The Research On The Mechanism Of Neomangiferin Effect Osteolytic Disease

Bone hoemeostasis is a dynamic equilibrium of bone resorption and bone formation,maintained primarily by osteoblast osteogenesis and osteoclast bone resorption.Osteoblasts,as the main function cells of bone formation,are mainly derived from mesenchymal progenitor cells in the matrix of periosteum and bone marrow.Mature osteoblasts are mainly distributed on the surface of bone producing mucopolysacchride,glycoprotein,collagen fibers and collagen,etc,which promote the mineralization of extracellular bone matrix into osteoblasts.In the process of bone remodelling,the interaction between osteoblasts and osteoclasts take place in the basic multicellular units.Through direct contact secretion of paracrine factors and the interaction between cells and bone matrix,the interaction is regulated and then the remodelling process of bone is precisely affected.In order for homeostasis,the osteoblast secrete a large number of cytokines during differentiation,and these cytokines have the action to initiate the differentiation and functional change of osteoclasts and secrete Type I collagen chemical factor,osteocalcin of osteoclast progenitor cell migration regulation intervention.Osteoclasts are mononuclear macrophages derived from myeloid progienitor cells in the bone marrow that fuse with each other and are polynuclear giants cells.Mature osteoblasts mainly perform the function of bone dissolution and absorption and also have biological functions such as the regulation of bone angiogenesis,the regulation of bone formation,the regulation of haemopoiesis and the hormonal action of osteocalcin.It is a kind of polynuclear giant cell with bone resorption function.Osteoclasts are regulated by a variety of hormones including osteo-protective hormone,RANK ligand,parathyroid hormone,calcitonin from thyroid and growth factor interleukin-6.Osteoblasts need receptor activators(RANKL)with nuclear factor ligand and macrophage colony stimulatng factor(M-CSF)to form and differentiate.These membrane binding proteins are produced by adjacent stromal cells and osteoblast.Among them,M-CSF and RANKL are directly involved in regulation.M-CSF(or CSF-1)is a haemopoietic growth factor involved in the proliferation and differentiation of monocytes,macrophages and bone marrow progenitor cells.Under the endocrine stimulation of parathyroid hormone M-CSF released by osteoblasts has paracrine effect on osteoclasts to include the proliferation and differentiation of mononuclear macrophages towards osteoblast precursor cells,and further stimulate the expression of specific nuclear factor kappa B receptor activator(RANKL)in osteoblast precursor cells,leading to osteolysis and resorption decomposition.As an apototic regulatory gene RANKL a binding ligand of OPG and also a ligand of the receptor RANK.RANKL were members of the family of tumor necrotic factor(TNF)ligand belong to type II membrane proteins,it has certain effect on the immune system also plays an important role in the process of bone regeneration.RANKL regulates the protein levels of ID4,ID2 and cyclin D,to control cell proliferation.After the binding of RANKL with its specific receptor RANK under synergistic effect of M-CSF,RANKL plays a decisive role in the further differentiation and bone resorption of osteoclasts.Animal studies have shown that targeting the relevant target genes in mice leads to osteoclast deficiency and severe osteosclereosis.In mice lacking RANKL or its receptor,RANK,the t-lymphocytes and b-lymohocytes are deficient in early differentiation and fail to form lobule-alveolar breast structures during pregnancy.It was found in the experiment that during pregnancy,the RANK-RANKL signaling pathway is involved in regulating the differentiation and activity of osteoclasts which plays a role in the regulation of bone calcium release and is a key mediator of bone resorption and bone density.There was no linear correlation between the expression level of RANKL and the action of the ligand high protein expression of RANKL is commonly found in the lungs,thymus and lymph nodes in the bone marrow,stomach,peripheral blood,spleen,placenta,white blood cells,skeletal muscle,the expression of RANKL is the most important but plays a key role in bone metabolism.Osteoclast activity is trigerred by RANKL binding on the surface of osteoblasts to the surface-binding receptor,the activator of the osteoclast’s nuclear factor kappa b(NF-κ B).Recent studies have shown that bone cells are a major source of RANKL in postnatal bones.And the excessive formation of RANKL or activity associated with a variety of degenerative bone disease,such as come from inflammation(rheumatic arthritis,inflammatory bone disease,aseptic prosthetic loosening after artificial joint replacement),post-menopausal osteoporosis and bone solubility of the damage caused by cancer(such as bone destruction caused by tumor,multiple myeloma)can lead to obvious bone loss.RANKL-mediated