The Study Of Melatonin In Alleviating Osteoporosis By Regulating Balance Between Osteogenesis And Osteoclastogenesis

BackgroundMelatonin is a neurohormone involved in a large number of physiological activities,especially bone homeostasis.Osteoporosis is a metabolic bone disease characterized by bone mass decrease and microarchitectural deterioration as the result of imbalance between resorption and formation,which frequently increases bone fragility and fracture risk,and also predisposes us to an inappropriate situation for maxillofacial bone defect repair or dental implant placement.The epidemiological survey suggests that melatonin supplement can improve bone mass profoundly in postmenopausal women.However,how melatonin directs bone remodeling and the role of bone marrow mesenchymal stem cells(BMMSCs)in regulating melatonin-mediated bone formation-resorption balance still remain undefined.AimUsing the bone formation-resorption balance as the entry point,on the basis of female osteoporosis mouse model,this study aimed: 1.To identify the effects ofmelatonin treatment on osteoporosis.2.To explore the influences and molecular mechanisms of melatonin in regulating BMMSCs' osteogenesis.3.To clarify the role of melatonin on regulating BMMSC-mediated osteoclastogenesis,and the detailed mechanisms.Materials and Methods1.Osteoporosis models were established with female C57BL/6J mice by ovariectomy.After melatonin treatment,bone microstructure was observed using ?CT and H&E staining.What's more,osteogenic markers,osteoclastogenic markers and inflammatory cytokines in bone tissue and serum were detected using q RT-PCR,immunohistochemistry and ELISA.2.In order to identify which melatonin receptor(MT receptor)plays the main role in responding to melatonin,the MT receptors were selectively knocked down by lentiviral sh RNA transfection.ALP staining,Alizarin Red S staining,q RT-PCR,ELISA and western blot were used to detect the effects of melatonin and MT receptors on regulating BMMSCs' osteogenesis,and to explore the complex interplays among melatonin,MTs and NF-?B signaling pathway.Moreover,the NF-?B inhibitor JSH-23 was also employed to unveil the role of this classical pathway in osteoporosis.3.Both direct-contact and indirect-contact co-culture systems were established to gain a better understanding of the interaction patterns between BMMSCs and osteoclast precursors.Besides,the osteoclastogenic differentiation and maturation of osteoclast precursors were assessed by TRAP staining and western blot.In addition,the role of melatonin during the process was thoroughly investigated.Results1.After melatonin administration,the expressions of osteogenic markerssignificantly improved,the levels of osteoclastogenic markers and inflammatory cytokines obviously decreased in bone tissue and serum,and the bone mass loss and microstructure disorder in osteoporotic mice were profoundly reversed.2.Melatonin significantly promoted the osteogenesis of BMMSCs at a low physiological concentration(10-100 n M),presented with enhanced of ALP level and mineralization of BMMSCs,and selective elevations of Runx2,Osterix and OCN expressions,without any impact on Col-I.3.The MT2 receptor played the main role in melatonin regulating BMMSCs' osteogenesis.We found that,the elevation of osteogenic markers(including increased ALP,Runx2,Osterix and OCN expressions,and augmented cell mineralization)of BMMSCs with melatonin treatment was almost cancelled after MT2 knockdown,but nor did it happen after MT1 knockdown.4.The MT2-mediated NF-?B inactivation was responsible for melatonin-regulated osteogenesis.After BMMSCs were treated with melatonin,the NF-?B signaling was obviously suppressed,as phosphorylated I?B?,p65,IKK?/? dramatically decreased.Furthermore,the inhibitory effects of melatonin on NF-?B activation was obviously weakened in MT2-knockdown BMMSCs,but not in MT1-knockdown cells.What's more,we found JSH-23 could add extra positive effects of melatonin on BMMSCs' osteogenic differentiation.5.Melatonin did not have direct effects on osteoclastogenic differentiation of osteoclast precursors.However,melatonin successfully inhibited the production of RANKL and increased the ratio of opg/rankl in BMMSCs via down-regulating MT2-mediated NF-?B pathway.6.Melatonin indirectly inhibited the process of osteoclastogenesis by regulating BMMSCs' paracrine secretion function through MT2-mediated NF-?B pathway.Inindirect-contact co-culture system filled with BMMSCs and osteoclast precursors,melatonin significantly suppressed the osteoclastogenesis,nor did in the direct-contact co-culture system.In addition,keeping the same pace with the production pattern of RANKL in BMMSCs,the inhibitory effects on osteoclastogenesis by melatonin was promoted by JSH-23,unaffected by MT1-knockdown,but significantly reduced by MT2-knockdown.ConclusionOur study identifies that melatonin will probably become a potential therapeutic medicine for postmenopausal osteoporosis in the future,as melatonin exerts both anti-inflammation and bone-protective effects in osteoporotic mice.Mechanistically,to our knowledge,it is the first study demonstrating that MT2-mediated NF-?B signaling pathway is involved in the osteogenic and anti-osteoclastogenic effects of melatonin.What's more,melatonin indirectly inhibits osteoclastogenesis via affecting BMMSCs' paracrine cytokine secretion,and RANKL-RANK is probably one of the most principal paracrine axes linking BMMSCs and osteoclast precursers.Taken together,we demonstrate that,melatonin could successfully alleviate osteoporotic process by up-regulating BMMSCs' osteogenesis and indirectly down-regulating BMMSC-mediated osteoclastogenesis via inactivating MT2-mediated NF-?B signaling pathway in physiological environment.