Diosmetin Inhibits Osteoclast Formation And Differentiation And Prevent LPS-induced Bone Loss In Vivo

Background and aims: Osteolytic bone diseases are closely linked to the over-activation of osteoclasts and enhancement of bone resorption.It has become a major health issue in orthopedic practice worldwide.Inhibition of osteoclasts is proposed to be the main treatment for osteolytic disorders.Diosmetin(DIO)is a natural flavonoid with properties of antioxidant,anti-infection and anti-shock.The effect of DIO on osteoclast differentiation is poorly understood.In this research project,we found that DIO could inhibit osteoclastic formation induced by RANKL in a dose-dependent manner.The expression of the osteoclast differentiation marker gene,Ctsk,NFATc1,Acp5,Ctr,Atp6v0d2,Mmp9 were also decreased by the treatment of DIO.In addition,DIO attenuated the formation of actin ring and the ability of bone resorption.Further,Western blotting showed that DIO inhibits the phosphorylation of MAPK signaling pathway induced by RANKL,accompanied with the downregulation of NFATc1 and c-Fos expressions.We also found that DIO could reduce the accumulation of reactive oxygen species(ROS)induced by RANKL.In vivostudy revealed that DIO can significantly reduce LPS-induced osteolysis in mice.Collectively,our study shows that DIO can inhibit osteoclast formation and activation and could serve as a potential therapeutic drug for osteolytic bone diseases Experimental methods:(1)In vitro experiment: 1)6-week-old C57BL/6J mice were sacrificed and bone marrow cells obtained by exposure of medullary cavity of femur and tibia and cultured in α-medium supplemented in order to induce osteoclast differentiation,BMMs were cultured in α-MEM supplemented with RANKL and M-CSF in the absence or presence of DIO.The cells were fixed in4 % formalin for 20 minutes and osteoclast formation was investigated by TRAP staining.TRAP-positive multinucleated cells with more than 3 nuclei were calculated as osteoclast cells.Total RNA was extracted from the cells,Real-time PCR was used to analyze the expression of osteoclast specific genes in this study.BMMs were cultured in 96-well plates with complete α-MEM medium containing M-CSF and RANKL and treated with 5 μM or 10 μM DIO for 5 days.When osteoclast formation was observed,cells were fixed with 4%paraformaldehyde for 15 mins at room temperature and permeabilized with0.1% triton X-100 in PBS for 5 mins.The cells were blocked by 3% BSA in PBS for 30 mins.Next,cells were incubated with 0.2% phalloidin in PBS at room temperature for 60 mins.DAPI staining was performed to observe the nucleus.Multiple image of the cells was taken using a fluorescent microscope.To perform the resorption pit assay,the reactive oxygen species(ROS)levels in the cells were determined by reactive oxygen species assay kit with 2,7-dichlorodihydrofluorescein diacetate(DCFH-DA),the images were obtained by fluorescence microscope.BMMs were placed on a calcium phosphate(CaP)-coated 48-well plate and cultured with M-CSF,RANKL,and treated withDIO until generate multinucleated osteoclasts.CCK-8 Kit was employed to detect the cytotoxicity of DIO on BMMs.2)the effects of DIO on the phosphorylation of important signal proteins in RANKL-induced NF-κB and MAPK signaling pathway were detected by western blot assay.(2)In vivo experiments: An LPS model were established to measure the osteolysis suppressing effect of DIO.Result: We found that DIO effectively suppresses phosphorylation of MAPK signaling pathway induced by RANKL.But has no effect on NF-κB signaling pathway.Next,we evaluated the expression levels of RANKL-induced transcription factors NFATc1 and c-Fos,which are critical to the terminal differentiation of osteoclasts.By western blotting analyses,we found that the protein expression of NFATc1 was suppressed,whereas the expression of c-Fos was mainly inhibited relative to control group.These results revealed that DIO inhibits the protein expression of key transcription factors NFATc1 and c-Fos.The mRNA expression of osteoclastic-specific genes,DIO was able to inhibit the resorption activity of osteoclast accompanied with the impairment of F-actin ring formation in vitro.The signal intensity and quantity of ROS positive cells after DIO treatment showed a decreasing trend in a concentration-dependent manner.Furthermore.Consistent with the in vitro results,DIO inhibited LPSand lipopolysaccharide(LPS)-induced bone resorption by suppressing the osteoclastogenesis.Conclusion: our study has demonstrated for the first time that DIO has the capacity of diminishing osteoclast differentiation and osteoclast resorption activity,mainly by inhibiting MAPK signaling pathway,rather than NF-κB signaling pathway.In addition,the inhibition of ROS by DIO can also play a role in suppressing osteoclast formation.Further,DIO displays ananti-inflammatory and bone-protective effect on the LPS-induced osteolysis in mice.These results indicate that DIO might have potential clinical application value for osteoclast related diseases.