The Mechanism Of Artesunate In The Prevention And Treatment Of Osteoclast-related Osteolysis Disorders

Background and aims: osteoclasts are the only cells with bone resorption in human body.The abnormal number and function of osteoclasts are closely related to diseases such as osteoporosis,osteoarthritis,tumor bone destruction and so on.Although many drugs to prevent and cure osteoclast-related bone loss,there are still some adverse reactions in long-term use.Therefore,there is an urgent need to seek or develop new drugs that can effectively inhibit the activation of osteoclasts and have a definite effect,reasonable price,and less side effects.Traditional Chinese medicine and its active components are becoming a hot spot in the research of anti-bone loss drugs because of their wide range of materials and small side effects.Artesunate(Art),the water-soluble derivative of artemisinin has been investigated owing to its anti-malarial properties.However,its effects in osteoclastogenesis havenot yet been reported.In this study,we found that artesunate can effectively inhibit osteoclast differentiation and bone resorption,and bone loss in osteolysis mice.Experimental methods:(1)In vitro experiment: 1)Bone-marrow macrophages(BMMs)were obtained from femurs and tibias of6-week-old C57/BL6 mice.In order to induce osteoclast differentiation,BMMs were cultured in α-MEM supplemented with RANKL and M-CSF in the absence or presence of Art.The cells were fixed in 4 % 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.To perform the resorption pit assay,BMMs were placed on a calcium phosphate(CaP)-coated 48-well plate and cultured with M-CSF,RANKL,and treated with Art until generate multinucleated osteoclasts.Cell viability was determined using the MTS cytotoxicity assay.2)the effects of artesunate 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: A mice calvarial osteolysis model and OVX model were established to measure the osteolysis suppressing effect of Art.Result: In this study,Art was shown to inhibit the nuclear factor-B ligand(RANKL)-induced osteoclastogenesis,the mRNA expression of osteoclastic-specific genes,and resorption pit formation in a dose-dependent manner in primary bone marrow-derived macrophages cells(BMMs).Furthermore,Art markedly blocked the RANKL-inducedosteoclastogenesis by attenuating the phosphorylation of p38,degradation of IκB and phosphorylation of NF-κB p65.Consistent with the in vitro results,Art inhibited OVX-and lipopolysaccharide(LPS)-induced bone resorption by suppressing the osteoclastogenesis.Conclusion: Together our data demonstrated that Art inhibits RANKL-induced osteoclastogenesis by suppressing the p38-MAPK signaling pathway and NF-κB signaling pathway and that it is a promising agent for the treatment of osteolytic diseases.