Mechanism Of Action Of Protein Kinase C Inhibitor G?6983 On Osteoclasts

Background and aims: At present,artificial joint replacement is the most effective treatment for severe bone and joint disorders.Aseptic loosening of artificial joints is the most common cause of replacement failure.At present,effective treatment for aseptic loosening of artificial joints is still undergoing artificial joint revision surgery.However,its mechanism remains unclear.The mainstream view is that the occurrence of osteolysis is related to the wear particles generated around the prosthesis.These wear particles can directly or indirectly promote the abnormal formation of osteoclasts and activate the bone resorption function of osteoclasts,causing osteolysis around the prosthesis,and the final result is loosening of the prosthesis.The only cells with bone resorption function in the human body are osteoclasts.The abnormal production and function of osteoclasts are closely related to the aseptic loosening of artificial joints,osteoarthritis and osteoporosis.At present,there are many drugs on the market that prevent bone loss due to excessive activation of osteoclasts in the market for abnormal osteolysis,but many long-term applications still find many adverse reactions.Therefore,the development of new anti-osteoclast drugs is currently a hot topic.G?6983 is an inhibitor of five PKC isoforms that can enter cells by simple diffusion.In this study,we found that G?6983 can effectively inhibit osteoclast differentiation and bone resorption,and can resist osteolysis caused by titanium particle wear in the mouse calvarial model.Experimental methods:(1)In vitro experiments: 1)To determine the effect of G?6983 on osteoclast differentiation and bone resorption,bone marrow-derived macrophages(BMMs)were cultured in vitro.The concentration of G?6983 interfered with RANKL-induced osteoclast differentiation.After TRAc P staining,the formation of osteoclasts(cells with ? 3 cells)was observed and observed.The effect of drugs on the proliferation of BMMs was detected by MTS assay.Different concentrations of G?6983 were used.After 6 days of intervention with RANKL to induce osteoclasts,the expression of osteoclast-specific genes was determined by Real-time PCR.The hydroxyapatite plate was used for bone resorption experiments to analyze the effect of G?6983 on bone resorption of osteoclasts..2)To investigate the regulation of G?6983 on RANKL-induced osteoclast-associated signaling pathway,we used Western blot to detect G?6983-related proteins in NF-?B and MAPK signaling pathways induced by RANKL(NF-?B,JNK,p38,ERK)The effect of phosphorylation.After confirming that G?6983 can affect RANKL-induced osteoclast differentiation and bone resorption,we further analyzed the effects of G?6983 on osteoblast differentiation and bone formation.(2)In vivo experiment: Titanium-induced mouse skull osteolysis model: 8-week-old male C57 mice were randomly divided into 4 groups,sham operation group,positive group,and low dose group(G?6983 2.5 mg/kg).High dose group(G?6983 5mg/kg).After G?6983 intervention for 14 days,the calvarial bones of the mice were collected,and the results of micro-CT and pathological tissue analysis were analyzed.RESULTS:(1)In vitro experiments: 1)G?6983 inhibited osteoclast differentiation and function,and had no toxic effects on osteoclast precursor cells;2)G?6983 had no effect on osteoblast differentiation and bone formation.(2)In vivo test: G?6983 is resistant to osteolysis of mouse skull induced by titanium particles.Conclusion: G?6983 can resist the osteolysis of mouse skull induced by titanium particles by inhibiting the function of osteoclast differentiation and the function of bone resorption.