| Part 1.Inhibitory effects of aspirin on bone loss in murine calvarial osteolysis Objectives:To examine whether aspirin can suppress wear debris-induced bone destruction.Methods:All mice were divided into four groups:sham,Ti group,low-dose aspirin group(Ti/5 mg/kg/d aspirin)and high-dose aspirin group(Ti/30 mg/kg/d aspirin).The titanium particles were suspended in sterile normal saline.To establish murine calvarial osteolysis,the titanium particles were injected onto the calvaria of male C57BL/6J mice separately.The sham group was injected with normal saline on the surface of the calvaria,however aspirin treatment group were treated with various doses of aspirin(5 and 30 mg/kg)by gavage daily.After two weeks,mice were sacrificed and the skull of each murine was removed.The scan and reconstruction of collected calvariae by micro-CT and3-dimensional(3D)analyses of the skull in osteolysis was performed.H&E and TRAP staining was applied to analyze the calvarial absorptive area and the number of osteoclasts.Results:Compared with the sham group,3D reconstruction demonstrated that Ti group had more pores in quantity.However,aspirin treatment reduced the number of pores of calvaria osteolysis in a dose-dependent manner.In addition,the 3D reconstruction of calvarial osteolysis showed that aspirin enhanced bone volume(BV),bone volume/total volume(BV/TV)and bone mineral density(BMD)in region of interest.H&E staining showed that the eroded surface area and the bone thickness(BT)in Ti group were(0.1363±0.0071)mm~2 and(0.088±0.0032)mm,respectively.However,the eroded surface area and the bone thickness in the high-dose aspirin group were(0.0517±0.0033)mm~2,(0.1963±0.0130)mm(P<0.01).Collectively,aspirin suppressed bone loss in osteolysis model.TRAP staining analysis showed that the numbers of osteoclasts in calvarial histological sections of mice treated with low-and high-dose aspirin were(20.67±2.333)and(12.67±1.453),both of which were significantly lower than those of Ti group(35.67±4.333)(P<0.05).Conclusion:Aspirin abolished titanium particle-induced calvarial bone destruction by inhibiting the proliferation of osteoclasts.Part 2.The suppression and underlying mechanism of aspirin on osteoclast differentiation Objectives: To detect effects of aspirin on RANKL-induced osteoclast differentiation and its potential mechanisms.Methods: Bone marrow-derived macrophages(BMMs)and RAW264.7 cells were applied to examine the effects of aspirin on RANKL-induced osteoclast formation.After mice were sacrificed,BMMs from the femur and tibia were cultured.BMMs were cultured in plates and then cultured for CCK-8 assay to examine the viability of BMMs cell with aspirin treatment.In addition,BMMs were seeded in 24-well plates and cultured for 4 hours with essential medium,M-CSF(30 ng/ml)and different concentrations of aspirin(0,0.028,0.28,1.4 m M)for 5 days.The medium was changed every three days.After culture for five days,TRAP staining was used to assess the effect of aspirin on RANKL-induced osteoclast differentiation.In addition,BMMs cells were reseeded into Osteo Assay Plate for resorption pit assay and phalloidin staining,which aims to determine the effect of aspirin on the morphology and function of the osteoclastic F-actin ring.RT-PCR was applied to investigate effects of aspirin on the osteoclast-related genes.Furthermore,the effect of aspirin on the curcial signaling pathways(MAPKs,NF-κB and PI3K/Akt)during osteoclastogenesis was investigated by western blot analysis.In addition,oxidation of RANKL-induced ROS was quantified by DCF-DA with fluorescent microscopy and flow cytometry.Then,western blot analysis was used to determine the impact of aspirin on NOX-4 during osteoclast formation.Meanwhile,expression of NOX-4 in the murine calvaria of various group were analyzed by immunohistochemical.Results: In CCK-8 assay,there is no toxicity for BMMs with aspirin treatment(0.0028-1.4 m M).After 5 days,TRAP staining showed a large number of mature osteoclasts in RANKL group,whereas aspirin decreased the number of mature osteoclasts in aconcentration-dependent manner.The numbers of TRAP-positive multinucleated osteoclasts in the groups of 0.28 and 1.4 m M aspirin were(48.67 ± 5.487)/cm2 and(13 ± 2.646)/cm2,which was significantly less than the number in the RANKL group(189.3 ± 12.24)/cm2.Compared with non-aspirin group,the absorption area of 0.28 and 1.4 m M aspirin-treated groups were decreased by 56.75% and 64.33%,respectively(P<0.05).Phalloidin staining for F-actin ring showed that aspirin inhibited the morphology and number of osteoclastic F-actin rings.RT-PCR demonstrated that aspirin suppressed the expression of osteoclast-related genes.In addition,western blot analysis demonstrated aspirin significantly down-regulated the expression of RANKL-induced p-ERK,p-JNK,p-p38,c-Fos and NFATc1,while the expression of p-IκBα,p-p65,p-AKT1 was not significantly affected by aspirin.Fluorescent microscopy and flow cytometry analysis revealed that aspirin suppressed RANKL-induced ROS generation.And western blot analysis demonstrated that aspirin inhibited RANKL-induced NOX-4 expression.Meanwhile,immunohistochemical analysis showed that aspirin inhibited NOX-4 production in osteolysis model.Conclusion: Aspirin inhibits osteoclast differentiation,morphology and function by inhibiting MAPKs and NOX-4. |
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