| BackgroundGlucocorticoids(GC)can exert non-specific anti-inflammatory,antiviral,anti-shock and immunosuppressive pharmacological effects and are therefore widely used in anti-inflammatory therapy,organ transplantation,autoimmune diseases and tumor treatment.However,the incidence of glucocorticoid-induced osteoporosis(GIOP),which is one of its major side effects,is increasing year by year and has become a pressing clinical problem.As the most common secondary osteoporosis,GIOP is a metabolic bone disease characterized by reduced bone mass,destruction of bone tissue microarchitecture,decreased bone density,increased bone fragility,and increased fracture risk,and the pathogenesis and prevention of GIOP are receiving increasing attention.Eldecalcitol(ED-71),a new active vitamin D analogue,is a first-line anti-osteoporosis drug and has been extensively studied in the treatment of postmenopausal osteoporosis,but its role in improving GIOP needs to be further confirmed.The aim of this study is to clarify the role of a new active vitamin D analogue,Eldecalcitol(ED-71),in improving GIOP and the related mechanisms.The expected results will provide a new theoretical basis for the clinical prevention of glucocorticoid osteoporosis and have positive scientific significance.Materials and methods1.Effect of ED-71 on bone mass,osteoblast status and osteoclast status in GIOP mice7-week-old male wild C57BL/6 mice were used to construct a glucocorticoid osteoporosis mouse model using methylprednisone(MPED)and dexamethasone(DEX),respectively,and were randomly divided into three groups:1)control group;2)glucocorticoid group with intraperitoneal injection of methylprednisone 10 mg/kg/day for 4 weeks or dexamethasone 1 mg/kg/day for 8 weeks;3)ED-71 group with intraperitoneal injection of methylprednisone 10 mg/kg/day or dexamethasone 1 mg/kg/day,both administered orally to ED-71 at 50 ng/kg/day.group,intraperitoneal injection of MPED 10 mg/kg/day or DEX 1 mg/kg/day,both administered ED-71 orally at 50 ng/kg/day.after 4 or 8 weeks of treatment,the femurs of C57BL/6 mice were taken and used as the objects for morphological evaluation.Bone histological changes were detected by Micro-CT and hematoxylin-eosin(HE)staining.The rate of new bone formation was detected by calcein/tetracycline labeling.To comprehensively evaluate the bone histopathological changes caused by glucocorticoids and evaluate the preventive effect of Eldecalcitol on glucocorticoid osteoporosis.2.Effect of ED-71 on osteogenic differentiation in GIOPIn vivo,osteogenic differentiation in C57BL/6 mice was detected by immunohistochemistry(IHC)staining.Pre-osteoblast cell line MC3T3-E1 cells were treated with DEX and ED-71,and untreated MC3T3-E1 cells were used as controls.Osteogenesis was induced by adding mineralization induction solution.The osteogenic differentiation ability of MC3T3-E1 cells was assessed by alkaline phosphatase(ALP)staining and alizarin red(AR)staining.Real-time quantitative polymerase chain reaction(qRT-PCR)and Western blot were applied to detect the expression of osteogenic differentiation-related factors.The effects of DEX and ED-71 on the differentiation and mineralization levels of osteoblasts and the role played by ED-71 in the prevention of glucocorticoid osteoporosis were evaluated together.3.The specific mechanism of ED-71 promoting osteogenic differentiation through Notchl/Wnt-β-catenin signaling in GIOPThe analysis of literature showed that Notch and Wnt/β-catenin signaling pathways may play an important role in the promotion of osteoblast differentiation by ED-71,and relevant in vitro experiments were conducted to verify this.MC3T3-E1 cells were cultured in vitro,stimulated with dexamethasone or/and Eldecalcitol,and the expression of Notch and Wnt/β-catenin signaling pathway-related factors were detected by immunofluorescence,qRT-PCR and Western Blot.Subsequently,MC3T3-E1 cells were given Wnt signaling inhibitor(XAV939)or made to overexpress Notch l,respectively,and continued to be stimulated with dexamethasone or/and Eldecalcitol,and the transduction of Notch and Wnt/β-catenin signaling pathway was detected by qRT-PCR and Western Blot,as well as osteogenesis-related factors.Meanwhile,the osteogenic differentiation and mineralization ability of MC3T3-E1 cells were assessed by alkaline phosphatase staining and alizarin red staining again.Results1.ED-71 preventd bone loss,promoted bone formation and inhibited bone resorption in GIOP miceThe cancellous bone mass in the femur of C57/BL6 mice administered with glucocorticoids was significantly lower than that of the control group,with sparse arrangement of trabeculae,reduced number and thickness,increased separation,and slowed rate of new bone formation;further administration of ED-71 increased cancellous bone mass,increased number and thickness of trabeculae,reduced separation,and increased rate of new bone formation,which to some extent rescued the glucocorticoid-induced bone loss.In addition,glucocorticoid administration reduced the number of ALP-positive osteoblasts and TRAP-positive osteoclasts,whereas ED-71 administration partially increased the number of ALP-positive osteoblasts while further reducing the number of TRAP-positive osteoclasts.2.ED-71 improved the inhibitory effect of glucocorticoid on osteoblast differentiationThe immunoreactivity of osteogenic differentiation-related factors RUNX2,OCN and COL1 in femoral tissues of C57/BL6 mice was significantly reduced after glucocorticoid administration compared with the control group,however,the expression levels of RUNX2,OCN and COL1 were restored after further administration of ED-71.Also,after osteogenic induction,ED-71 resulted in increased mRNA and protein levels of ALP,RUNX2,OCN and COL1 in MC3T3-E1 cells and a significant increase in the positive expression regions of ALP staining and AR staining.3.ED-71 promoted osteogenic differentiation by regulating Notch1/Wnt-β-catenin signaling pathway in GIOP stateCompared with the glucocorticoid group,administration of ED-71 increased Notchl levels in MC3T3-E1 cells,as well as Wnt pathway-related factors Wnt5a,Frizzled4 and GSK3-β-p,which also had significantly higher expression levels of downstream β-catenin as well as activated β-catenin.In addition,the expression of Notchl pathway and Wnt-β-catenin pathway-related factors decreased after the addition of Wnt signaling inhibitor XAV939 or overexpression of HES1,a molecule downstream of Notch1,and the expression of osteogenic differentiation-related factors RUNX2,OCN and COL1 was reduced,while the positive expression regions of ALP staining and AR staining were significantly reduced.Conclusion1.ED-71 prevented bone loss,promoted bone formation and inhibited bone resorption in GIOP mice.2.ED-71 improved the inhibitory effect of glucocorticoid on osteoblast differentiation.3.ED-71 promoted osteogenic differentiation of MC3T3-E1 cells via Notch1/Wnt-β-catenin signaling pathway. |
PDF Full Text Request