OPN And N-glycosylation Activate NF-?B Pathway To Regulate Bone Remodeling In Periapical Periodontitis

I.ObjectivePeriapical periodontitis is caused by pulpal infection.The major clinical manifestation of periapical periodontitis is the destruction of the apical alveolar bone.Bacterial lipopolysaccharide(LPS)is the main cause of periapical periodontitis because it stimulates the body's local immune response.A large number of inflammatory cells are activated after the release of inflammatory mediators,cytokines and enzymes,leading to the destruction of the apical tissue,the formation of inflammatory granulation tissue,and alveolar bone destruction.The dynamic balance between osteoblasts and osteoclasts is the critical link in the process of periapical bone remodeling.OPN is a secretory glycosylated extracellular matrix protein with many biological activities.It is a multifunctional matrix protein that is widely involved in the progression of bone-related diseases.A number of studies have shown that OPN promotes osteoclast adhesion and improves the osteocytes activity of osteoclasts.When the OPN gene was knocked out,the number of osteoclasts decreased significantly.Many studies have examined the role of cytokines in pathogenesis at the periapical circumference,but there has been little research into the role of OPN in periapical periodontitis.In the present study,we investigated the function of OPN and analyzed the relationship between OPN and chronic periapical periodontitis.We provide evidence that OPN is involved in the pathogenesis of this disease.At present,the research on the pathogenesis of periapical periodontitis mainly focuses on the exploration of cytokines,but there are few studies on the role of OPN in periapical periodontitis.This study intends to collect human chronic periapical periodontitis lesions,detect whether OPN is expressed in chronic apical periodontitis,and analyze the relationship between OPN and other inflammatory factors.Establish a mouse model of periapical periodontitis and analyze it.The relationship between OPN and bone destruction in periapical periodontitis.In addition,the effects of different concentrations of LPS on osteoblasts and osteoclasts cultured in vitro were studied,and the effects of different expression levels of OPN on LPS-mediated inflammation microenvironment regulation of osteoblast differentiation were investigated.The protein of the mutant OPN-sugar chain was constructed to study the effect of N-glycan on the function of OPN protein.To investigate the role of OPN N-glycosylation in the activation of NF-?B pathway in the inflammatory microenvironment to regulate bone remodeling in periapical periodontitis.Provide experimental basis for the pathogenesis of periapical periodontitis.II.MethodsA.The expression and role of OPN in periapical periodontitis1.The expression and role of OPN in human periapical periodontitis(1)Real-time PCR was used to detect the gene expression of OPN and bone destruction marker in human periapical periodontitis;(2)Immunohistochemistry and Western Blot were used to detect the expression of OPN and bone destruction markers in human periapical periodontitis.2.The expression and role of OPN in human periapical periodontitis(1)Establishing a mouse model of periapical periodontitis and establishing a successful animal model by Micro-CT;(2)Total RNA was extracted from the periapical tissues of mice,and the expression of OPN and bone destruction markers in mouse periapical periodontitis was detected by Real-time PCR.(3)Immunohistochemistry was used to detect the protein expression of OPN and bone destruction marker in mouse periapical periodontitis.B.OPN in the LPS-mediated inflammation microenvironment regulate the proliferation and differentiation of osteoclasts and osteoblasts1.Observation and identification of cell morphology after induction of osteoclasts and osteoblasts(1)After in vitro culture of RAW264.7 cells and addition of osteoclast inducer,the osteoclasts were successfully induced by light microscopy,Real-time PCR and TRAP staining;(2)After in vitro culture of MC3T3-E1 cells,the osteoblasts were induced,and the osteoblasts were successfully induced by alizarin red assay,Real-time PCR and ALP.2.Effects of LPS on proliferation and differentiation of osteoclasts and osteoblasts(1)After using different concentrations of LPS to act on the induced RAW264.7 and MC3T3-E1 cells,the CCK8 experiment was used to screen the final optimal experimental concentration;(2)The pre-experimental 100 ng/ml LPS was applied to the induced RAW264.7,and the effects of TRAP on the proliferation and differentiation of osteoclasts were detected by CCK8 and ALP assays(3)1000 ng/ml LPS screened by pre-experiment was applied to MC3T3-E1 after induction,and the effects of LPS on proliferation and differentiation of osteoblasts were detected by CCK8 and ALP assays(4)Real-time PCR and Western Blot were used to detect the expression of LPS in osteoclasts and osteoblasts.3.Effects of different expression levels of OPN on proliferation and differentiation of osteoclasts and osteoblasts(1)Verification of transfection efficiency of interfering OPN;(2)Verification of transfection efficiency of overexpressing OPN;(3)The effect of different expression levels of OPN on the proliferation of osteoclasts and osteoblasts was detected by CCK8 assay(4)The effects of different expression levels of OPN on the differentiation of osteoclasts and osteoblasts were detected by TRAP and ALP experiments.C.OPN regulates the expression of bone destruction related factors through NF-?B signaling pathway1.Add