A Preliminary Study On The Effects Of Chemerin/ChemR23 On Cementoblasts Function And Inflammatory Root Resorption

Inflammatory root resorption(IRR)is a pathological process in which the volume of root resorption is larger than the volume of root restoration in an inflammatory environment.Severe inflammatory root resorption may cause loss of tooth.Periodontitis,apical periodontitis,and inappropriate orthodontic treatment may all be predisposing factors for inflammatory root resorption.However,its inherent mechanism and prevention methods have not yet been clear.Cementoblasts are located on the surface of cementum,which can regulate the expression of genes related to osteoclastogenesis and osteogenesis,promoting or inhibitting root resorption,and can also form cementum to repair root resorption lacuna.Therefore,factors that can affect cementoblasts function can affect root resorption.Chemerin is a newly discovered chemotactic protein.Its interaction with ChemR23 not only plays an important role in the migration of macrophages and dendritic cells to inflammation sites,but also plays an important role in inducing the differentiation and formation of osteoclasts.Studies have found that inflammatory factors can affect the differentiation and mineralization of cementoblasts.According to this,there is less evidence about whether Chemerin promotes cementoblasts secretting inflammatory factors and then affects its function,thereby promoting the occurrence of inflammatory root resorption.The purpose of this experiment is to explore the role of Chemerin/ChemR23 in cementoblasts function and inflammatory root resorption to provide new evidence for the mechanism of inflammatory root resorption and provide a new target for clinical treatment.The experiment is divided into two parts: in vitro and in vivo:Ⅰ.The study of effect of Chemerin on cementoblasts function and internal mechanismPart Ⅰ: The effect of Chemerin/ChemR23 on cementoblasts functionObjective: According to stimulation by Chemerin in vitro,explore the effect of Chemerin on cementoblasts functionMethods: Firstly,25 ng/ml Chemerin was used to stimulate cementoblasts for 12 h,24 h and 48 h.The cell proliferation was detected by CCK-8.The number of apoptotic cells was examined by flow cytometry.The expression of OPG,RANKL,Cathepsin K and Runx2 m RNA was examined by RT-PCR.The protein levels of cleaved-caspase3,Cathepsin K and Runx2 was examined by Western blot.Secondly,cells were treated with Chemerin in combination with si ChemR23.The expression of OPG,RANKL,Runx2,Osterix,BSP and OCN m RNA was examined by RT-PCR.The protein expession of Cathepsin K,Runx2,and cleaved-caspase3 were by Western blot.Result: After treatment with Chemerin for 12 h,proliferation ability of cementoblasts decreased significantly.Flow cytometry showed that the percentage of apoptotic cells increased in a time-dependent manner following Chemerin(25 ng/ml)treatment.After 24 h of Chemerin treatment,the expression of the osteoclastic differentiation markers Cathepsin K and RANKL increased,whereas that of osteogenic differentiation marker Runx2 and OPG significantly decreased(P<0.05).The protein levels of cleaved-caspase3 and Cathepsin K increased while Runx2 decreased.The difference was statistically significant(P<0.05).To investigate whether Chemerin affects cementoblasts function through binding with ChemR23,we transfected OCCM-30 cells with si RNA against ChemR23.Compared with the control group,the expression of OPG,Runx2,Osterix,BSP and OCN m RNA in cementoblastss increased,and the expression of RANKL decreased upon si ChemR23 transfection.The difference was statistically significnt(P<0.05).At the same time,the protein levels of cleaved-caspase3 and Cathepsin K decreased with knocking out ChemR23,the protein level of Runx2 showed the opposite pattern.Conclusion: Chemerin promotes the apoptosis of cementoblasts,the expression of osteoclast-related genes and inhibits the differentiation and mineralization of cementoblasts by combining with ChemR23.Part Ⅱ: The role of inflammatory factor and related signaling pathways in Chemerin/ChemR23 regulating cementoblasts functionObjective: To explore the role of inflammatory