Effects Of Niclosamide And Its Derivatives DK-520 On RANKL-Induced Osteoclast Early Fusion And Differentiation

Osteoporosis is currently rated by the World Health Organization（WHO）as the second most harmful disease to human health,after cardiovascular disease.With the high urbanization brought about by reform,the increasing process of population aging and the widespread popularity of unhealthy lifestyles,the prevalence of osteoporosis is increasing.According to the results of the latest Osteoporosis epidemiological survey released by the National Health Council and the Centers for Disease Control and Prevention in October 2018,the prevalence of osteoporosis in people over 50 years of age in China is 19.2%,and the prevalence rate over 65 years is 32%,with female prevalence rates as high as 51.6%.Brittle fractures caused by osteoporosis seriously reduce the quality of life of patients and the 1 years after the fracture of the death rate increased up to 20%.It brings a heavy economic burden to both families and society.Therefore,it is of great social significance to pay attention to the prevention and treatment of osteoporosis and prevent the occurrence of fractures.Bone is constantly remodeled through osteoclasts and osteoblasts.The number and activity of osteoclasts,the only cells in the body that can perform bone absorption,play an important role for normal bone turnover and bone metabolism.The differentiation of osteoclasts depends on the receptor Activator of Nuclear factor Kappa B Ligands（RANKL）and the receptor Activator of Nuclear factor Kappa B（RANK）,which mediate and activate signaling pathways and downsteam transcription factors.RANKL regulates the differentiation of osteoclasts by mediating the expression of downstream signaling molecular nuclear transcription factor Kappa B（NF-κB）,C-jun,activated T-cell nucleus factor C1（NFATC1）and P38/c-fos（cellular oncogene Fos）.In addition,transcription factor PU.1 is directly combined with NFATC1 promoter to increase NFATC1 expression and promote the development of osteoclasts.The fusion of osteoclast progenitor cells（OCPs）is a necessary condition for osteoclast formation,RANKL induces the expression of dendritic cell-specific transmembrane protein（dendritic cell-specific transmembrane protein,DC-STAMP）.NFATC1 promotes Osteoclastogenesis and the differentiation of osteoclasts by combining with DC-STAMP to increase cell fusion between OCPs.ATPase mediates the external acidification of cells and plays an important role in the formation and fusion of osteoclasts.At present,the drugs of osteoporosis are still mainly to inhibit osteoclast bone resorbing,such as bisphosphonates.However,there are concerns of higher prices,poor patient compliance,digestive tract mucosal side effects,mandibular necrosis and atypical fractures with long-term treatment.Therefore,the development of new methods for treating osteoporosis is of paramount importance.Niclosamide has been widely used in antihelminthic therapy against intestinal infection of tapeworm for more than 30 years,with positive efficacy and small side effects.In recent years,Niclosamide has attracted wide attention as a potential anticancer drug,and targets multiple signaling pathways such as Wnt,NF-κB,Notch and STAT-3,interfering with cell metabolism,affecting cell growth rate,and even inducing cell death.Moreover,it can inhibit the maturation of dendritic cells induced by lipopolysaccharide and the expression of various cytokines by inhibiting c-fos,c-jun and c-myc,and inhibit the proliferation of osteosarcoma cells.These pathways and cytokines also play an important role in osteoclast-mediated bone resorption.Thus,we inferred that the Niclosamide may inhibit osteoclast differentiation and function and provide new ideas for the treatment of osteoporosis.DK-520 is a acyl derivative of Niclosamide,which can be metabolized to Niclosamide in vivo and improves the oral blood concentration and bioavailability.DK-520 also extend the duration of exposure and have no observable adverse effects over three weeks of oral dosing.By exploring the effects of Niclosamide and DK-520 on the differentiation of osteoclasts and the effects on the expression of main molecules PU.1,DC-STAMP and ATPase that regulate osteoclast differentiation and fusion,this paper clarifies the main molecular mechanisms of Niclosamide and DK-520 inhibitory effect on osteoclasts.It is expected to provide a new direction for the clinical treatment of osteoporosis and achieve the goal of "new use of old medicine".Part Ⅰ: Osteoclasts induced by RANKL and M-CSF Objective: In this part,we extracted the mononuclear macrophages from bone marrow,induced and cultured