Preliminary Study Of The Notch1 Signaling Pathway Involved In The Alveolar Bone Resorption During Tooth Movement In Rats

With the improvement of living standard and the popularization of oral health knowledge,the public is paying more and more attention to facial beauty and oral health.More and more patients require orthodontic treatment to improve facial beauty and oral function.However,many patients with malocclusion tend to be deterred from the 2-3 years of orthodontic treatment,and then choose to give up orthodontic treatment or choose other treatment methods.How to find an efficient and safe method to accelerate tooth movement in orthodontic treatment is one of the hot and difficult points in orthodontic research in recent years.To solve this difficulty,the key point is to clarify the biological mechanism that regulates the alteration and remodeling of periodontal tissues during orthodontic tooth movement(OTM).Since the birth of the orthodontic discipline,the domestic and foreign scholars have done a lot of research on the mechanism of periodontal tissue remodeling and reconstruction during tooth movement,and found that many factors are involved in the tooth movement process,including the Wnt/β-catenin signal,P13K/AKt/m TORsignal,the RANK-RANKL-OPG and Caspase-3 signals are involved in the reconstruction process of periodontal tissue.Wnt/β-catenin signaling is mainly involved in the process of osteoblasts in response to orthodontic mechanical stress.Wnt ligands such as Wnt3 a and Wnt10 b regulate bone mass and mineralization by inducing alkaline phosphatase(ALP)activity and osteoblast formation;PI3K/AKt/m TOR signal is involved in the synthesis and transcription of protein in periodontal ligament cells such as fibroblasts and osteoblasts during OTM,preparing for periodontal tissue remodeling;RANK-RANKL-OPG interaction inhibits osteoclast generation and bone resorption during OTM;Caspase-3 signal is involved in the apoptosis process during OTM.In recent years,more and more studies have found that the Notch signaling pathway plays a key role in the process of cell-to-cell signaling,especially in bone metabolism.Notch signaling has the potential to regulate the differentiation and function of osteoblasts and osteoclast lineage cells,and plays a key role in skeletal development,cartilage formation and the like.Inactivation of Notch1 in osteoblasts increases bone resorption and leads to bone loss by inhibiting the expression of osteoprotegerin(OPG).Notch1 inhibits osteoclastogenesis through direct and indirect mechanisms,while inducing increased production of OPG.Inactivation of Rbpjκ by Notch receptor in bone marrow cell culture resulted in enhanced osteoclastogenesis,suggesting that Rbpjκ is an inhibitor ofosteoclastogenesis and that the role of Notch1 in osteoclast differentiation is mediated by classical mechanisms.OTM is the periodontal tissue remodeling process mediated by orthodontic mechanical load,in which bone remodeling is the focus,including bone resorption and bone formation.Many studies have been conducted on the role of Notch signaling in the development of bone loss cells involved in bone immune regulation.However,whether Notch1 signal conduction is involved in the orthodontic-induced alveolar bone resorption remodeling process during OTM and its mechanism is still unclear.In this study,we established the OTM model with the SD rats and used HE,TRAP and immunohistochemical staining techniques to investigate whether the Notch1 signaling pathway is involved in the alveolar bone resorption process during OTM,and conducted a preliminary study on the remodeling process of alveolar bone resorption during OTM by using Notch1 signaling pathway inhibitor DAPT.Objective: To detect the expression of the Notch1 signaling pathway-related molecules(Notch1 and Jagged1)during rat OTM process,and conducted a preliminary study on the remodeling process of alveolar bone resorption during OTM by using Notch1 signaling pathway inhibitor DAPT.To explore the mechanism of Notch1 signaling pathway involved in orthodontic tooth movement.METHODS: In this experiment,sixty healthy,adult,and male SD(Sprague-Dawley)rats were randomly divided into groups A and B: 30 in each group.Each group was randomly divided into 6 groups: 0 day group,1 day group,4 days group,7 day group,10 day group and 14 day group,5in each group.The OTM model was established in the left maxillary force of the rats in group A as the Loaded group;the right side was not forced,as the Unloaded group;the upper left maximal force of the group B was used to establish the OTM model,and the right side was not forced,and at the same time,the flexure of mucosa of both maxillary first molar were injected with 0.6ml/kg DAPT solution,starting from the application of force(0 days),every two days: the left side is the Loaded+DAPT group,the right side is the Unloaded+DAPT group.After the rats were sacrificed at the corresponding time points,the bilateral masses including the maxillary first and