The Mechanism Of Vitexin Inhibiting Osteoclast Differentiation And Its Therapeutic Application

ObjectiveBone remodeling is a dynamic ongoing process mediated by the balanced actions of the bone forming osteoblasts and bone resorbing osteoclasts.Perturbations in this balance leading to excessive osteoclast-mediated bone resorption is the cause of many osteolytic diseases.It is imperative that novel agents are identified that can regulate abnormal osteoclast formation and activity for the effective and safe treatment of osteolytic diseases with minimal adverse effects.In the present study,we aimed to explore the effects of vitexin on osteoclast differentiation and the therapeutic role of vitexin on the osteoclast-related osteolytic diseases.MethodsPrimary murine BMM cells were isolated from 6-week-old C57BL/6 mice by flushing the marrow from the femurs and tibiae.And osteoclast differentiation were induced by the stimulation of receptor activator of nuclear factor-κB ligand(RNAKL)and macrophage colony-stimulating factor(M-CSF).First,the CCK-8 assay was performed to determine the non-toxic dose of vitexin on BMMs.Next,the effect of vitexin on the differentiation of osteoclast was evaluated by TRAP staining(TRAP-positive cells with more than 3 nuclei were counted as osteoclast).Then,bone resorption assay and F-actin ring staining assay were used to determine the effect of vitexin on the function of osteoclast.Furthermore,q-PCR were used to test the expression of osteoclast-specific genes including Acp5,Ctsk,Fos,Nfatc1,Dcstamp and Atp6v0d2.And western blotting was performed to examine the impact of vitexin on MAPKs and NF-κB signaling pathways.Finally,a murine model of lipopolysaccharide(LPS)-induced calvarial osteolysis was established to explore the therapeutic potential of vitexin.The mice were randomly divided into four groups:Sham-operated(injection with PBS only),LPS(5mg/kg body weight),low dose vitexin(LPS with injection of 5mg/kg vitexin),and high dose vitexin(LPS with injection of 15mg/kg vitexin).Mice received subcutaneous injections over the sagittal midline suture of the calvaria under light anaesthesia for 7 days.At the end of the experimental period,all mice were sacrificed.The calvarias were excised,fixed in 4%PFA for 3 days and processed for micro-computed tomography(μCT)and histological analysis.ResultsWe demonstrated that BMMs exposed to 50μM or less of vitexin for 48 or 96hrs showed no cytotoxic effect However,a time-dependent decrease in cell viability was observed in cells treated with 100 μM of vitexin.M-CSF-dependent BMMs that were stimulated with RANKL in the presence of vitexin showed a dose-dependent decrease in their ability to respond to RANKL.This resulted in less TRAP-positive multinucleated osteoclasts being formed at the end of the 6 day culture.In addition,treatment of cells with vitexin on day 2 to day 4(D2-D4;mid stage)or from day 4 to day 6(D4-D6;late state)of the 6 day period,resulted in a decrease in the number of TRAP-positive osteoclasts formed.However,treatment of cells on the first 2 days of RANKL stimulation(D0-D2;early treatment)did not significantly alter the number or size of TRAP-positive osteoclasts formed.Since vitexin inhibited RANKL-induced osteoclast differentiation,its effect on in vitro osteoclast bone resorption was further examined.The F-actin ring staining assay showed that smaller and fewer F-actin rings were observed in vitexin treated cells as compared with untreated controls.The pit formation assay indicated that the in vitro bone resorptive capacity of vitexin-treated osteoclasts were significantly reduced when compared with untreated controls.And the q-PCR results suggested that treatment of vitexin significantly inhibited the expressions of RANKL-induced osteoclast marker genes including Acp5,Ctsk,Fos,Nfatcl,Dcstamp,Atp6v0d2 when compared with untreated controls.Additionally,the activation phosphorylation of MAPK members ERK and p38 occurs rapidly and within 5mins of RANKL stimulation,peaking at 15min before returning to basal levels of phosphorylation.In contrast,treatment with vitexin markedly inhibited the phosphorylation of ERK and p38.Furthermore,to examine the effects of vitexin on c-Fos and NFATcl expression,BMMs were stimulated with RANKL without or with vitexin for 1,3 and 5 days.RANKL robustly induced the expression of c-Fos protein over the 5 day culture period.Treatment with vitexin however downregulated c-Fos expression by approximately half on day 3 of RANKL stimulation.Similarly the induction of NFATcl in response to RANKL during the 5 day period was significantly inhibited following treatment with vitexin,With promising anti-osteoclastogenic and anti-resorptive effects,we next explored the potential protective effect of vitexin on LPS-induced calvarial osteolysis in vivo.The 3D micro-computed tomographic reconstructions showed extensive bone loss in mice injected with LPS only relative to PBS-treated sham controls.In contrast,treatment with vitexin(low-and high-dose groups)strongly suppressed LPS-induced osteolytic bone loss.Quantitative morphometric analyses further showed the protective effect of vitexin treatment on preventing the loss of bone volume/tissue volume(BV/TV),and decrease number and percentage of bone porosity in a dose-dependent manner.Histological assessments further demonstrated the protective effect of vitexin on osteolytic bone erosion.Consistent with marked reduction in inflammatory bone loss and a dose-dependent increase in bone volume,the number of TRAP-positive multinucleated osteoclasts were significantly reduced following vitexin treatment.This inhibitory effect on osteoclasts was further confirmed by histomorphometric analysis which showed a dose-dependent decrease in the total number of TRAP-positive osteoclasts,as well as the number of osteoclasts per bone surface.Conclusion1.Vitexin can inhibit osteoclast differentiation in vitro.2.Vitexin can suppress the RANKL-induced activation of ERK and p38 MAPK signaling pathways during osteoclast formation.3.Vitexin can protect against LPS-induced bone loss in vivo...