Role And Mechanism Of Melatonin Synergistically With BMP9-induced Osteogenic Differentiation Of Mesenchymal Stem Cells

[OBJECTIVE] Mesenchymal stem cells(MSCs)have the potential for self-renewal and multi-directional differentiation,and can differentiate into osteoblasts,adipocytes and chondrocytes,which play a vital role in maintaining the dynamic balance of bone metabolism.Melatonin is a hormone secreted by the pineal gland.In addition to the well-known diurnal regulation function,melatonin can regulate bone metabolism by regulating the lineage stereotypes and differentiation signaling pathways of MSCs,but the mechanism of action is still unclear.Our previous study found that melatonin can promote the expression of osteogenic differentiation markers of MSCs induced by BMP9(Bone morphogenetic protein 9),so hypothesis: melatonin promotes BMP9-induced osteogenic differentiation of mesenchymal stem cells.This study aims to further validate this hypothesis.[METHODS] 1.Whether melatonin can promote the elevation of osteogenic differentiation markers induced by BMP9 at various stages.Mesenchymal stem cells C3H10T1/2 cells were treated according to the control group,melatonin group,BMP9 group,BMP9 and melatonin co-culture group.Through the quantitative detection and staining qualitative detection of early osteogenic markers alkaline phosphatase activity,the effects of melatonin and/or BMP9 on early osteogenesis of mesenchymal stem cells were observed;through the late osteogenic markers Osteocalcin and Osteopontin was stained with immunohistochemistry to observe the effects of melatonin and/or BMP9 treatment on the late osteogenesis of mesenchymal stem cells.Alizarin red S staining was performed to observe the formation of mineralized nodules in mesenchymal stem cells.Impact.2.According to the above grouping,the in vitro culture of fetal rat limbs and stem cell implantation in nude mice further verified whether melatonin and BMP9 treatment can synergistically induce osteogenesis.The limbs of E18.5 fetuses were taken in a clean bench,and the skin and muscles were preserved.After the corresponding treatment,the limbs were observed under a fluorescence microscope,and then the limbs were fixed for H&E staining to observe the cartilage formation of the fetus limbs;C3H10T1/2 cells were treated with melatonin and/or BMP9,induced in osteogenic induction medium for 7days,implanted in 4w male nude mice,and 6 weeks later,ectopic bone was removed for Micro-CT scan and reconstruction analysis,H&E staining and Masson’s staining to observe the formation of ectopic bone.3.To explore the possible mechanism by which melatonin promotes BMP9-induced osteogenic differentiation of mesenchymal stem cells.Immunofluorescence and Western blotting were used to evaluate the expression and localization of BMP9 and its downstream Smad1/5/8 after treatment with melatonin and/or BMP9.The melatonin and BMP9 promoted mesenchyme by Western blot.Changes in the AMPK/β-catenin pathway in stem cell osteogenic differentiation.Subsequent silencing of AMPK or β-catenin by si-RNA,Western blotting and alkaline phosphatase activity assays were performed to further verify that the AMPK/β-catenin pathway promotes osteogenic differentiation of mesenchymal stem cells in combination with melatonin and BMP9.[RESULTS] 1.Melatonin combined with BMP9 can significantly enhance the expression of osteogenic markers in different stages of mesenchymal stem cells.Ad BMP9 adenovirus can effectively infect C3H10T1/2 cells;melatonin alone can increase the alkaline phosphatase activity of mesenchymal stem cells in a dose-dependent manner;according to the control group,melatonin group,BMP9 group,BMP9 and fading The melanin co-culture group treated C3H10T1/2 cells,and the results showed that melatonin combined with BMP9 group could significantly enhance the alkaline phosphatase activity of mesenchymal stem cells,and its activity was significantly higher than that of the other three groups.Alkaline phosphatase staining results The same as the activity test;the results of immunohistochemical staining of osteogenic markers osteopontin and osteocalcin were similar to those of alkaline phosphatase,and melatonin significantly enhanced the expression of osteopontin and osteocalcin of BMP9;The results of staining the cells with mineral matrix alizarin red S further confirmed this: melatonin combined with BMP9 group formed the most mineralized nodules,the most obvious;2.melatonin effectively enhanced BMP9 in fetal rat limbs Osteogenesis induction in culture and stem cell transplantation experiments in ectopic bone formation in nude mice.In the in vitro limb culture experiment of fetal rats,the melatonin combined with BMP9 group showed higher fluorescence intensity than the BMP9 group,indicating that new bone formation was more active;H&E staining showed that melatonin combined with BMP9 group had Active growth plate area,higher secondary cell density and higher trabecular maturity.In stem cell transplantation experiments with ectopic bone formation,we observed that melatonin combined with BMP9 treatment resulted in increased bone mass and relative bone density increased by 30% compared with BMP9;Masson staining showed that melatonin enhanced BMP9 induction.Mineralized bone matrix.3.Melatonin promotes the possible mechanism of BMP9-induced osteogenic differentiation of mesenchymal stem cells.Immunofluorescence and Western blotting showed that melatonin increased the expression of BMP9 in mesenchymal stem cells,increased the expression of p-Smad1/5/8,and induced the transfer of p-Smad1/5/8 from the cytoplasm to the nucleus;Phosphorylation of the AMPKα Thr172 site and phosphorylation of theβ-catenin Ser552 site in melatonin plus BMP9 group were significantly increased compared with melatonin alone or BMP9 alone;followed by treatment with AMPKαsi RNA or β-catenin si RNA C3H10T1 / 2 cells,si AMPKα pretreatment successfully blocked the β-catenin phosphorylation induced by melatonin and BMP9 co-treatment;in addition,si AMPKα or siβ-catenin pretreatment significantly inhibited the alkali induced by the synergistic induction of melatonin and BMP9 Increased sex phosphatase activity.[CONCLUSION] 1.Melatonin can promote BMP9-induced osteogenic differentiation of mesenchymal stem cells.2.Melatonin combined with BMP9 can directly up-regulate BMP9 and its downstream targets,and promote the transfer of p-Smad1/5/8 from cytoplasm to nucleus.3.The AMPK /β-catenin pathway is at least partially involved in the osteogenic differentiation induced by melatonin combined with BMP9.