hyperfunction of osteoclasts is closely related to various types of pathological osteolysis,and RANKL/RANK/OPG signaling system is a typical pathway for RANKL to participate in bone reconstruction,so inhibiting the activity of RANKL osteoclasts and bone resorption is a reasonable choice for the treatment of osteoclast-related osteolysis diseases.Therefore,RANKl as a therapeautic target has a broad application prospect in clinical treatment of related osteolytic diseases,which is conductive to the exploration of specific targeted therapy.With the development of traditional Chinese medicine(TCM),monomer or compound preparation of TCM has been applied in clinical and achieved good results.Chinese herbal monomers used in anti-osteoporosis bone also become one of the hot spots in current research.Mango glycoside was a special aryxanthane compound and had extensive and good biological activity.Neomangiferin(7-o-d-glucopyranosyl-mangiferin)is a monomer of traditional Chinese medicine,which is isolated from the fruits and leaves of the mangoes belonging to the sumac family.It has the effects of anti-oxidation,anti-inflammation,anti-virus,immune regulation and anti-tumor.Studies have confirmed that the neomagniferin has strong chemicl activity and can be used for the prevention and treatment of diabetic nephropathy.Many pharmacological experiments have proved that neomagniferin can correct the imbalance of REDOX inhibit the generaton and accumulation of AGE,inhibit the activity of plyol pathway and regulate lipid metabolism,so as to combat the development of diabetic nephropathy and improve renal function.Neomangiferin can protect vascular endothelial cells,and improve total SOD activity in cells.It can effectively inhibit the oxidative damage of cells and further clarify the new pathway and treatment mechanism of neomangiferin in the treatment of cardiovascular disease and diabetic vascular complications.However,no related studies have been reported on the prevention and treatment of osteoclast related osteolysis diseases by neomangiferin.In this study,the effect of neomangiferin on osteoclast formation differentiation and osteolysis resorption was studied by modern cell and molecular biology experiments and possible molecular mechanism was also discussed.In additon,an animal model of osteoclast-related osteolytic disease-rat skull osteolysis model and rat osteoporosis model were built up to further explore the role of neomangiferin in the prevention and treatment of osteolytic disease.This experiment is designed in the following four parts.Part I Analysis of the effect of neomangiferin on osteoclast differentiation induced by bone marrow macrophages in vitro and osteoclast bone resorption function.Objective To study the effect of neomangiferin on differentiation of bone marrow macrophages into osteoclasts in vitro.Methods Bone marrow mononuclear cells(BMMs)from tibia and femur were isolated from C57BL/6 mice aged 8 weeks.M-CSF and RANKL were used to stimulate and induce bone marrow mononuclear cells to differentiate and amplify toward osteoclasts.The ability of bone marrow mononuclear cells to differentiate into osteoclasts was determined by cell morphology observation tartrate acid phosphatase staining and bone lysis and absorption.In order to detect the non-toxic effect of neomangiferin on osteoclast precursor cells,we performed a cytotoxicity test.To determine the effect of neomangiferin on osteoclasts,we used different concentrations of neomangiferin in vitro to intervene in the differentiation of BMMs into osteoclasts induced by RANKL and TRAP staining was used to detect the differentiation of osteoclasts,and cells with 3 or more nuclei were counted.Neomangiferin of different concentrations was added again to intervene in the differentiation process of osteoclasts induced by RANKL,and the expression level of osteoclast related specific genes was determined by real-time PCR.Then bone resorption experiment was carried out to study the effect of neomangiferin on osteoclasts and bone lysis and resorption.In order to investigate the regulatory effect of neomangiferin on the relevant signaling pathways of RANKL-induced osteoclast formation and differentiation and bone resorption,we applied western blot to detect the effect of neomangiferin on the phosphorylation level of important signaling proteins related to RANKL induced osteoclast NF-κB and MAPK signaling pathway.Results 1.Neomangiferin has an inhibitory effect on the formation and differentiation of osteoclasts and the higher the concentration of the drug,the more obvious the inhibitory effect onthe differentiation of osteoclasts and the concentration-depentend inhibition of RANKL-induced osteoclast formation.2.The MTS results showed that activity of BMM cells was not significantly affected by the concentration of neomangiferin.3.Compared with the positive control group(0 μM),the number of osteoclasts and the area of occluded bone in the high-concentration neomangiferin(2.5 μM)group were