Mechanism Of Deguelin In Preventing And Treating Osteolysis Induced By Titanium Particles

Background and aims:One of the important reasons for the failure of total hip arthroplasty is periprosthetic osteolysis stimulated by titanium(Ti)particles and subsequent aseptic loosening.The purpose of this study was to investigate the effect of Deguelin(Deg)on osteoclast differentiation and osteolysis-mediated by Ti particles.We’ve demonstrated that Deg,a natural component extracted from leguminous plants,had significant inhibitory effect on the dissolution of mice’s skull stimulated by Ti particles.Further histological analysis showed that the key to prevent osteolysis induced by Ti particles was to inhibit the differentiation and function of osteoclasts.We found that Deg could suppress osteoclastogenesis,bone resorptive function and F-actin ring formation by the induction of receptor activator of nuclear factor-kB ligand(RANKL).In addition,Deg could significantly inhibit the expression of osteoclast specific genes,including V-ATPase D2(Atp6v0d2),calcitonin receptor(Ctr),cathepsin K(Ctsk)and matrix metalloproteinase-9(Mmp9).Our experiments have also confirmed that Deg inhibited osteoclastogenesis and function via attenuating the AKT and MAPK signaling pathways.Collectively,these data indicated that Deg was a potential natural agent for the prevention and treatment of periprosthetic osteolysis and other osteoclast-related osteolytic diseases.Experimental methods:(1)In vitro experiment:In order to determine the effect on the differentiation and function of the osteoclast,we take 4-6-week-old C57/BL6 mice to take the bone marrow-derived mononuclear macrophages(BMMs)from the femur and the tibia.First of all,the effect of rattan on the toxicity of bone marrow-derived mononuclear macrophages was excluded,and we used the CCK8 kit to test the proliferation of mononuclear macrophages from bone marrow.After that,we treated RANKL-induced BMMs to the osteoclast in different concentrations,and through the anti-tartrate-acid phosphatase staining(TRAP staining),the morphological status of the osteoclast differentiation was observed.TRAP staining was positive and the number of nuclei in the nucleus was considered to be osteoclast,and the osteoclast was counted as a result of the observation of the effect of the viberine on the differentiation of osteoclasts.The expression of osteoclast-specific gene was determined by Real-time PCR.In ord to study that regulation and regulation of the related signal pathway of the effect of rattan on the differentiation of osteoclast and the function of bone absorption,we use the Western blot method to study the effect of rattan on the differentiation of osteoclast and the function of bone resorption.The effect of rattan on the phosphorylation of important signaling proteins of the signal pathway of NF-B,MAPK,PI3K-AKT signal induced by RANKL was detected.Based on the early experiments to determine the effect of the rattan to the osteoclast differentiation and the bone resorption induced by RANKL,we further analyze the effect of the viberine on the dissolution of the cranium of the mouse induced by the titanium particles.(2)In-vivo experiment:In order to exclude the effect of estrogen on osteoclast,in vivo,8-10-week-old male C57/BL6 mice were randomly assigned to four experimental groups,and the control group(PBS group)was selected.Titanium-particle,low-drug concentration(Ti-particle+low-concentration Deguelin),high-drug concentration(Ti-particle+high-concentration Deguelin)were collected.After 17 days,the cranium of the mouse was collected,and the experimental results were analyzed by the micro-CT scan and the morphological analysis of the pathological section.Result:(1)in vitro experiment:1)Deguelin has obvious inhibitory effect on osteoclast differentiation and has no toxic effect on osteoclast precursor cells,2)Deguelin can effectively inhibit the expression of osteoclast-specific gene,and it has no toxic effect on osteoclast precursor cells,2)Deguelin can inhibit osteoclast-specific gene expression.3)Deguelin can inhibit bone resorption by inhibiting the formation of F-actin ring in osteoclasts;4)the differentiation of osteoclasts was regulated by PI3K-AKT and p38 gene pathway.(2)in vivo experiment:Deguelin could effectively prevent and treat the damage of skull cap induced by titanium particles in mice.Conclusion:Deguelin Can effectively inhibit the differentiation of osteoclast and the bone absorption function,thereby preventing and treating the osteolysis of the periprosthetic bone induced by the titanium particles.