The Mechanism Of Eldecalcitol Improving Postmenopausal Osteoporosis By Regulating Oxidative Stress

BackgroundPostmenopausal osteoporosis(PMOP),one of the most common types of osteoporosis,is caused by insufficient estrogen production starting from perimenopause,which leads to increased loss of bone density and fracture risk in women.PMOP has become a an increasingly severe social problem as the population aging.Eldecalcitol(ED-71)is the third generation of active vitamin D analogue with long half-life and low side effects.It plays a positive role in improving PMOP by inhibiting bone resorption and promoting bone formation,but the exact mechanism remains to be clarified.In addition,oxidative stress is considered to be the culprit of PMOP,and its role in the pathogenesis of PMOP has received more and more attention.Combined with our previous findings that ED-71 has antioxidant function in the process of improving periodontal inflammation,this study speculated that the inhibition effect of ED-71 on oxidative stress under PMOP may be an important way to improve PMOP.Therefore,on the basis of ED-71 improving bone loss caused by PMOP,this study further clarified its specific mechanism of regulating bone formation and bone resorption by inhibiting oxidative stress,hoping to provide guidance for the clinical use of PMOP.Materials and methods1.ED-71 improved osteoporosis status in ovariectomized(OVX)rats(1)Evaluation of the effects of ED-71 on bone mass in OVX ratsA rat OVX model was established to simulate postmenopausal osteoporosis.30 ng/kg ED-71 was given orally once a day for 8 weeks.The body weight of the rats was recorded weekly.The bone mass,histological parameters and state of new bone formation of rat femur were evaluated by Micro-CT,hematoxylin-eosin(HE)staining and MASSON staining.(2)Evaluation of the effects of ED-71 on the function of osteoblast and osteoclastTartrate resistant acid phosphatase(TRAP)staining and cathepsin K(CtsK)immunohistochemical staining were used to observe the osteoclast status in the femoral region of rats.Immunohistochemical staining of alkaline phosphatase(ALP),osteocalcin(OCN)and collagen(COL)I was performed to observe the osteoblast function.2.ED-71 improved PMOP by regulating oxidative stress(1)Evaluation of the antioxidant effect of ED-71 during proventing PMOPBone tissue and serum were extracted,and the oxidative stress levels of OVX rats were analyzed by bone tissue real-time polymerase chain reaction(qRT-PCR),Western Blot and serum malondialdehyde(MDA)detection.RAW264.7 cell line and rat bone marrow mesenchymal stem cells(BMSCs)were cultured in vitro,and H2O2 was added to simulate oxidative stress in osteoporosis.The effects of ED-71 on oxidative stress were detected by DCFH-DA staining and qRT-PCR.(2)The effects of ED-71 on osteogenic differentiation and osteoclast differentiation under oxidative stressALP staining,Western Blot,qRT-PCR and DCFH-DA staining were used to detect the osteogenic differentiation ability and oxidative stress level of BMSCs in Sham and OVX groups.The effects of ED-71 on osteogenic differentiation of BMSCs were detected by alizarin red staining and Western Blot.H2O2 was added to RAW264.7 cells to simulate the oxidative stress state in osteoporosis.The effects of ED-71 on osteoclast differentiation were detected by TRAP staining,F-actin staining,Western Blot and qRT-PCR.3.The mechanism of ED-71 promoting bone formation by regulating BMSCs senescence under oxidative stress(1)The effect of ED-71 on cell senescence in OVX ratsThe senescence of rat femur cells was observed by immunohistochemical staining in vivo.The femur tissues of rats were extracted,and the expression of age-related factors in bone tissues in different groups were evaluated by tissue qRT-PCR and Western Blot.(2)The regulatory effect of ED-71 on the senescence of OVX rat BMSCsBMSCs were extracted and cultured in vitro.The BMSCs senescence in Sham group and OVX group was evaluated and the regulatory effects of ED-71 on OVX rat BMSCs senescence were observed by β-galactosidase(β-gal)staining,qRT-PCR,Western Blot and immunofluorescence staining.(3)Clarify the relationship between BMSCs senescence and osteogenic differentiation under oxidative stressBMSCs were induced to osteogenic differentiation,and the expression of osteogenic factors was detected by Western Blot to explore the role of cell senescence in ED-71 promoting osteogenic differentiation of BMSCs.H2O2 was used to regulate cellular oxidative stress,and ROS and senescence levels were detected by DCFH-DA staining and β-gal staining to explore whether ED-71 regulates cell senescence through antioxidant.(4)Explore the mechanism of ED-71 promoting bone formation by regulating BMSCs aging The expression of SIRT1-Nrf2 signal was detected by qRT-PCR,Western Blot and immunofluorescence staining.The signaling pathway was validated by the use of SIRT1 inhibitor EX-527 and Nrf2 inhibitor ML-385.Western Blot,DCFH-DA staining and β-gal staining were used to investigate whether SIRT1-Nrf2 signal was involved in the regulation of oxidative stress,cell senescence and osteogenic differentiation by ED-71.4.The mechanism of ED-71 inhibiting bone resorption through regulating EphrinB2-EphB4 signal under oxidative stress(1)Observe the effect of ED-71 on EphrinB2 in osteoclastsThe effect of ED-71 on the expression of EphrinB2 in rat femur region was observed by immunohistochemical staining.A H2O2 stimulation model was established to simulate osteoporosis in vitro,and osteoclast differentiation of RAW264.7 cells was induced.The expression of EphrinB2 in osteoclasts was detected by qRT-PCR and Western Blot.EphrinB2 was knocked down by siRNA,and the differentiation of the osteoclasts was detected by TRAP staining,f-actin ring staining,qRT-PCR and Western Blot.