Effects Of Artesunate On Osteoclasts And The Molecular Mechanisms

BackgroundThe metabolism of bone is the balance between resorption of bone by osteoclasts and the synthesis of bone matrix by osteoblasts. However, when the balance is broken, it may lead to bone disease.The osteoclast is a unique bone-resorbing cell with bone resorption activity in vivo. Bone damage caused by excessive differentiation and activation of osteoclasts leads to some common diseases, such as OP (osteoporosis), RA (rheumatoid arthritis), OA (osteoarthritis), etc. At present, drugs used to treat these diseases mainly include corticosteroids or nonsteroidal anti-inflammatory drugs, antibiotics and immunosuppressive agents etc. However, these agents described as above can not ameliorate the bone destruction and some of them will cause adverse reactions, such as inducing uterine cancer, ovarian cancer and breast cancer and increase the risk of cardiovascular disease to some degree. What is more, some traditional anti-inflammatory agents such as dexamethasone, Methotrexate (MTX) etc. can even promote osteoclast formation and bone destruction. Developing an agent targeting inhibition of osteoclast activation is considered to be the most effective to reverse the bone destruction and the pain caused by osteoclast-related diseases.Osteoclasts are differentiated from hematopoietic macrophage/monocyte lineage precursor cells in several steps, including proliferation, differentiation, fusion and activation. RANKL (receptor activation of nuclear factor NF-κB ligand) and M-CSF (macrophage-colony stimμlating factor) are essential for osteoclastogenesis. During RANKL-induced osteoclastogenesis, the binding of RANKL to its receptor RANK (receptor activation of nuclear factor NF-κB) leads to the recruitment and activation of adaptor TRAF6 and triggers activation of MAP kinases (ERK, JNK and p38) and transcription factors such as NF-κB, AP-1, and NFAT. The activation of these transcription factors can consequently regulate the osteoclast differentiation by up-regulating the expression of osteoclast-associated genes.Lipopolysaccharide (LPS) is a main component of the Gram-negative bacterial cell wall. It is well-known that LPS is an important pathogen in inflammatory bone loss and the first potent bone resorbing factor in vitro. LPS induces bone loss, which is associated with the differentiation and activation of osteoclasts. The mechanisms of LPS inducing the differentiation and activation of osteoclasts show as follow. LPS enhances RANK signal and COX-2 expression to promote the differentiation and activation of osteoclasts. LPS may induce production of various cytokines such as TNF-α, which induce the differentiation and activation of osteoclasts. Moreover, LPS may also initiate osteoclast formation and facilitate the fusion and survival of osteoclasts.Artesunate is a semi-synthetic derivative from artemisinin, one of the most effective drugs for clinical treatment of falciparum malaria with low toxicity. However, in recent years, more and more studies showed that artesunate has the inhibitory effect of anti-inflammation. Artesunate can suppress NF-κB, IκB, TNF-α, IL-6 and Th response to ameliorate the colitis, sepsis and inhibit arthritis, adjuvant arthritis or arthritis osteoporosis induced by type Ⅰ collagen. Some of these diseases above belong to osteoimmunology diseases, which caused by excessive activation of osteoclasts, increase of bone destruction. However, there were no reports on whether artesunate can influence osteoclastogenesis and ameliorate osteoclast-related diseases. In this study, we established RANKL-induced and LPS-induced osteoclast culture system in vitro, ovariectomy-induced osteoporosis animal model and LPS-induced acute bone destruction model to investigate the regulating effects and molecular mechanisms of artesunate on osteoclasts.There are three sections in this study.1.Effects of artesunateon RANKL-induced osteoclasts and ovariectomy-induced osteoporosis mouse model.2.The molecular mechanisms of action of artesunate on RANKL-induced osteoclasts.3. Effects of artesunate on LPS-induced osteoclasts and acute bone destruction mouse model and the molecular mechanisms.Methods1. Using MTT method to investigate the cytotoxicity of varying concentration of artesunate (3.125,6.25 and 12.5 μM) on RAW264.7 cells for 8 h,24 h,48 h