The Molecular Mechanism Of Aspirin Inhibiting Osteoclastogenesis

Background The dynamic balance of osteoclast and osteoblast is a critical factor of bone homeostasis, and the formation and function of osteoclast are based on the fusion of macrophage precursor cell. Meanwhile, RANKL, a tumor-necrosis factor(TNF) related cytokine and a key factor of osteoclast differentiation, when bonding with RANK, stimulates cytoplasmic tumor-necrosis factor receptor-associated factor 6(TRAF6) and correspondingly activates the downstream signaling pathways, such as p38 MAPK, c-jun-N-terminal kinase(JNK), nuclear factor-kappa b(NF-κB) and extra-cellular signal-regulated kinase(ERK), As the result, through a series of activation, osteoclast progenitors fuse into mature multi-nucleated osteoclasts, expressing a variety of protein makers, like CTSK, TRAP, CTR and MMP-9. Osteoporosis is regarded as a metabolic disease, with characteristics of bone mass loss and fracture risk increase, which is an acknowledged public health problem in an aging society. Several anti-resorptive agents such as bisphosphonates, calcitonin and estrogen, have been widely applied on the treatment of osteoporosis, however, each ofthem has its clinical limitation and side-effects such as induction of breast cancer, osteonecrosis and vaginal bleeding. Thus, a much safer therapeutic strategy is required to prevent and/or treat lytic bone diseases including osteoporosis. Aspirin is a widely-used and safe compound applied as an effective antipyretic, analgesic and anti-inflammatory drug. However, more effects of aspirin needs to be found. Besides, based on the epidemiological study and our preliminary research, it is proved that aspirin has the effect of anti-postmenopausal osteoporosis in ovariectomized rat model, which indicate a possible clinical application for aspirin in the prevention of bone loss. Yet its detailed molecular associated mechanism has not been clarified.Objective The purpose of this study was to assay the effects of different concentrations of aspirin on the differentiation of osteoclast in RANKL-induced RAW264.7 cells, and the expression of NF-κB and MAPKs signaling pathways in osteoclasts, to investigate the possible molecular mechanism of aspirin in prevention and treatment of osteoporosis, so that to provide a new theoretical basis for the innovation application of aspirin to prevent and treat postmenopausal osteoporosis.Methods RAW264.7 cells were cultured and passaged in conventional mehtods, when growth up to 80% confluence, cells were harvested and used for experiments. ②The experiment is divided into six groups: group A was negative control group which was conventional culture medium didn’t contain aspirin and RANKL; group B was positive control group which was conventional culture medium contain 100ng/ml RANKL; Group C-F were the experimental groups added different concentrations of aspirin(group C 0.25 mmol / L, group D 0.5mmol / L, group E 1.0mmol / L, group F 1.5mmol / L), and all containing 100ng/ml RANKL. ③ Raw264.7 cells in logarithmic phase were seeded on 96-well plates at a density of 104 Per well and co cultured with different concentrations of aspirin group for 24 hour, then were used to the conventional MTT detecting the relative number of live cells. ④Raw264.7 cells in logarithmic phase were seeded on slice in 96-well plates at adensity of 2×105 Per well, Cultured in the common medium for 18 hour, and incubated with each group for 5 days, then were stained by Tartrate-Resistant Acid Phosphatase(TRAP). The three or more nuclei of TRAP-positive cells were considered osteoclast in slice observed under an optical microscope and counting(arbitrarily selected three regions and averaged). ⑤Raw264.7 cells in logarithmic phase were seeded on 6-well plates at a density of 2×105 Per well, cultured in the common medium for 18 hour, and incubated with each group for 5 days, then osteoclast marker genes(TRAP, MMP-9, CTSK and CTR) m RNA expression were detected by the method of Real Time PCR. ⑥ Raw264.7 cells in logarithmic phase were seeded on 6-well plates at a density of 2×105 Per well, cultured in the common medium for 18 hour, and incubated with each group for 2 hours, then Western blot assay of three NF-κB signaling molecule protein(IκB-α, p50, p65 and p-IκB-α, p-p50, p-p65) and three MAPKs channel signaling molecule protein(ERK, p38, JNK and p-ERK, p-P38, p-JNK) expression in osteoclasts. ⑦Raw264.7 cells in logarithmic phase were seeded on slice in 6-well plates at a density of 2×105 Per well, cultured in the common medium for 18 hour, and incubated with each group for 2 hours, then detected the NF-κB P65 translocation to nucleus by immunofluorescence method.Results:(1) Within the concentration range of aspirin using in the culture, aspirin have no cytotoxicity to these cells.(2) Each group had lots of polynuclear large cells And with the intervention of aspirin concentration increased, the number of osteoclasts gradually reduced in a dose-depended manner.(3) After induced by RANKL, the m RNA expression of osteoclast markers TRAP, CTSK, MMP-9, and CTR, increased respectively, however, decreased for aspirin intervention, and the higher solubility of aspirin, the less expression of the markers, which is called dose-depended manner.(4) In RAW264.7 cells induced by RANKL, aspirin significantly inhibited the degradation of IκB-α and its phosphorylation. In addition, we also researched thephosphorylation of p50 and p65, and found that aspirin significantly reduced p50 and p65 phosphorylation.(5)Aspirin, in RANKL-induced RAW264.7 cells, significantly inhibited the expression of p-ERK, p-P38 and p-JNK, but has little effect on the expression of the non-phosphorylated ERK, JNK and p38 protein.(6)Compared with the untreated cells, P65 obviously translocated from the cytoplasm to the nucleus in the RANKL induced cells. The cells pretreated by 1m M aspirin, showed the inhibition effect of P65 translocation.Conclusion: Aspirin inhibits the osteoclast formation in RANKL-induced RAW 264.7 cells. Aspirin also reduces the expression of the osteoclast marker genes in the RANKL-induced osteoclast. In addition, aspirin inhibits the activation and phospholation of P38, JNK, ERK, and NF-κB moleculars. The translocation of P65 from cytoplasm to nucleus is reduced by aspirin in RANKL-induced RAW264.7 cell line. Although the in vivo effect of aspirin for the treatment ofosteoporosis needs more clinical research to prove, the results of our current study suggests that aspirin may be a potentially effective drug for the treatment of osteoporosis.