visibly decreased,and the difference was statistically significant(P <0.05).4.Real-time PCR results showed that the expression levels of cathepsin K,Matrix Metallo Proteinase-9(MMP-9)and TRAP were lower in osteoclasts treated with the neomangiferin(2.5 μM)group than that in the positive control group(0μM).5.The analysis of experimental results showed that neomangiferin could effectively hinder the phosphorylation of RANKL-induced NF-κB signaling pathway and related proteins of MAPK signaling pathway in osteoclasts.Conclusion RANKL-induced osteoclasts formation and bone resorption were inhibited in a dose-dependent manner by neomangiferin which may be related to the phosphorylation of NF-κB and MAPK signaling pathways.The second part The effect of neomangiferin on osteolysis and resorption induced by ovariectomyObjective To observe the effect of mamgiferin on osteolysis and resorption induced by ovariectomyMethods Female C57BL/6 mice were selected and ovariectomized to establish an animal model of osteolysis.Bone morphological parameters were compared and analysed by micro-CT,and the knee joints of mice were analysed by pathological HE staining and TRAP staining.To study the effect of neomangiferin on osteoporosis after ovary resection were mainly manifested as decreased trabecular bone missing bone or discontinuous bone connection.We established a mouse model of ovariectomized mice to stimulate post menopausal osteoporosis and tested the effect of neomangiferin on osteoporosis osteolysis in vitro.After the model mice the right tibia was selected for micro-CT scanning.It was observed that the number of tibia trabeculae in the model group and the control group was less than that in the negative group.while the number of trabeculae in the estrogen group and the low/high dose of neomangiferin group was higher than that in the model group.At the same time,the number of bone trabeculae increased in the high-dose group compared with the low dose group.Three-dimensional recostruction of the mouse tibia showed that neomangiferin could significantly reduce bone loss in ovariectomised model,the higher the concentration of neomangiferin,the stronger the preventive effect.Pathologic examination showed that neomangiferin could significantly reduce the number of osteoclasts on the surface of trabecular bone per unit area that the contact area of osteoclasts.Conclusion Neomangiferin inhibit the osteolysis induced by ovariectomy in mice.The third part The effect of neomangiferin on UHMWPE abrasion granule induced osteolysis in miceObjective To study the effect of neomangiferin on UHMWPE abrasion granule induced osteolysis in mice.Methods C57BL/6 male mice were selected and UHMWPE wear particles were implanted under the skull periosteal to established the osteolysis model.Mice were divided into 4 groups and given differnt treatment factors.After 3weeks,the levels of osteoclast related receptors(OSCAR)and type I collagen c-terminal peptide(CTX-1)of peroosteal arterial blood enzyme-linked immunosorbent assay(ELISA)were detected.Then the mice were sacrified and the skull was separated for micro-CT analysis of the osteolysis.Then HE staining,TRAP-resistant acid phosphatase staining were used to observe the skull dissolution.Results Micro-CT analysis indicated that neomangiferin could significantly inhibit UHMWPE abrasion granule induced skull resortion and sissolution and inhibit osteoclast formation in mice(P<0.05).Histopatological analysis indicated that neomangiferin significantly inhibited the release of inflammatory cytokines TNF-α,IL-1β.Conclusion Neomangiferin has a significant anti-inflammatory effect can induce bone lysis around the prosthesis induced by UHMPE particles.The fourth part The effect of neomangiferin on osteoblast differentiation and gene expressionObjective To investigate the effect of neomangiferin on the formation and differentiation of primary osteoblasts and the expression of osteoblast-related genes in mice.Methods BMMs from healthy C57BL/6 mice were extracted,and the digested BMMs was cultured in DMEM containing 10% FBS,100U/ml penicillin and100mg/ml streptomycin.The original generation of OB respectively innoculated in 24 holes cell culture plate and the osteoblast induced liquid medium stimulate osteoblast differentiation,intervention with different concentrations of neomangiferin,ALP activity of adherent cells was determined by alkaline phosphatase kit after 7days of cell culture.On the twenty first day Alizarin red kit was used to detect the mneralization of adherent cells in each group was not different after the interventin with different concentrations of neomangiferin.Alizarin red staining showed that the mineralized area was similar between the control group and the neomangiferin treatment group.RT-PCR showed that neomangiferin had no effect on the expression level of OB related genes different time points.Conclusion: Neomangiferin had no effect on the formation of mouse osteoblasts and bone mineralization in vitro.