and 7 d, and then determine the maximal concentration applying to other experiments in vitro. A culture system of osteoclast formation induced by RANKL from RAW264.7 cells or bone marrow macrophage (BMMs) was established in vitro. The effects of varying concentration (3.125,6.25 and 12.5μM) of artesunate on RANKL-induced osteoclast differentiation from RAW264.7 cells and BMMs were assessed via tartrate-resistant acid phosphatase staining (TRAP) staining assay, which was further confirmed by examining expression of osteoclast-related genes such as c-Src, Fra-2, TRAP, β3-Integrin, Cathepsin K, MMP-9, DC-STAMP and Atp6v0d2 using Real-time PCR. The effects of varying concentration (3.125,6.25 and 12.5μM) of artesunate on osteoclast resorption activity were evaluated by bone resorption pits assay.The ovariectomized mouse model was made and osteoporosis was induced by ovariectomy. Using zoledronic acid as positive control, the effect of artesunate on osteoclast-related bone destruction in ovariectomized mouse was evaluated through examining the values of bone parameters including BMD, BV/TV, Tb.N and Tb.Sp of mouse femur and the cytokines such as RANKL, OPG, RANKL/OPG and ALP.2. Studies on the underlying molecular mechanisms of artesunate action on suppresing RANKL-induced TRAP-positive osteoclast formation and bone resorption. Firstly, Real-time PCR assay, Western blot analysis and luciferase reporter gene assay were carried out to determine expression levels of mRNA of transcriptional factor NFATc1, NFATcl protein and luciferase reporter gene. Effects of artesunate on the activation of nuclear transcription factor NFATcl were assessed at mRNA, protein and transcriptional activity levels. Furthermore, we measured the protein expression of NFATcl signaling pathway upstream molecule (calcineurin) by Western blot analysis. Fluo-3/AM-ester was used as Ca2+ probe to detect the effect of artesunate on the intracellular calcium ion signal characteristics in RANKL-induced RAW264.7 cells with Laser Scanning Confocal Microscope (LSCM). We also examined the effect of artesunate on RANKL-induced phosphorylation of PLCyl via Western blot and the expression of signaling molecules (RANK, TRAF6), upstream of RANKL/RANK signaling pathway by Real-time PCR and Western blot respectively. Finally, we investigate the effect of artesunate on NF-κB transcriptional activity by the luciferase activity assays of NF-κB.3. A culture system of osteoclastogenesis induced by LPS from RAW264.7 cells was established in vitro. The effects of different concentrations of (3.125,6.25 and 12.5μM) artesunate on LPS-induced osteoclastogenesis from RAW264.7 cells were assessed via TRAP staining. To further verify inhibitory effect of artesunate on osteoclastogenesis, the expression of osteoclast-related genes (Fra-2, TRAP, β3-Integrin, Cathepsin K, DC-STAMP and Atp6v0d2) induced by LPS was detected. Using ELISA technique, we investigated the effect of varying concentrations of artesunate (3.125,6.25 and 12.5 μM) on the release of TNF-a in LPS-induced RAW264.7 cells in vitro. To make model of LPS-induced acute bone damage in mouse, LPS (5 mg/kg) was given by intraperitoneal injection at 0 and 4 d respectively. Further, the mouse was treated with artesunate (i.g.10 mg/kg/d) for a total of 8 d. The effect of artesunate on osteoclast-related bone destruction in ovariectomized mouse was evaluated through examining the values of serum TNF-a, the cytokines such as RANKL, OPG, RANKL/OPG and ALP and bone parameters of mouse femur including BMD, BV/TV, Tb.N and Tb.Sp.Studies on the underlying molecular mechanism of artesunate action on suppresing LPS-induced osteoclastogenesis were taken as follow. Firstly, we evaluated the effect of artesunate on the nuclear transcription factor NFATcl activation through assessing expression of mRNA, protein and transcriptional activity of NFATcl by Real-time PCR, Western blot and luciferase reporter gene assay, respectively. Furthermore, we measured the protein expression of NFATcl signaling pathway upstream molecule (calcineurin) by Western blot analysis. Fluo-3/AM was used as Ca2+ probe to detect the effect of artesunate on the intracellular calcium ion signal characteristics in LPS-induced RAW264.7 cells with Laser Scanning Confocal Microscope (LSCM). We also examined the effect of artesunate on LPS-induced phosphorylation of