Mechanism Of Cimifugin Inhibiting Titanium Particle-related Osteolysis

Objective:Wear particles from prosthetic wear after arthroplasty can cause periprosthetic osteolysis,which is the most common cause of aseptic loosening of the prosthesis long after surgery,thereby reducing prosthetic longevity and patient quality of life.There are no satisfactory preventive drugs for aseptic loosening of the prosthesis,and prosthetic joint revision surgery is risky and expensive.The search for herbal extracts that can be used to prevent periprosthetic osteolysis is a current research hotspot,and China has very rich herbal resources that offer the possibility of finding these drugs.Previously,researchers have found that cimifugin have inhibitory effects on signaling pathways related to osteoclast differentiation and function in studies on cimifugin.In contrast,osteoclasts are the only bone resorbing cells,and their massive aggregation and activation at the bone-prosthesis or bone-cement interface is an important cause of periprosthetic osteolysis.Therefore,the purpose of this study was to investigate the effect of cimifugin on the differentiation and function of osteoclasts and the mechanism of action therein,and to verify the effect of cimifugin on titanium particle-induced osteolysis in a mouse cranial bone model,providing a pre-study basis for the use of cimifugin in clinical prevention of periprosthetic osteolysis caused by wear particles.Method:1.The non-cytotoxic concentration of ascomycin was detected by CCK-8 assay,and then the differentiation of osteoclasts in vitro after the addition of cimifugin was detected by osteoclast formation assay in the range of non-cytotoxic concentration.Bone fragment resorption assay and immunofluorescence assay were used to detect the changes of osteoclast bone resorption function and F-actin ring after the addition of cimifugin.2.Quantitative real-time polymerase chain reaction(q RT-PCR)analysis was introduced to detect the expression of specific genes in osteoblasts after the addition of cimifugin treatment,followed by Western blotting and luciferase reporter gene analysis to detect the regulatory effects of cimifugin in NF-κB and MAPKs signaling pathways.3.A mouse skull osteolysis model induced by titanium particles was used to observe the effect of cimifugin on titanium particle-induced osteolysis by micro-CT histomorphometric analysis and immunohistochemical staining.Results:1.The cellular activities of RAW264.7 cells and bone marrow mononuclear macrophages were not significantly decreased after 96 h of cimifugin treatment at concentrations of 320 μmol/L and below.The number of osteoclasts,colony area,area of resorption pits formed on bovine bone fragments,and the number and area of F-actin rings of osteoclasts decreased after treatment with cimifugin in this concentration range,and the greater the cimifugin concentration,the greater the degree of decrease.2.After cimifugin treatment,the transcript levels of key osteoclast factors and marker genes such as c-Fos,Nfatc1,Acp5,Calcr,Ctsk,Dc-stamp and Traf6 in osteoclasts were reduced,the activation level of NF-κB signaling pathway was decreased,the phosphorylation of key protein IκBα on it was delayed,but the peak phosphorylation was increased,while the expression levels of related proteins on MAPK signaling pathway were not significantly changed.3.Treatment with cimifugin gavage resulted in a reduction in the number of osteoclasts induced by titanium particles implanted in the skull of mice,as well as a reduction in skull bone destruction.Conclusion:1.Non-cytotoxic concentrations of cimifugin inhibited RANKL-induced osteoclast differentiation and bone resorption,and the inhibitory effect was positively correlated with the concentration.2.The mechanism of action of cimifugin to inhibit osteoclastogenesis is inhibition of IκBα phosphorylation in the NF-κB signaling pathway.3.Cimifugin inhibits titanium particle-induced osteoclast aggregation and calvarial bone erosion in mice.