The Effects And Mechanism Of Naringin On The Co-culture System Of Osteoblast And Osteoclast And Endothelial Progenitor Cells

Objective Naringin is an important component of the traditional Chinese medicine Rhizoma Drynariae.Evidence indicated that naringin plays an important role in promoting osteoporotic fracture.In vivo studies,naringin can promote osteoblast(OB)activity and inhibit osteoclast(OC)bone resorption.However,previous studies did not consider the mutual coupling effect of OBs and OCs in vivo.The effects of naringin on the OBs or OCs lonely did not reflect the real situation.Co-culture with OBs and OCs by Transwell loculus can simulate the microenvironment in vivo.Recently,it was suggested that bone regeneration was closely related to angiogenesis.In addition,our previous studies have shown that naringin can promote angiogenesis in early period in the healing process of osteoporotic fracture.While angiogenesis wa mainly mediated by the endothelial progenitor cells(EPCs)homing to injured site.Therefore,we speculate that naringin could promote EPCs differentiated into endothelial cells(ECs),and promote early vascularization of bone fracture healing.The purpose of the current studies:(1)Transwell was used to establish osteoblasts-osteoclasts co-culture model,different doses of naringin was performed to observe of osteoblastic bone formation and osteoclasts bone resorption effect.PCR and Western-blot techbology were used to investigate potential mechanism of naringin on OBs and OCs.(2)Isolation of rat bone marrow EPCs,immunofluorescence and morphological methods were used to identify the culturing cells were EPCs.MTT assays was used to identify the optimal concentration of naringin.Matrigel matrix was used to observe the effect of naringin on differentiation of EPCs.Meanwhile,to explore the role of SDF-1?/CXCR4/PI3K/Akt axis in the process of naringin on EPCs differentiation.Methods: 1.MC3T3-E1 and RAW264.7 cell lines were used to establish the co-culture model,the ratio of OB and OC 2:1 as referenced.And then the upper and lower chambers of Transwell were seeded with OB and OC respectively.There were a total of four groups(naringin group with 2ng/m L,20ng/m L and 200ng/m L naringin)and control group.Seven days later,TRAP staining and bone absorption area were used to identify the bone resorption ability.RTPCR and q RTPCR were used to analyses the relative expression of bone osteoprotegerin(OPG),receptor activator of nuclear factor-? B ligand(RANKL)and the ratio of OPG/RANKL and osteoclast differentiation genes NFATc-1,NFATc-2 and RANK genes.Meanwhile,the relative expression of OPG,RANKL and RANK were by tested by Western-blot.2.Four weeks' old Sprague Dawley(SD)rats were obtained and the femur and tibia were obtained in a relative gnotobasis environment.Then the bone marrow was rushed and isolated by the mononuclear cells separating medium.EPCs were then authenticated.MTT assay was used to determine the optimal dose of naringin for EPCs.PCR and Western-blot was used to identify the relative expression of SDF-1? and CXCR-4 in the EPCs when adding the naringin.Meanwhile,the downstream signaling way PI3 K and Akt was also identified.Matrigel matrix was used to investigate the angiogenesis ability that identified by the tube area and length in the Matrigel matrix.Then the CXCR4 receptor antagonist AMD3100 and PI3 K inhibitor LY294002 was respectively incubated and the length and area of tube formation chang.Meanwhile,PCR and Western-blot was used to identify the relative expression of SDF-1?,CXCR-4,PI3 K and Akt changes.Data analysis was performed by the software of SPSS 20.0 and graph was drawed by the Graphpad 6.01 software.Results: 1.Naringin can promote the expression of OPG and RANKL in MC3T3-E1,and naringin can also improve the ratio of OPG/RANKL,the optimal dose was set at the dose of 20ng/m L.TRAP positive cells in the naringin groups was less than control group,the difference was significantly significance(P<0.05).Meanwhile,naringin inhibits the gene expression of osteoclast differentiation,NFATc-1 NFATc-2 and promote the expression of RANK.The difference was statistically significant when compared with control group(P<0.05).2.MTT results indicated that the optimal dose of naringin on the proliferation of EPCs was 500ng/ml,500ng/ml of naringin can promote blood vessel formation reflected by the tube area and the length in the matrix when compared with the control group,the difference was statistically significant(P<0.05).PCR and Western-blot results showed that naringin at the dose of 500ng/ml can promote the gene and protein expression of SDF-1? and CXCR-4,the difference was statistically significant(P<0.05).Compared with the control group,when incubated with AMD3100,the area and length of the blood vessels on the matrix adhesive was decreased.Compared with the group of AMD3100,naringin combined with AMD3100 can increase the angiogenesis area and length,the differences were statistically significant(P<0.05).Compared with LY294002 group,naringin combined with LY294002 can increase the EPCs tube formation,the difference was statistically significant(P<0.05).Conclusion 1.In the co-culture system,naringin can promote osteoblast activity and meanwhile inhibit osteoclastic bone resorption ability.The mechanism may through regulating the ratio of OPG/RANKL.2.Naringin can promote EPCs proliferation and angiogenesis ability in vitro,these effects possibly by through regulating the signaling pathway of SDF-1?/CXCR4/PI3K/Akt.