PLCyl via Western blot and the expression of upstream signaling molecules (TLR4, TRAF6) of signaling pathway by Real-time PCR and Western blot respectively. Finally, we investigate the effect of artesunate on NF-κB transcriptional activity by the NF-κB luciferase activity assays to determine whether artesunate could impact on NF-κB signaling pathway.Results1. Artesunate (0-12.5 μM) had no significant cytotoxicity to RAW264.7 cells within 7 d except for 94.55%(P<0.05),85.61%(P<0.05) and 91.60%(P<0.05) survival rate of RAW264.7 cells impacted by 12.5 μM artesunate at 24 h,48 h and 7 d respectively. Varying concentration of artesunate (3.125,6.25 and 12.5 μM) could significantly inhibit RANKL-induced OCs differentiation from RAW264.7 (F=124.566, P<0.001) or BMMs (F=79.919, P<0.001) in a concentration-dependent manner in vitro. The results of bone absorption pit assay show that varying concentration of artesunate (3.125,6.25 and 12.5 μM) could significantly decrease the bone resorption area in a concentration-dependent manner (F=50.855, P=0.003). We also found that artesunate could significantly inhibit the expression of RANKL-induced osteoclast-related genes such as c-Src(F=245.807, P<0.001), Fra-2 (F=102.631, P<0.001), β3-Integrin (F=21.97, P<0.001), Cathepsin K (F=510.220, P<0.001) and MMP-9 (F=88.322, P<0.001) by Real-time PCR. Moreover,12.5 uM artesunate could also significantly inhibit the mRNA expression of TRAP (F=403.031, P<0.001), DC-STAMP (F=23.369, P=0.001) and Atp6v0d2 (F=427.932, P<0.001).Through Micro-CT scanning analysis of mouse femur imaging features, we found that 30 mg/kg artesunate and zoledronic acid could significantly ameliorate osteoporosis in ovariectomized mice with increasing bone volume, the number of trabecular bone and trabecular bone density.These results above suggest that artesunate could rescue bone loss in osteoporosis mice and protect bone matrix. Analyzing the parameters of the mouse femur, we also found that both artesunate and positive control drug zoledronic acid could significantly increase bone mineral density (F=62.597, P<0.001), bone volume fraction (F=32.563, P<0.001), trabecular number (F=18.005, P<0.001), decrease trabecular separation (F=25.557, P< 0.001). Meanwhile, artesunate could reduce serum RANKL level (F=l 1.208, P<0.001), TRAP-5b enzyme activity (F=6.937, P<0.001) and RANKL/OPG ratio (F=22.399, P<0.001) but increase OPG level (F=24.557, P<0.001) and ALP activity (F=3.201, P<0.01) in ovariectomized mice.2. The mechanism research found that both 3.125 μM (P<0.001) and 12.5 μM (P<0.001) artesunate could concentration-dependently inhibit the mRNA expression of RANKL-induced NFATc1 gene (F=123.524, P<0.001). Varying concentration of artesunate (3.125、6.25 and 12.5 μM) could significantly increase the expression of p-NFATc1 protein in cytoplasm and suppress the expression of NFATcl protein in nucleus. That was, artesunate could significantly inhibit RANKL-induced nuclear translocation of NFATc1. Both varying concentration of artesunate (3.125、6.25 and 12.5 μM) and 1 μM CsA, the positive control drug, could significantly reduce the expression of NFAT luciferase reporter gene increased by RANKL stimulation (F=26.590, P<0.001). Taken together, these results show that artesunate could inhibit the RANKL-induced activation of nuclear transcription factor NFATc1. Artesunate (3.125-12.5 μM) significantly inhibited the upregulation of PP2B-Aa, upstream molecular of RANKL signal pathways for NFATc1. Both 3.125 and 12.5 μM artesunate could significantly reduce the intracellular Ca2+ concentration, especially 12.5 μM artesunate and positive control drug CsA reducing intracellular Ca2+ concentration closed to the control group (F=19.570, P<0.001). (3.125-12.5 μM) artesunate concentration-dependently inhibited the level of p-PLCyl protein, upstream molecular of Ca2+ signaling pathway. Artesunate could inhibit the expression of gene and protein of TRAF6, upstream signaling molecule of RANKL/RANK signaling pathway (F=41.395, P<0.001). Artesunate (12.5 μM) could also significantly inhibit the mRNA expression of upstream signaling molecule RANK (F=30.819, P<0.001). BAY11-7082, NF-κB positive control inhibitor, at the concentration of 5 μM could significantly inhibit the increase of expression of NF-κB reporter gene stimulated by RANKL (inhibition of approximately 50%,P<0.05). However, only 