Experimental Research In Vitro On Effect Of Lanthanum Chloride Through NF-κB Signal-pathway To Regulate RANKL-induced Differentiation And Maturation Of Osteoclast

Objective: Using experiment in vitro to investigate the effect of a certainconcentration of lanthanum chloride on RANKL-induced differentiation and theexpressions of osteoclastogensis related to signal factors in the level of protein andphosphorylation. To giving theoretic basis and experimental reference about effect oflanthanum on osteoclastogensis and the further using of lanthanum elements in thefuture.Methods: Extract the Bone Marrow-derived Macrophage(BMM) in vitro,afterwards using Receptor Activitor of Nuclear Factor-kappaB Ligand(RANKL) andMacrophage-Colony Stimulating Factor(M-CSF) to induce the differentiation of it invitro. Test the cytotoxicity of different concentrations of lanthanum chloride by usingCell Counting Kit-8and after that choosing the appropriate concentration used in ourfollowing experiments. Deviding cells randomly into5groups according to ourdesign and the result of CCK-8:Blank control gourp (gourp A)、Positive control group（group B）、Low concentration of lanthanum chloride group（50μmol/L，group C）、Middle concentration of lanthanum chloride group（100μmol/L，group D）、Highconcentration of lanthanum chloride group（200μmol/L，group E）.Testing the cellnumbers of mature osteoclast and the dyeing area of each group by using TartrateResistant Acid Phosphatase method.Western Blot was employed to test the variationsof the NF-κB signaling pathway related factors（IκBα、p65）after a certain lanthanumchloride was joined, in the level of protein and phosphorylation, and the variations oftranscription factors Nuclear Factor of Activated T-cells1(NFATc1) in the level ofprotein.Results: Tested by CCK-8method at24、48、72、96hours time points provedthat concentration below800μM showed no cytotoxicity to BMM cells and the cellssurvived normally,and there is no statistically significant difference(p＞0.05).Nonetheless when concentration was more than800μM,there showed abviouslycytotoxicity(p＜0.05). The TRAP staining results indicated that there was no mature osteoclast createdin group A，whereas group B、C、D、E created mature osteoclast more or less and therewas statistically significant difference(p＜0.05).Compared with group B， thedifferentiation of osteoclast in group C、D、E were inhibited to varying degrees,andthe differences were statistically significant(p＜0.05).And there were statisticallysignificant differences between groups among groupC、D and E(p＜0.05).Western Blot showed that lanthanum chloride can inhibit the activation ofNF-κB signaling pathway at the early stage,and decrease the expressions of Nuclearfactor of activated T cells-1，an important transcription factor protein of osteoclast.Conclusion: The appropriate concentration of lanthanum chloride used in ourexperiment showed no cytotoxicity to cells,and can inhibit the RANKL-inducedosteoclastogensis,and decrease the expression of Nuclear Factor of ActivatedT-cells1， an important transcription factor protein.There existed a dose-effectrelationship in some range of concentrations of lanthanum chloride.The mechanismmay be related to that lanthanum chloride can inhibit the activation of NF-κBsignaling pathway of osteoclast.