second molars and the surrounding alveolar bone were removed.The distance between the first and second molars was used to evaluate the OTM distance.HE staining was used to observe the morphological changes of periodontal tissue during OTM and TRAP staining to observe the changes of osteoclasts.The expression of Notch1,Jagged1,OPG,RANK and RANKL during tooth movement was observed by immunohistochemical staining,and the average optical density(MOD)value of immunohistochemical stained sections was measured byImage-Plus Pro 6.0 software.All data was analyzed using SPSS 20.0software.Result:1.OTM distance: In the two groups of the Loaded group and Loaded+DAPT group,from the first day of loaded force,the OTM distance increases with the increase of the loaded time.Except for 0 days,the OTM difference between the Loaded group and the Unloaded group and the Loaded+DAPT group and the Unloaded+DAPT group were statistically significant(P<0.05).On days 10 and 14,the OTM distance in the Loaded group was significantly less than that in the Loaded+DAPT group(P <0.05).2.HE staining: In the Unloaded group and Unloaded+DAPT group,the surface of rat molar root and alveolar bone was smooth,and the width of periodontal ligament(PDL)was basically unchanged.From the 4th day,in the Loaded group and Loaded+DAPT group observed that the compression side width of the PDL was narrower than that of the tension side.With the increase of the application time,in the Loaded group and Loaded+DAPT group,the compression side width of the PDL was narrower than that of the Unloaded group and Unloaded+DAPT group.Osteoclasts and bone resorption pits were observed on the pressure side,while osteoblasts and bone formation were observed on the tension side.3.TRAP staining: In the Unloaded group and Unloaded+DAPT group,no obvious TRAP-positive multinucleated osteoclast reabsorption cavities were observed on the alveolar bone surface.On the 4th day,TRAP positive polynuclear osteoclast resorption cavities were observed on the surface of the alveolar bone on the compression side of the Loaded group and Loaded+DAPT group,and new cementoid like structures were observed on the surface of the cementum.On days 4,7,10 and 14,the number of TRAP-positive cells in the Loaded group and Loaded+DAPT group were significantly higher than that in the Unloaded group and Unloaded+DAPT group(P < 0.05).On days 10 and 14,the number of TRAP-positive cells in the Loaded+DAPT group was higher than that in the Loaded group(P < 0.05).4.Immunohistochemical staining: After the establishment of OTM model,with the increase of time,the positive expression of Notch1,Jagged1,RANKL and RANK in the compression side of the Loaded group increased gradually,and the expression of OPG gradually decreased;There was no significant change in the positive expression of Notch1,Jagged1,RANKL,RANK and OPG in the compression side of the Unloaded group.The expression of Notch1 and OPG in the compression side of the Loaded+DAPT group gradually decreased,Jagged1 The expression level increased slightly on the 4th and 14 th day,and the expression levels of RANKL and RANK gradually increased.The expression level of Notch1 inthe compression side of the Unloaded+DAPT group gradually decreased,and the expression levels of Jagged1,RANKL,RANK and OPG showed no significant changes.At other time points except 0 days,the expression of Notch1,Jagged1,RANKL and RANK in the compression side periodontal membrane of the Loaded group was significantly higher than the Unloaded group(P<0.05),except for 0 and 1 days,the expression of OPG in the Loaded group was significantly lower than that of the Unloaded group(P<0.05).At other time points except 0 days,the expression of Notch1 in the Loaded+DAPT group was significantly lower than that in the Loaded group(P<0.05),and the expression of Jagged1 in the Loaded+DAPT group was significantly lower than that in the Loaded group on the 1st,7th,10 th and 14 th day(P<0.05).The expression level of RANKL in the Loaded+DAPT group was significantly higher than the Loaded group on the 4th,7th and 10 th day(P<0.05).The expression level of OPG in the Loaded+DAPT group on the4,7 and 10 days were significantly lower than the Loaded group(P<0.05).The expression levels of RANK in the Loaded+DAPT group and the Loaded group were statistically significant except 0 and 14 days(P<0.05).Except for 0 and 1 day,the expression level of Notch1 and RANK in the Loaded+DAPT group were significantly higher and the expression of OPG was significantly lower than the Unloaded+DAPT group(P<0.05);exceptfor 0 days,the expression of RANKL in the Loaded+DAPT group was significantly higher than the Unloaded+DAPT group(P<0.05).Conclusion: The expression of Notch1 signaling pathway-related proteins Notch1,Jagged1 and OPG,RANK,RANKL were associated with the remodeling of periodontal bone tissue after orthodontic treatment in rats.These observations suggest that Notch1 signaling pathway may play a role in the remodeling of alveolar bone during OTM.