NF-?B inhibitor to detect whether OPN passes NF-?B signaling pathway2.Western Blot method was used to detect the expression of osteoclast and osteoblast related factors in OPN through NF-?B signaling pathway3.Immunofluorescence method to detect OPN promotes NF-?B nuclear transfer in osteoclasts and osteoblastsD.The effect of OPN N-glycosylation modification on the biological characteristics of osteoblasts and osteoclasts mediated by LPS.1.Identification of OPN N-glycosylation sites by mass spectrometry2.The point mutation was used to mutate the asparagine at position 79 of the OPN protein of the overexpressing plasmid to glutamine,and the above plasmid was transformed into osteoblasts and osteoclasts3.CCK8 detection of OPN N-glycosylation modification on the proliferation of osteoblasts and osteoclasts;4.Western Blot was used to detect the expression of OPN N-glycosylation on osteoclasts and osteoblasts.5.Western Blot detection of OPN N-glycosylation modification activates NF-?B signaling pathway to regulate osteoblasts and osteoclasts;6.Immunofluorescence assay for OPN N-glycosylation promotes nuclear transfer of NF-?B in osteoclasts and osteoblasts.III.ResultsA.The expression and role of OPN in periapical periodontitis1.The expression and role of OPN in human periapical periodontitis(1)The expression of OPN and the expression of genes and proteins in osteoporosis were increased in osteoporosis markers NF-?B,Cathepsin K and MMP9;(2)The expression of OPN was correlated with osteoclast markers NF-?B and Cathepsin K,but not with MMP9.2.The expression and role of OPN in mouse periapical periodontitis.(1)Micro-CT was used to detect the establishment of mouse apical periodontitis animal model.Micro-CT showed that the mouse periapical inflammation model was successfully constructed.The results showed that the amount of apical bone destruction in mice increased with time in 1-4 weeks,and there was no significant difference in 3-4 weeks.(2)The expression of OPN and the expression of genes and proteins in the periapical periodontitis of mouse osteoclasts NF-?B,Cathepsin K and MMP9 were increased;(3)The expression of OPN was correlated with osteoclast markers NF-?B,Cathepsin K and MMP9.B.OPN in the LPS-mediated inflammation microenvironment regulate the proliferation and differentiation of osteoclasts and osteoblasts1.Observation and identification of cell morphology after induction of osteoclasts and osteoblasts(1)After in vitro culture of RAW264.7 cells and addition of osteoclast inducer,the osteoclast induction success was identified by light microscopy,Real-time PCR and TRAP staining;(2)After in vitro culture of MC3T3-E1 cells with osteoblast induction agent,osteoblast induction was successfully identified by alizarin red assay,Real-time PCR and ALP.2.Effects of LPS on proliferation and differentiation of osteoclasts and osteoblasts(1)After using different concentrations of LPS to induce RAW264.7 and MC3T3-E1 cells,the CCK8 assay was used to screen the subsequent experimental concentration of 100 ng/ml LPS for RAW264.7 and 1000 ng/ml LPS after induction.MC3T3-E1 after induction;(2)100 ng/ml LPS was applied to RAW264.7 after induction,and LPS promoted osteoclast proliferation and differentiation;(3)1000 ng/ml LPS acts on MC3T3-E1 after induction,and LPS inhibits osteoblast proliferation and differentiation;(4)LPS can increase the expression level of osteoclast and osteoblast bone destruction marker3.Effects of different expression levels of OPN on the proliferation and differentiation of osteoclasts and osteoblasts.(1)The transfection of si-OPN and over-OPN was confirmed by fluorescence microscopy and Real-time PCR;(2)OPN can promote osteoclast proliferation and differentiation,but inhibit osteoblast proliferation and differentiation.C.OPN regulates the expression of bone destruction related factors through NF-?B signaling pathway1.OPN regulates the expression of osteoclast and osteoblast related factors through NF-?B signaling pathway;2.OPN promotes nuclear transfer of NF-?B in osteoclasts and osteoblasts.D.The effect of OPN N-glycosylation modification on the biological characteristics of osteoclasts mediated by LPS.1.Identification of OPN N-glycosylation site by mass spectrometry as position 79;2.OPN N-glycosylation modification can promote osteoclast proliferation and inhibit osteoblast proliferation;3.OPN N-glycosylation modification regulates the expression of osteoclast and osteoblast related factors through NF-?B signaling pathway;4.OPN N-glycosylation modification promotes nuclear transfer of NF-?B in osteoclasts and osteoblasts.IV.Conclusions1.OPN is involved in the progression of chronic periapical periodontitis in humans and mice;2.The expression of OPN in apical periodontitis with bacterial infection was correlated with osteoclast markers NF-?B,Cathepsin K and MMP9,indicating that the occurrence and development of bone tissue destruction in apical periodontitis may be related to OPN.3.OPN affects osteoclasts and osteoblasts involved in bone remodeling.In LPS-mediated inflammation microenvironment,OPN can promote the proliferation and differentiation of osteoclasts and inhibit the proliferation and differentiation of osteoblasts;4.OPN may play a dual role in bone metabolism,making it a bridge for communicating OC and OB and participating in bone remodeling;5.LPS as endotoxin can activate NF-?B signaling pathway through OPN;6.OPN can accelerate the nuclear transfer of NF-?Bp65 in osteoblasts and osteoclasts;7.The OPN N-glycosylation site is the 79th;8.OPN N-glycosylation is essential for the biological function of OPN proteins.