factor,P38 MAPK、Erk1/2 MAPK and P13K-Akt pathways in Chemerin/ChemR23 regulating cementoblasts function.Methods: Firstly,10/25/50 ng/ml Chemerin was used to stimulate cementoblasts for 24 h.The expression of TNF-α and IL-6 was examined by RT-PCR and Elisa.Secondly cells were treated with Chemerin in combination with si ChemR23.The expression of TNF-α and IL-6 was dectected by RT-PCR and Elisa.The activity of MAPK and PI3K-Akt pathways were evaluated by Western blot.Finally,the cementoblasts were pretreated with specific inhibitors of P38,Erk1/2 MAPK and PI3K-Akt for 1 h,and then the cells were stimulated with 25ng/ml Chemerin for 24 h.Expression of TNF-α and IL-6 were detected by RT-PCR and Elisa.Western blot dectected protein levels of Cathepsin K,Runx2 and cleaved-caspase3.Result: The result of RT-PCR and Elisa showed that the expression of TNF-α and IL-6 increased in a concentration-dependent manner following Chemerin treatment.After knocking out ChemR23,we found the expression of TNF-α and IL-6 decreased.At the same time,the levels of phosphorylated Erk1/2,P38 MAPK and Akt were significantly increased upon Chemerin treatment(25 ng/ml,24 h)but decreased in cementoblastss transfected with si ChemR23.Western blot analysis revealed that PD98059,SB203580,and LY294002 reversed the suppression of Runx2 expression.Compared with PD98059 and LY294002,SB203580 considerably enhanced the expression of Cathepsin K.However,both PD98059 and SB203580 reversed the change in cleaved-caspase3.The RT-PCR and Elisa results revealed that all inhibitors suppressed the expression of TNF-α and IL-6 after Chemerin treatment.Conclusion: Chemerin/ChemR23 induced TNF-α and IL-6expression dependent on Erk1/2,P38 MAPK and PI3K-Akt signaling pathway activation,thereby regulating cementoblasts function.Ⅱ.The effect of Chemerin/ChemR23 on inflammatory root resorptionObjective: Construct an in vivo model of inflammatory root resorption to verified that Chemerin/ChemR23 promotes the occurrence of inflammatory root resorption.Method: 6 wild-type mice and 12 Chemerin overexpressing mice were used in vivo experiment.The inflammatory root resorption model was established by applying a force of 50 g to left first molar of the mice.Among them,6 Chemerin overexpressing mice were injected with ChemR23 antagonist(300 μg/kg/day)in the periodontal ligament of the first molar on both sides.All mice were divided into three groups:wild-type mice,Chemerin overexpressing mice and Chemerin overexpressing mice injected with ChemR23 antagonist.In the statistical analysis of experimental results,they were divided into four groups:wild-type noload group(WT-blank),wild-type load group(WT-load),Chemerin overexpression load group(TG-load),Chemerin overexpression injected ChemR23 antagonist load group(TG-anti ChemR23-load).After 7days of loading,the mice were sacrificed.Micro-CT was used to scan the first molars roots of mice.The data was imported into Mimics to measure and analyze the volume of root resorption lacuna.The specimens of mice first molar roots were made into paraffin sections for immunohistochemical staining of TNF-α,IL-6,Cathepsin K and Runx2.Result: The data of Micro-CT showed that among four groups,the volume of root resoprtion lacuna was largest in TG-load group,and the difference was statistically significant(P<0.05).When expression of ChemR23 decreased,the volume of root resorption lacunae decreased.The results of immunohistochemistry showed that compared with the WT-blank group,the expression of TNF-α,IL-6,and cathepsin K close to the distobuccal root(mesial side)of the first molar was higher in the WT-load group than in the WT-blank group.When Chemerin overexpressed,the expression of TNF-α,IL-6 and Cathepsin K close to the distobuccal root(mesial side)of the first molar was higher,while the expression level of Runx2 was the lowest among all groups.When the expression of ChemR23 was down-regulated,the expression of TNF-α,IL-6,Cathepsin K and Runx2 close to the distobuccal root(mesial side)of the first molar was reversed.Conclusion: Chemerin/ChemR23 promote inflammatory root resorption which may have close association with the increase of inflammatory factor(TNF-α、IL-6).