50μM artesunate could suppress the increase of RANKL-induced NF-μB reporter gene expression and 3.125-25 μM artesunate revealed no inhibitory effect. Therefore, suppression of the osteoclast formation and osteoclast resorption by artesunate might be independent on inhibiting NF-κB signaling pathway.3. Results of TRAP stain assay show that varying concentration of artesunate (3.125,6.25 and 12.5 μM) could significantly inhibit LPS-induced OCs differentiation from RAW264.7 in a concentration-dependent manner in vitro (F=67.221, P<0.001). Varying concentration of artesunate (3.125、6.25 and 12.5 μM) could significantly inhibit the level of TNF-a in supernatant of LPS-stimulated RAW264.7 in concentration-dependently manner (F=117.712, P<0.001). Artesunate could significantly inhibit the expression of LPS-induced osteoclast-related genes including Fra-2 (F=17.440, P<0.001), TRAP (F=40.550, P<0.001), Cathepsin K (F=26.400, P<0.001), DC-STAMP (F=12.870, P=0.002) and Atp6v0d2 (F=161.500, P<0.001) by Real-time PCR. Moreover,12.5 μM of artesunate could also prominently suppress the level of β3-Integrin mRNA expression (F=30.900, P<0.001). Through Micro-CT scanning analysis of mouse femur imaging features, we found that 10 mg/kg artesunate can recover the reduction of bone mass in model mice stimulated by LPS. Through the analysis of mouse femoral bone parameters, we found that treatment with 10mg/kg artesunate could significantly enhance the bone mineral density (F=7.217, P=0.014), bone volume fraction (F=6.621, P=0.017), trabecular bone number (F=6.561, P=0.018) and decrease trabecular separation (F=5.778, P=0.024). In addition, artesunate (10 mg/kg) can significantly reduce serum TNF-α(F=10.85, P=0.004).The results concerning on mechanisms of artesunate suggest that 3.125 and 12.5 μM artesunate concentration-dependently could inhibit the level of LPS-induced NFATc1 gene expression (F=90.330, P<0.001). Artesunate (3.125-12.5 μM) could concentration-dependtly increase the protein expression of cytosolic p-NFATc1 and suppress the protein expression of nuclear NFATcl significantly. That was, artesunate could significantly inhibit LPS-induced nuclear translocation of NFATcl. Treatment with artesunate (12.5 μM) and CsA (1 μM) could significantly down-regulate the expression of LPS-induced NFAT luciferase reporter gene (F=26.321, P<0.001). All in all, these results show that artesunate could inhibit the LPS-induced activation of nuclear transcription factor NFATc1. Artesunate (3.125-12.5 μM) significantly inhibit the upregulation of PP2B-Aa. Treatment with 3.125(P<0.001) and 12.5(P<0.001) μM artesunate significantly reduced the intracellular Ca2+ concentration concentration-dependently (F=38.618, P<0.001). (3.125-12.5μM) artesunate concentration-dependently inhibited the level of p-PLCyl protein, upstream molecular of Ca2+ signaling pathway. The gene and protein expressions of the upstream signaling molecule TRAF6 of signaling pathway were also inhibited (F=24.862, P<0.001). Treatment with 12.5 μM artesunate could significantly inhibit the gen expression of upstream signaling molecule TLR4 (F=9.558, P=0.014). BAY11-7082, NF-κB positive control inhibitor, at the concentration of 5 μM could significantly inhibit the increase of NF-κB reporter gene expression stimulated by LPS (P<0.001). However, only 25 (P,0.05) and 50 μM (P<0.01) artesunate could suppress the increase of RANKL-induced NF-κB reporter gene expression and 3.125-25 μM artesunate revealed no inhibitory effect (P>0.05), which suggest that inhibitory of artesunate on NF-κB signaling pathway might not contribute to suppressing osteoclast formation and bone resorption.Conclusions1. Artesunate could significantly suppress osteoclastogenesis induced by RANKL and LPS, and its bone resorption function.2. Artesunate could ameliorate bone destruction caused by excessive activation of osteoclasts in both ovariectomized mice and LPS-induced acute bone destruction mice, and enhance bone density. Artesunate could potentially have therapeutic applications to bone destructive diseases such as postmenopausal osteoporosis.3. The adapter molecule TRAF6 and nuclear transcription factor NFATc1 were mainly potential targets for artesunate.Artesunate might suppress osteoclast formation and bone resorption activity via inhibiting TRAF6-Ca2+-calcineurin-NFATcl signaling pathway.