Mechanism Of Bone Resorption Supernatant Involved In Regulating Eldecalcitol(ELD)to Promote New Bone Formation

Background and purpose Active vitamin D(la,25(OH)2D3)is the basic drug for clinical intervention in osteoporosis and its complications currently,which can not only greatly reduce the fracture incidence of postmenopausal osteoporosis and senile osteoporosis but also has important clinical therapeutic value for steroid osteoporosis.In recent years,many active vitamin D analogues have optimized the pharmaceutical properties on the basis of the original efficacy,which brings new ideas for the treatment of osteoporosis.Among them,the well-represented Eldecalcitol(ELD)has a prolonged half-life in the blood,which greatly improves the bioavailability.And at the same time,it can make up the Vitamin D-caused defects in calcium and phosphorus imbalance by reducing the inhibition of parathyroid hormone,optimizing the regulation of bone metabolism.Since it began to be used in the clinical treatment of osteoporosis in 2011,it has obtained a good clinical evaluation.Nevertheless,in order to better understand and apply the pharmacological effects of ELD in improving osteoporosis,the relevant mechanisms still need further investigation.As the main functional cells in the process of bone remodeling,there is a close mutual coupling relationship between osteoclasts and osteoblasts.In the preliminary work,the research team determined that there was also a morphological positional relationship between osteoclasts and pre-osteoblasts,and demonstrated the presence or absence of osteoclasts during bone metabolism,which directly affected the proliferation and differentiation of pre-osteoblasts and the biological activity of osteoblasts.Based on the preliminary work,this topic intended to explore the role of bone resorption supernatant in the treatment of Eldecalcitol in osteoporosis and promote the formation of new bone through related experimental design and related mechanisms.The expected results may provide new targets and ideas for the development of therapeutic agents for osteoporosis,which will have a positive scientific significance.Materials and Methods1.The first part of the study was to construct the osteoporosis models caused by postmenopausal estrogen deficiency in older women and tumor chemotherapy drugs through operating explore ovariectomy(OVX)surgery and giving the tumor chemotherapy drug cyclophosphamide(CTX)in rats.And rats tibia was obtained as a subject for histomorphological evaluation.Hematoxylin and eosin(HE)staining was used to compare the morphological changes of bone tissue;serum markers of bone metabolism were detected by ELISA;tartrate-resistant acid phosphatase(TRAP)staining was applied to detect the presence of osteoclasts;immunohistochemical staining was used to detect the expression of alkaline phosphatase(ALP),cathepsin K(CK),matrix metalloprotein 9(MMP9)and Osteopontin(OPN).Bone histopathological changes caused by estrogen deficiency and cyclophosphamide application and changes in bone tissue after applying ELD was detected using the methods above,which in order to explore the therapeutic effect of ELD on primary osteoporosis(estrogen deficiency)and secondary osteoporosis(drug application).2.The second part of the study in vitro induced the mouse monocyte macrophage cell line RAW 264.7 to differentiate into functional osteoclasts and obtained differentiated osteoclasts culture supernatant(OCS).At the same time,the differentiated osteoclasts and bovine bone samples were co-cultured to obtain bone resorption culture supernatant(BRS).Subsequently,mouse-derived pre-osteoblasts(MC3T3-E1 cell line)were treated with two supernatants(OCS and BRS)and ELD in combination.The proliferation activity of MC3T3-E1 cells was evaluated by Cell Counting Kit-8,and the osteogenetic activity of MC3T3-E1 cells was detected by alkaline phosphatase(ALP)kit,while the expression of bone-related proteins was evaluated by reverse transcription quantitative(RT-q)PCR and Western blot analysis.The role of osteoclasts and osteoclastic bone resorption in the process of ELD improving osteoporosis and promoting new bone formation was comprehensively evaluated.3.In the third part of the study,through bioinformatics prediction and comprehensive literature analysis speculated that BMP-2,TGF-β and TCD may play an important role in the formation of new bone in ELD,and verified by relevant in vitro experiments.The mouse MC3T3-E1 pre-osteoblasts