The Effects And Molecular Mechanisms Of Silica Nanoparticles On Osteoclastogenesis

Background and objectives:In physiological condition,osteoclasts and osteoblasts maintain a dynamic balance whereby osteoclasts resorb worn bone and osteoblasts synthesize new bone.The imbalance of bone turnover results in bone loss and development of osteoporosis.Silica nanomaterials attract much attention due to their unique physicochemical properties and biological activities.In this study,fluorescent silica nanoparticles(SiNPs)with a size of 50 nm were synthesized by modified St(?)ber method to investigate their effects and molecular mechanisms on osteoclastogenesis.Methods:1.RANKL-induced RAW264.7 cells were used as pre-osteoclasts.Biosafety of SiNPs was evaluated through cytotoxicity assay.Phagocytosis of SiNPs was observed with fluorescence microscope.Intracellular distribution of SiNPs was observed with confocal microscope.Effects of SiNPs on osteoclastogenesis were detected with TRAP staining.Effects of SiNPs on m RNA and protein expression of osteoclast-related genes were detected with Real-Time PCR and Western blot.2.SiNP effects on expression of p62 and Lc3 b were detected through Real-Time PCR and Western blot.M-SiNPs intracellular distribution was observed with transmission electron microscopy.Relationship between SiNPs and autophagosomes was observed with confocal microscope.Interaction between M-SiNPs,p62 and LC3B-II proteins was detected through Pull-down assay.3.Effects of SiNPs on expression of MAPK and NF-κB signaling pathway proteins were tested through Western blot.Relationship between ERK signaling pathway and expression of p62 and Lc3 b was detected with Western blot and Real-Time PCR.Effects of SiNPs on TRAF proteins were detected with Western blot and immunofluorescence assay.Results:1.SiNPs showed no cytotoxicity.SiNPs were rapidly internalized by pre-osteoclasts.Intracellular distribution of SiNPs was associated with lysosomes.SiNPs reduced the number of TRAP-positive osteoclasts.SiNPs inhibited the expression of Nfatc1,c-Fos,Rank,Trap and Oscar m RNA.SiNPs inhibited protein expression of whole-cell NFATC1 and nuclear c-Fos/c-Jun.2.SiNPs increased m RNA and protein expression of p62 and Lc3 b.M-SiNPs were encapsulated by bilayer membrane structures.SiNPs colocalized with LC3 B protein and increased the number of LC3 B dots.Pulldown samples with M-SiNPs contained p62 and LC3B-II proteins.3.SiNPs increased ERK phosphorylation and inhibited JNK phosphorylation.SiNPs decreased protein expression of whole-cell pIKKα/β and p-IκBα,as well as nuclear p50,p65,p52 and Rel B.ERK inhibitor U0126 blocked the effects of SiNPs on p62 and Lc3 b expression.SiNPs stabilized TRAF3 protein but had no effect on TRAF2 and TRAF6.Conclusion:In pre-osteoclasts,50 nm SiNP increased p62 and Lc3 b expression through inducing ERK signaling pathway,were linked with p62 and LC3B-II proteins in autophagosomes,inhibited NF-κB signaling pathway through blocking TRAF3 protein degradation,and decreased nuclear cFos/c-Jun through downregulating JNK signaling pathway,resulting in inhibition of RANKL-induced osteoclastogenesis.