Sabutoclax Inhibits Osteoclast Differentiation And Associated Osteolytic Diseases

Objectives: The maintenance of human bone homeostasis hinges on the balance between osteoclast and osteoblast function.Osteoclasts,whose main function is bone resorption,are the major cells involved in bone remodeling and pathological bone destruction in vivo.Differentiation and over activation of osteoclasts may lead to related osteolytic diseases.Therefore,it may play an important role in the prevention and treatment of related osteolytic diseases by inhibiting the differentiation and function of osteoclasts.Therefore,the search for effective drugs that target osteoclasts to treat osteolysis related diseases is a research hotspot in recent years.In this study,we investigated the effects and mechanisms of sabutoclax on animal models of osteolytic diseases by investigating the molecular biological mechanisms by which sabutoclax inhibits osteoclast differentiation and function in vitro,and then exploring whether sabutoclax could be a novel treatment for osteoclast related osteolytic diseases.,in vivo.Methods:(1)The potential role of sabutoclax in osteoclast related osteolytic diseases was analyzed based on the related database of network pharmacology.(2)Bone marrow macrophages(BMMs)of C57 BL / 6 mice were extracted and induced by RANKL,and then treated with different concentrations of sabutoclax.The effect of sabutoclax on osteoclast differentiation was analyzed by tartrate resistant acid phosphatase(TRAP)staining.The cytotoxicity of sabutoclax on osteoclast precursor was determined by CCK8 method,and IC50 was calculated Methods: osteoclasts were stained with cyclic peptide/DAPI to observe F-actin ring;Bmms were inoculated into bone slices to observe the effect of sabutoclax on bone resorption function;QRT PCR was used to detect the expression of genes related to sabutoclax on osteoclasts,including DC-STAMP,c-Fos,Trap,Ctsk,MMP9 and NFATc1;double luciferase gene report and immunofluorescence staining experiment were used to explore the inhibitory effect of sabutoclax on NFATc1 nuclear transfer in osteoclasts;Western blot was used to detect the effect of sabutoclax on RANKL induced signal pathways,including NF-κB,MAPK and other signal pathways,in order to find the target of sabutoclax in osteoclasts.(3)We established an animal model of LPS induced calvarial osteolysis in mice,which was divided into four groups,including sham operation group,positive group,low drug group and high drug group.Different concentrations of sabutoclax were used to intervene for 7 days.Micro CT scanning was used to analyze the calvarial osteolysis in each group,and the number of osteoclasts in each group was statistically analyzed by pathological section staining.(4)We used CCK8 test,cell scratch test and Transwell test to evaluate the proliferation,migration and invasion of cells,to illustrate the effect of sabutoclax on the metastasis of MDA-MB-231 cells.We detecte the apoptosis of MDA-MB-231 cells by flow cytometry.QRT-PCR and Western blot were used to evaluate the effects of sabutoclax on apoptosis genes and related signaling pathways of MDA-MB-231 cells.We injected breast cancer MDA-MB-231 cells into the tibial plateau of nude mice to establish the osteolysis model of breast cancer metastasis.They were divided into control group,positive group,low sabutoclax group and high sabutoclax group.After 28 days of intervention with different concentrations of sabutoclax,micro CT scanning was used to analyze the osteolysis of tibia of nude mice in each group,and the pathological sections were stained for statistical analysis.Results:(1)We found that sabutoclax may act on osteoclast related osteolytic diseases by network pharmacology study.(2)Cytotoxicity experiments showed no toxic effect on osteoclast precursor cells at concentrations lower than or equal to 0.5μm for 48 h of sabutoclax,with an IC50 of 0.89 μm,respectively.Osteoclast differentiation experiments showed that sabutoclax(0.1,0.2,0.3,and 0.4 μ m)inhibited osteoclastogenesis in a dose-dependent manner.The results of F-actin staining showed that sabutoclax inhibited F-actin ring formation in a dose-dependent manner.The results of statistical analysis also supported this conclusion.The results of bone resorption experiments showed that sabutoclax(0.2 and 0.4μm)inhibited osteoclastic bone resorption area in a dose-dependent manner.PCR assays showed that,compared with the control group,sabutoclax inhibited the expression of osteoclast related genes DC-STAMP,c-Fos,TRAP,Ctsk,MMP9 and NFATc1 in a dose-dependent manner.Dual luciferase gene reporter and immunofluorescence staining assays confirmed that sabutoclax inhibited the nuclear translocation of NFATc1 in osteoclasts.Results of Western blot showed that sabutoclax was able to inhibit the phosphorylation of ERK protein,thereby impairing the activation of MAPK signal transduction pathway.In vitro osteoclastogenesis and bone resorption were markedly attenuated.(3)Sabutoclax injections in mice protected them from lipopolysaccharide(LPS)-induced skull destruction.Micro-CT showed that sabutoclax reduced LPS induced calvarial osteolysis as indicated by increased perforation rate,number of bone pits,and bone volume fraction.Pathological tissue section results showed that sabutoclax decreased the number of osteoclasts in the osteolytic area of the skull.(4)Sabutoclax can treat tibial osteolysis induced by bone metastasis of breast cancer cells in nude mice in vivo.Sabutoclax inhibited proliferation,migration and invasiveness of breast cancer cells,and it induced apoptosis of MDA-MB-231 cells as well as decreased expression of apoptotic genes and upregulation of anti apoptotic genes,along with inhibition of PI3K/Akt protein phosphorylation signaling.Conclusions:Sabutoclax inhibited RANKL induced osteoclast formation and bone resorption in vitro and LPS and breast cancer MDA-MB-231 cell induced osteolysis in vivo by regulating ERK/NFATc1 signaling.In conclusion,based on the favorable biosafety profile of sabutoclax,this study provides,for the first time,a novel therapeutic option for the clinical treatment of osteolytic diseases.Our study suggests that sabutoclax has a potential therapeutic effect on osteoclast related osteolytic diseases.