Melatonin Attenuates Peripheral Osteolysis Via Inhibition Of NF-?B Signaling Pathway

Part 1.Establishment of mice osteolysis skull model and evaluation of inhibitory effects of melatonin on osteolysisObjectives: To explore the effects of melatonin on wear debris-induced inflammatory bone erosion.Methods: Establishment of mice osteolysis skull model: 60 male 7-8-week-old C57BL/6 mice were randomly divided into 4 groups: normal control group(control),skull grafted with Ti particles only group(vehicle),skull grafted with Ti particles then Intraperitoneally injection of low-dose melatonin group(low),skull grafted with Ti particles then Intraperitoneally injection of high-dose melatonin group(high).From the first day postoperatively,mice in control group received no further intervention,high-,low-dose melatonin group were intraperitoneally injection of 200 microliters of PBS containing 50mg/kg and 5mg/kg melatonin.Vehicle group were administrated equal amount of PBS only.2 weeks post-operatively,skull were harvested for micro-CT,histological stains and Immunohistochemistry analyses.Supernatant of skull medium for enzyme-linked immunosorbent assay(ELISA).Results:3D construction of ?CT showed that resorption lacuna was intensively distributed on the calvarial in the vehicle group,while bone erosion was obviously abrogated dose-dependently in the low-and high melatonin groups.Quantitively,bone mineral density(BMD)and bone volume/total volume(BV/TV)were significantly elevated after melatonin treatment.In the meanwhile,H&E staining of bone erosion and the thickness of the periosteum is significantly reduced after melatonin administration.Furthermore,TRAP stain shows number of osteoclast was45.81±5.72?14.63±3.91 in ROI,which was significantly different from that in the vehicle group((66.40±10.43).Additionally,immunohistochemical analysis revealed that melatonin inhibits the inflammatory response of wear debris-irrigated osteolysis.Consistent with IHC results,ELISA results suggest that level of inflammatory factors including IL-1?,IL-6,TNF-? in the vehicle group are 182.5±16.73 pg/ml,182.76±13.97 pg/ml,298.34±24.63 pg/ml.while level of such factors exhibited significantly dose-dependent reduction in the low-melatonin group(namely,145.76±12.33 pg/ml,144.38±14.38 pg/ml,167.03±17.06 pg/ml).Conclusion: Melatonin has obvious inhibition of periprosthetic osteolysis,and this inhibitory effect were mediated by inhibiting inflammatory reaction and expression of osteoclasts.Part 2.Culture of bone marrow-derived macrophages(BMMs)and investigation of melatonin inhibit osteoclastic differentiationObjectives: To investigate effects of melatonin on differentiation and function of osteoclasts as well as possible mechanisms.Methods: Bone marrow-derived macrophages(BMMs)were cultured and cultured with different concentration melatonin(0,0.25,0.5,1m M)for 4 hours.Then M-CSF(30ng/ml)and RANKL(50ng/ml)were added.5 days later,Tartrate-resistant acid phosphatase(TRAP)staining was performed to evaluate whether differentiation of osteoclast precursor cells was altered by melatonin.In addition,bone absorption test was used to detect the activity of osteoclasts after treatment with melatonin.Western Blot was performed to detect key proteins of signaling pathway involved in regulation of osteoclast-activation.Results: After cultured with RANKL and M-CSF for 5 days,BMMs were then stained for TRAP,results revealed that purple red mature osteoclasts were intensively visible in RANKL group,and count of mature osteoclast in 0.25-1m M melatonin groups revealed dose-dependently decrease,and Image J showed there were 330.03±20.12 /cm2?130.35±21.25 /cm2 ? 65.33±11.93 /cm2 osteoclasts,respectively.There was significant reduction in comparison of RANKL group(228.3 ± 22.4 /cm2)(p<0.01).Furthermore,bone absorption area in 0.25 and 1 m M melatonin group revealed 47.33% ?63.67% decrease in comparison with control group when OAP was cultured with different concentration of melatonin for 5 days(p<0.05).And the morphology and number of F-actin was dramatically altered by melatonin in a concentration-dependent manner.Western blot showed the presence of RANKL and Ti particles irrigated significant expression of p-I?B??p-P65?c-Fos as well as NFATc1,while level of these proteins was obviously inhibited by melatonin,However,phosphorylation of IKK??ERK?JNK?P38?AKT1 was not significantly altered by melatonin.Conclusion: Melatonin is effective in suppressing early differentiation of osteoclasts and its function.These results may due to the effect of melatonin on abrogating NF-?B signaling pathway as well as down-stream cascades.