The Role And Mechanism Of Galectin-3 In Chicken Osteoclast Differentiation Induced By 1?,25-?OH?₂D₃ In Vitro

1?,25-dihydroxyvitamin D3[1?,25-(OH)2D3],the main active form of Vitamin D(Vit D),is widely involved in the development,differentiation,growth and regulation of a wide variety of cells.Osteoclasts(OC)can dissolve bones and release calcium ions into the blood,which can not only maintain bone homeostasis,but also regulate calcium homeostasis in the body.Previous studies by our research group have shown that 1?,25-(OH)2D3 can regulate OC differentiation.The differential proteomics results indicate that OC is regulated by Galectin-3(Gal-3)in addition to the regulation by the classical nuclear factor-?B ligand(RANKL)/nuclear factor ?B receptor activator(RANK)/osteoprotegerin(OPG)regulatory axis,but the specific regulatory effect and mechanism are still unclear.In this study,bone marrow stromal cells(BMSCs)and Bone marrow mononuclear macrophage(BMMs)were co-cultured to establish an in vitro OC culture model of chicken,and the effects of 1?,25-(OH)2D3 on differentiation and function of chicken OC were observed.After hypothermia or overexpression of gal-3 in BMSCs,the role and mechanism of gal-3 in regulating chicken OC differentiation induced by 1?,25-(OH)2D3 were observed,which provided theoretical basis for clinical studies on skeletal and calcium metabolic disorders in poultry.1.Effect of 1?,25-(OH)2D3 on chicken OC differentiation in vivoTo investigate the effect of 1?,25-(OH)2D3 on OC differentiation in co-culture system,the number of OC,marker genes level,marker proteins level,secreted proteins level and bone resorption activity were ovserved by TRAP staining,qRT-PCR,Western blot,ELISA,and bone resorption activity identification.The results showed that the number of OC was maximum in BMSCs:BMMs co-cultured system at a density of 1:100 in the presence of 10-8 mol/L 1?,25-(OH)2D3.The expression level of CtsK,MMP-9,CA?,TRAP mRNA and protein,the secretory protein(CtsK,MMP-9)level and the activity of OC bone resorption were upregulated by 10-8 mol/L 1?,25-(OH)2D3 on day 4 and 7.It was shown that 1?,25-(OH)2D3 could promote the differentiation and function of chicken OC in co-culture system of BMSCs and BMMs in vitro.2.Effects of 1?,25-(OH)2D3 on the expression of galectin-3 in OC,BMSCs and BMMsTo investigate the effect of 1,25-(OH)2D3 on Gal-3 in OC,BMSCs and BMMs,the expression of OC Gal-3 mRNA and secretory protein was observed by qRT-PCR,ELISA and other detection methods.Expression of RANKL and OPG mRNA and secretory proteins in BMSCs,expression of Gal-3 mRNA in BMMs.The results showed that 1?,25-(OH)2D3 promoted the expression levels of Gal-3 mRNA and secreted protein Gal-3 in OC,BMCSs and BMMs.In addition,RANKL mRNA and secreted protein RANKL of BMSCs were up-regulated,while OPG mRNA and secreted protein OPG were down-regulated.Among them,1?,25-(OH)2D3 has the most obvious regulatory effects on BMSCs.It was shown that 1?,25-(OH)2D3 could affect the expression of gal-3 in OC,BMSCs and BMMs.3.The role of galectin-3 in the regulation of osteoclast differentiation by 1?,25-(OH)2D3In order to investigate the role of Gal-3 in regulation of OC differentiation by 1?,25-(OH)2D3.BMSCs treated by si-Gal-3 or EGFP-Gal-3 were co-cultured with BMMs.The number of OC,marker gene level and bone resorption activity were observed by TRAP staining,qRT-PCR and bone resorption activity identification.The results showed that OC differentiation was blocked,OC marker gene level and bone resorption activity were down-regulated after si-Gal-3 in BMSCs.The effects of 1?,25-(OH)2D3 on OC differentiation,OC marker gene expression and bone resorption activity were be alleviated.Howere,overexpression of Gal-3 in BMSCs promoted OC differentiation,marked gene expression,and inhibited bone resorptive activity.The results showed that gal-3 was involved in OC differentiation and function regulated by 1?,25-(OH)2D3 in vitro.4.The mechanism of galectin-3 involved in osteoclast differentiation induced by vitamin DTo investigate the molecular mechanism of Gal-3 in OC differentiation induced by vitamin D,this study conducted transcriptome sequencing analysis on BMSCs that overexpressed or knocked-down Gal-3.The results showed that 307 genes were significantly up-regulated and 253 genes significantly down-regulated after si-Gal-3 in BMSCs.After overexpression of Gal-3 in BMSCs,214 and 418 genes were significantly up-regulated and down-regulated respectively.KEGG analysis of differentially expressed genes in BMSCs after knocking-down or overexpression of Gal-3 revealed significant enrichment of cytokine-cytokine receptor interaction pathways,Ca2+signaling pathways and Wnt signaling pathways.qRT-PCR was used to identify genes in this enrichment pathways,the results showed that nitric oxide synthase(NOS2),slow excitation peptide B2 receptor(BDKRB2),endothelin B receptor(EDNRB),BMP and activin membrane bound inhibitor(BAMBI),DKK1 and Wnt inhibitors(WIF1)gene expression trends were consistent with transcriptome sequencing result.NOS2,BAMBI mainly play catalytic activity and protein function;BDKRB2 and EDNRB are mainly involved in transmembrane signal transduction and receptor binding.In conclusion,Gal-3 may participate in the differentiation of OC induced by 1?,25-(OH)2D3 through these target genes.