression. The expression of osteoclast-specific genes was measured by real-time PCR in preosteoclastic RAW264.7 cells after artesunate and LPS administration. Bars represent the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, compared with the LPS-treated group

Fig. 3

Artesunate inhibits LPS-induced activation of NFATc1. a The NFAT luciferase activity of NFAT–luc-RAW264.7 cells and b the NF-κB luciferase activity of NF-κB-luc-RAW264.7 cells were measured. c mRNA levels of NFATc1 were assessed by real-time PCR. d Protein levels of NFATc1 were detected by Western blot (left panel). The relative density of nuclear NFATc1 was calculated (right panel). Bars represent the mean ± SEM from three independent experiments. ^###P < 0.001, compared with the untreated group; *P < 0.05, **P < 0.01, ***P < 0.001, compared with the LPS-treated group

Fig. 4

Artesunate suppresses LPS-increased Ca²⁺ levels and calcineurin expression by decreasing the phosphorylation of PLCγ1. a LPS-induced Ca²⁺ influx was determined by confocal microscopy. b The fluorescence intensity in 200 s was analyzed to indicate intracellular [Ca²⁺]. Protein levels of c PP2B-Aα and d phosphorylated PLCγ1 were analyzed by Western blot. Bars show the mean ± SEM from three independent experiments. ^##P < 0.01, ^###P < 0.001, compared with the untreated group; *P < 0.05, **P < 0.01, ***P < 0.001, compared with the LPS-treated group

Fig. 5

Artesunate suppresses LPS-induced expression of TLR4 and TRAF6. a mRNA levels of TLR4 and TRAF6 were determined by real-time PCR. b Protein levels of TLR4 and TRAF6 were detected by Western blot (left panel). The relative TLR4 and TRAF6 densities were calculated (right panel). Bars show the mean ± SEM from three independent experiments. ^###P < 0.001, compared with the untreated group; **P < 0.01, ***P < 0.001, compared with the LPS-treated group

Fig. 6

Artesunate prevents LPS-induced osteolysis in vivo. a Distal femur images of micro-CT analysis. b Graphical depiction of trabecular bone parameters, including BMD, BV/TV, and Tb.N. and Tb.Sp. by micro-CT. c Histological changes of tibial sections were assessed by H&E staining (×100). d Serum levels of TNF-α were measured using an ELISA kit. Data are representative of two independent experiments, and bars show the mean ± SEM (n = 5). ^#P < 0.05, ^##P < 0.01, compared with the control group; *P < 0.05, **P < 0.01, compared with the LPS-injected group