(2)Observe the regulation effect of ED-71 on EphB4 in osteoblastsThe effect of ED-71 on the expression of EphB4 in rat femur was observed by immunohistochemical staining.MC3T3-E1 cells were stimulated with H2O2 in vitro,and osteogenic induction was performed.The expression of EphB4 in osteoblasts was detected by qRT-PCR,Western Blot and immunofluorescence.(3)Explore the specific mechanism of ED-71 inhibiting osteoclast differentiation in coculture modelA coculture model of MC3T3-E1 cells and RAW264.7 cells was established to simulate the direct contact between osteoblasts and osteoclasts.EphB4 antibody was used to block EphB4 in osteoblast to inhibit EphrinB2-EphB4 signal.Osteoclast differentiation was observed by F-actin staining.Results1.ED-71 improved bone loss in PMOP,inhibitd bone resorption and promoted bone formationCompared with Sham group,the bone mass of OVX group was decreased and the body weight was increased significantly.ED-71 treatment could inhibit the bone loss and weight gain of OVX rats and promote the regeneration of new bone.In vivo histological staining results showed that TRAP-positive and CtsK-positive osteoclasts were significantly increased in OVX group compared with Sham group,ALP positive osteoblasts were slightly increased,OCN positive expression was increased,and COLI deposition was decreased.ED-71 could decrease the number of osteoclasts in OVX rats,inhibit bone resorption,increase the positive expression of OCN and COLI,and promote bone formation.HE staining showed that ED-71 promoted "minimodeling" bone formation in the trabecular region of OVX rats.These results suggested that ED-71 could prevent OVX-induced osteoporosis in rats by both inhibiting bone resorption and promoting bone formation,which may be the result of ED-71 regulating bone remodeling and promoting "minimodeling".2.ED-71 affected PMOP bone formation and bone resorption by regulating oxidative stressThe level of oxidative stress in serum and bone tissue of OVX rats increased,and ED-71 inhibited oxidative stress.In vitro,ED-71 reversed the H2O2-induced increase in reactive oxygen species.The oxidative stress level of BMSCs in OVX group was increased and the osteogenic differentiation ability was decreased compared with Sham group.ED-71 could improve the osteogenic differentiation ability of OVX rat BMSCs.ED-71 also inhibited osteoclast differentiation induced by H2O2.3.ED-71 inhibited BMSCs senescence and promoted bone formation under oxidative stress through SIRT1-Nrf2 signalThe results of histological staining showed that although the expression of senescence-related factors increased in OVX group,the addition of ED-71 reversed the changes of these factors.The senescence ratio of BMSCs in OVX group was higher than Sham group after extraction and culture of BMSCs in vitro.ED-71 could significantly improve OVX-induced oxidative stress and cell senescence of BMSCs,and improve the osteogenic differentiation ability of OVX rat BMSCs.By adding H2O2 to enhance oxidative stress,the inhibition effect of ED-71 on BMSCs senescence was reversed.ED-71 also increased the expression of SIRT1 and Nrf2 in OVX rat BMSCs.After inhibiting SIRT1 or Nrf2,the inhibitory effect of ED-71 on oxidative stress and cell senescence of OVX rat BMSCs and the promotion effect on osteogenic differentiation were weakened.These results suggested that ED-71 could improve osteogenic differentiation of BMSCs by inhibiting cell senescence under oxidative stress,which might be played by regulating SIRT1-Nrf2 signal.4.ED-71 inhibited bone resorption by activating EphB4-EphrinB2 signal under oxidative stressUnder oxidative stress simulated by H2O2,accumulation of reactive oxygen species inhibited the expression of EphrinB2 in osteoclasts and EphrB4 in osteoblasts,while ED-71 improved their expression in vitro and in vivo.EphrinB2 was knocked down to reverse ED-71 inhibition of H2O2-induced osteoclast formation.ED-71 significantly inhibited the formation of osteoclasts in the coculture model of osteoblast and osteoclast,and the inhibitory effect was weakened by blocking the EphB4-EphrinB2 signal between osteoblast and osteoclast.These results suggested that ED-71 could inhibit oxidative stress-induced osteoclast differentiation by regulating the interaction between osteoblast and osteoclast.Conclusion1.ED-71 can improve weight gain and bone loss,inhibit osteoclasts and promote osteoblasts in OVX rats.2.ED-71 improves PMOP by regulating oxidative stress.3.ED-71 inhibits oxidative stress of OVX rat BMSCs by enhancing SIRT1-Nrf2 signal,thus inhibiting cell senescence and promoting osteoblast differentiation.4.ED-71 inhibits oxidative stress-induced osteoclast differentiation by enhancing the EphB4-EphrinB2 signal between osteoblast and osteoclast.