| Osteoclasts(OC),which has bone resorption activity,are multinuclear macrophages induced by macrophage colony stimulating factor(M-CSF)and nuclear factor B receptor activating factor ligand(RANKL).Vitamin D is a steroid hormone,which plays an important role in regulating calcium and phosphorus metabolism and maintaining bone homeostasis.1-Alpha,25-dihydroxy vitamin D3(1?,25(OH)2D3)is the hormonally active form of vitamin D3.Galectin-3(gal-3)is widely distributed in the body and has various functions.It can also regulate the osteoclastogenesis.Based on the differentiation of bone marrow mononuclear cells from C57BL/6 mice induced by 25 ngˇmL-1 M,CSF and 50 ngˇmL-1 RANKL in vitro,To study the effects of 1?,25(OH)2D3 on the osteoclastogenesis,bone resorption activity and the regulation factors,such as ?-catenin and gal-3,were investigated.Futhermore,the role of ?-catenin and gal-3 in osteoclastogenesis and bone resorption activity under the regulation of 1?,25(OH)2D3 were confirmed by activator or specific siRNA.These dates will provide theoretical basis for the study on the mechanism of 1,25-(OH)2D3 regulating calcium and phosphorus metabolism.1.The effect of 1?,25-(OH)2D3 on osteoclastogenesis and bone resorption activityIn order to reveal the effects of 1?,25-(OH)2D3 on mice osteoclastogenesis and bone resorption activity in vitro,the effects of 1?,25-(OH)2D3 on the cell proliferation were detected by CCK-8 and RTCA.TRAP staining was used to identincate osteoclast differentiation.Western blot was used to detect expression of marker proteins in osteoclastogenesis.Immunofluorescence and scanning electron were used to observe cell cytoskeleton and structure.The bone resorption area of pits was counted to investigate the effect of bone resorption activity by 1?,25-(OH)2D3.It was found that 10 nM 1?,25-(OH)2D3 had no significant effect on cell proliferation,but could significantly decreased the number of osteoclasts,inhibit the expression of bone resorption-related proteins and transcription factor proteins,the formation of actin rings and podosome belt and the bone resorption activity.It was shown that 1?,25-(OH)2D3 can inhibit mice OC differentiation and bone resorption activity in vitro.2.The effect of 1?,25-(OH)2D3 on gal-3,?-catenin and related signal pathway duringosteoclastogenesisTo investigate the effect of 1?,25-(OH)2D3 on gal-3,?-catenin and OC related signal in vitro,Western blot and immunofluorescence were used to detect differentiation-related proteins,qRT-PCR was used to detect related mRNA,The results showed that 1?,25-(OH)2O3 had no effect onRANKL-induced MAPKs,Akt,GSK-3? and CREB phosphorylation and the protein expression of c-fms and RANK.It was found that protein and mRNA expression of gal-3 increased and protein and mRNA expression of ?-catenin decreased during OC differentiation.la,25-(OH)2D3 up-regulated gal-3,?-catenin expression during OC difference,and 10 nM 1?,25-(OH)2O3 is the most effective concentration.It was shown that 1?,25-(OH)2D3 can regulate the expression of gal-3 and?-catenin expression during mice osteoclastogenesis in vitro.3.The role of ?-catenin in the inhibition of OC differentiation by 1?,25-(OH)2D3To investigate the role of ?-catenin in the inhibition of mice OC differentiation by 1?,25-(OH)2D3 in vitro,activate or knock down ?-catenin,using TRAP staining,Western blot,qRT-PCR and bone resorption assay were used to detect OC number,related proteins and bone resorption activity after activating or knock down p-catenin,The results showed that activating ?-catenin can inhibit OC formation,the expression of differentiation-related proteins and genes.Meanwhile,?-catenin knockdown promoted OC formation,but had no significant effect on inhibition of OC differentiation by la,25-(OH)2D3.It was shown that ?-catenm knockdown did not affect the inhibition of osteoclastogenesis by 1?,25-(OH)2D3.4.The role of gal-3 in the inhibition of OC differentiation by 1?,25-(OH)2D3To investigate the role of gal-3 in the inhibition of mice OC differentiation by 1?,25-(OH)2D3 in vitro,gal-3 of OC precursor cells was knocked out by siRNA,using TRAP staining,Western blot,qRT-PCR,bone resorption assay,immunoprecipitation and immunofluorescence double staining were used to detect OC number,related proteins and mRNA,bone resorption activity and the relationship between gal-3 and VDR bone resorption activity was also investigated.The results showed that gal-3 knockdown promoted osteoclastogenesis and attenuated the inhibition of OC differentiation by 1?,25-(OH)2D3.There was an interaction between gal-3 and VDR.It was shown that Gal-3 plays a role in the inhibition of OC differentiation by 1?,25-(OH)2D3.In conclusion,1?,25-(OH)2D3 can inhibit OC differentiation and bone resorption activity in vitro,and up-regulate the expression of both gal-catenin and gal-3.?-catenin knockdown did not affect the inhibition of osteoclastogenesis by 1?,25-(OH)2O3.The knockdown of Gal-3 promoted osteoclastogenesis and attenuated the inhibition of OC differentiation by 1?,25-(OH)2D3. |
PDF Full Text Request