cell line cultured in vitro was induced to differentiate in the medium containing the osteogenic induction solution for 7 days,and used CCK8,AKP activity assay,RT-PCR,Western-blot methods to detect the proliferation activity of osteoblasts,early differentiation and expression of key proteins in intracellular related signaling pathways in the presence or absence of BMP-2,TGF-β1 and TCD and to study the effects of BMP-2,TGF-β1,TCD alone and its synergistic influence with ELD on the proliferation and differentiation of osteoblasts.Among them,BMP-2 group detected the expression of osteoblasts differentiation marker proteins Runx2 and smad 1/5/9,p38 and JNK related signaling pathways by Western-blot.The expressions of Runx2,OCN,OPN and Collagen I were detected by RT-PCR in the TGF-β1 group.The expression levels of smad3,Akt,p38 and JNK signaling pathways in osteoblasts were detected by Western-blot.The TCD group detected the expression levels of osteoblasts differentiation marker proteins Runx2 and OCN by Western-blot,and studied the necessity of TCD for osteogenic differentiation through TCD-specific inhibitors,and studied whether TCD can affect osteoblasts differentiation through mTOR signaling pathway through the mTOR blocker Rapamycin.Results 1.After 4 weeks of ovariectomy in 8-week-old female rats,the number of trabecular bone was significantly reduced,and the animal model of osteoporosis caused by estrogen deficiency was successfully prepared.The results of HE staining of tibia tissue showed that compared with the control group,the proximal humerus of the OVX group showed a decrease in trabecular bone mass and an increase in osteoblasts alkaline phosphatase(ALP)activity.The expression of tartrate-resistant acid phosphatase(TRAP)and cathepsin K(CK)of osteoclasts were enhanced,which suggesting that the bone turnover rate in this region was higher than that in the control group.After Eldecalcitol(ELD)administration,the bone turnover rate decreased significantly,which was manifested by decreased ALP expression of osteoblasts and decreased TRAP staining of osteoclasts and expression of cathepsin K.And the bone mass of the beam was increased.For CTX group,two weeks after administration of cyclophosphamide,the tibial metaphyseal showed significant osteoporosis characteristics such as decreased number of trabecular bone and increased resolution,which suggesting successful construction of animal model.Application of ELD can well corrected the osteoporosis induced by CTX,which was manifested by an increase in bone mass,and the high bone turnover rate induced by CTX decreased to be similar to the normal control group.2.ELD reduced the survival rate of MC3T3-E1 cells and showed the characteristics of first inhibition and promotion after in osteoblasts differentiation.Osteoclasts culture supernatant(OCS)can change the inhibitory effect of ELD on the proliferation of pre-osteoblasts,but it does not show obvious regulatory functions in the differentiation of osteoblasts regulated by ELD.Osteoclast bone resorption culture supernatant(BRS)did not significantly improve the proliferation of osteoblasts by ELD inhibition,but significantly increased the extent of osteoblast differentiation by ELD promotion.3.BMP-2,TGF-β1 and TCD increased the survival rate of osteoblasts in a dose-dependent and time-dependent manner,that promoted osteogenic differentiation and synergistic effect with ELD.Western-blot results showed that BMP-2,TGF-β1 and TCD promoted the expression of Runx2,an osteogenic differentiation marker.BMP-2 can up-regulate the expression levels of smad1/5/9,p-p38 and p-JNK,and its synergistic effect with ELD was stronger.TGF-β1 significantly up-regulated the expression of smad3,p-Akt,p-p3 8,and p-JNK.Conclusion1.ELD could relieve the primary osteoporosis induced by estrogen deficiency and secondary osteoporosis caused by the tumor chemotherapy drug cyclophosphamide.2.ELD inhibited osteoblasts proliferation and early differentiation in vitro,but osteoclasts secretion medium and/or osteoclasts bone resorption release related factors could change the regulation of ELD on osteoblasts,especially bone resorption release related factors could significantly enhance effect of osteogenic differentiation promoted by ELD.3.BMP-2 promoted osteogenic differentiation through smadl/5/9,p38,JNK-related signaling pathways,TGF-β1 promoted the same phenomenon via smad3,Akt,p38,JNK signaling pathway,and the same as TCD via mTOR signaling pathway.