OCPs and mature osteoclasts with RANKL and M-CSF.In addition,we used TRAP staining and RT-PCR to identify the cells.We provided a research basis for the study of the function and differentiation of osteoclasts in vitro.Methods: Using healthy 4-8-week-old C57BL/6 mice,separating the tibia and femur,extracting mononuclear macrophages,adding RANKL and M-CSF to induce differentiation,observing cell morphology,identifying the morphology of osteoclasts through TRAP staining,and detecting the expression of osteoclast phenotype gene with RT-PCR.Results: After 3-6 days of induction,the presence of TRAP（+）multicore cells was seen,and the expression of osteoclast phenotype gene was found.Conclusion: Bone marrow mononuclear macrophages extracted from the femur and tibia of C57BL/6 mice can produce sufficient number of stable osteoclasts induced by RANKL and M-CSF.Part Ⅱ:Effect of Niclosamide and DK-520 on RANKL-induced osteoclast differentiation Objective: To extract bone marrow-derived mononuclear macrophages and RANKL and colony stimulating factor（M-CSF）was used to induce the OCPs and mature osteoclasts.The effects of two drugs on the formation of TRAP+ osteoclast progenitor cells and mature osteoclasts were evaluated by niclosamide and DK-520 intervention.Methods: Taking OCPS cells as the research object,detected the drug toxicity by CCK-8 and selected appropriate concentration for follow-up experiments.According to the type and concentration of the Niclosamide and DK-520,cells were divided into 5 groups,DMSO group,Niclosamide 0.75μM Group,Niclosamide 1.5Μm Group,DK-520 0.75μM Group and DK-520 1.5μM Group.To identify the time periods of suppressing differentiation of osteoclasts by Niclosamide and DK-520,we added these drugs in the different time of osteoclastogenesis: 0-1 days,1-2 days,2-3 days and 3-4 days.After 3 days of culture,the cells were stained using the TRAP kit.The difference of the number and size of OCPs and mature osteoclasts was compared between the intervention groups and control group.Results: Compared with control group,Niclosamide and DK-520 inhibited the information of TRAP+ multinucleated cells in a dose-dependent manner（p<0.05）.In addition,the results of time periods of suppressing differentiation of osteoclasts showed that Niclosamide and DK-520 inhibited RANKL-induced osteoclast differentiation in the early stage of the osteoclastogenesis（p<0.05）.Conclusion: Niclosamide and DK-520 significantly inhibited the differentiation of osteoclasts,especially in the early stage of osteoclastogenesis.Part Ⅲ :Effect of Niclosamide and DK-520 on RANKL-induced expression of DC-STAMP,PU.1 and ATPase V₀d₂ Objective: Cell fusion between OCPs is a key step in osteoclast differentiation,and the formation of multinucleated osteoclasts ensures the function of bone resorbing in osteoclasts.This part intends to investigate whether Niclosamide and DK-520 inhibit the formation and differentiation of osteoclasts by detecting the cell fusion genes in the early stage of osteoclast differentiation through the detection of PU.1,DC-STAMP and ATPase V₀d₂ gene and protein expression levels in the RANKL induced OCPs to mature osteoclasts,and to analyze the specific molecular mechanisms of the two drugs that affect the differentiation of osteoclast cells.Methods: Cells were derived into 5 groups according to the type and concentration of the intervention drugs,DMSO group,Niclosamide 0.75μM Group,Niclosamide 1.5Μm Group,DK-520 0.75μM Group and DK-520 1.5μM Group,and according to the time period 0-1 days,1-2 days respectively to use the drug treatment.Cells were seeded at a density 1×105/well in 6-well plates and added in RANKL and drugs intervention at 0-1,1-2days respectively.After 24 h,Total-RNA and cell protein were extracted from ccells.RT-PCR and Western blot were performed to examined the m RNA expression level of PU.1 and the protein level of DC-STAMP and ATPase V₀d₂.Results: Real time PCR results shown that Niclosamide and DK-520 significantly suppressed PU.1 gene expression,and the gene expression level of PU.1 was statistically lower than control group（p<0.05）.There was no difference in protein expression level of ATPase V₀d₂（p>0.05）.However,western blot results shown that Niclosamide and DK-520 inhibited DC-STAMP and ATPase V₀d₂ protein expression（p<0.05）.Conclusion: Niclosamide and DK-520 inhibited RANKL-induced the expression of transcription factor PU.1 and OCPs fusion mediating factor DC-STAMP in early stage of osteoclastogenesis.Supression on RANKL-induced OCPs fusion of Niclosamide and DK-520 may play an essential role in the decrease of osteoclasts differentiation,maturation and the fuction of bone resorbing.