The Mechanism Of Daphnetin In The Treatment Of Osteoclast-related Local Osteolysis Disorders

Background and aims: Inflammatory osteolysis caused by aseptic prosthesis loosening and infection around the prosthesis is a major complication of total joint replacement.At present,the main method for treating this complication is still the revision of total joint replacement surgery.The inflammatory reaction caused by bacterial endotoxin or wear particles derived from implants is the main cause of osteoclast formation and activity,and osteoporosis caused by inflammation.Activation of the cells and hyperactivity of the bone resorption function cause local osteolysis around the joint prosthesis to eventually lead to loosening of the prosthesis and cause surgical failure.Thus,drugs or compounds that reduce inflammation and inhibit osteoclast differentiation and inhibit excessive bone resorption provide a promising therapeutic approach to prevent aseptic prosthesis loosening of total joint replacement.Daphnetin is a natural coumarin derivative with anti-inflammatory effects and is clinically used to treat rheumatoid arthritis.In this study,we reported for the first time that Daphtein has a protective effect on lipopolysaccharide-induced inflammatory bone destruction in a mouse skull osteolysis model.This protective effect of daphnetin can be attributed inpart to its direct inhibition of RANKL-induced osteoclast differentiation,fusion and bone resorption in vitro.Biochemical analysis revealed that Daphnetin inhibited the activation of the ERK and NFATc1 signaling cascades.In summary,our results indicate that Daphnetin has the potential to treat inflammatory osteolysis as a natural compound.Experimental methods:(1)In vitro cell experiment.1)First,we explored whether Daphnetin inhibited the osteoclast differentiation.We cultured bone marrow macrophages washed out of the mouse bone marrow cavity for 4-5 days and then planted in96-well plates,stimulated in RANKL and MCSF.Next,different concentrations of daphnetin were used to observe the number of osteoclasts by Trap staining,and the effect of daphnetin on osteoclast formation was determined.Next,the effect of Daphnetin on the proliferation of murine bone marrow macrophages(BMMs)was examined by CCK8 assay.Finally,different concentrations of Daphnetin were used to stimulate RANKL-induced osteoclast differentiation,and Real-time PCR was used to detect the difference of osteoclast-related gene expression after the action of ruthenium.2)To explore the functional effects of ruthenium on osteoclasts by measuring the dimple area and osteoclast actin ring induced by hydroxyapatite bone resorption experiments.3)In order to explore the molecular mechanism of ruthenium on osteoclast differentiation and bone resorption,we examined the effects of Daphnetin on MAPK and NF-?B signaling pathways by Western blot,and then used luciferase assay to detect NFATc1 by luciferase assay.influences.Finally,the effects of Daphnetin on nuclear proteins NFATc1 and C-Fos were observed by Western blot.(2)In vivo animal experimentsThe use of daphnetin in LPS-induced osteolysis of the calvaria of mice,to explore the therapeutic effect of daphnetin on local osteolysis caused by inflammatory factors,and to verify the inhibition of osteoclast differentiation and function by ruthenium in animal experiments in vivo effect.Experimental results:(1)In vitro experiment1)Daphnetin has an inhibitory effect on the differentiation and formation of osteoclasts,and affects the expression of osteoclast-specific genes,and has no toxic effect on BMMs in this concentration range.2)Daphnetin inhibited the bone resorption function of osteoclast.3)Daphnetin suppressed osteoclast differentiation and bone resorption by inhibiting phosphorylation of ERK and nuclear protein NFATc1.(2)in vivo experimentDaphnetin has a therapeutic effect on LPS-induced local osteolysis in mice.RESULTS: Daphnetin treated LPS-induced osteolysis and RANKL-mediated osteoclast formation by inhibiting the ERK and NFATc1 pathways.