| Objective: Periodontitis is a chronic infectious disease of periodontal tissue caused by dental plaque biofilm.Periodontitis causes alveolar bone absorption,periodontal pocket formation and tooth loosening and falling off,which are the main causes of adult tooth loss.Dental plaque biofilm is the initial factor of periodontitis and periodontal infection,among which gram-negative anaerobic bacteria play an important role in periodontitis,and Porphyromonas gingivalis is one of the dominant bacteria in periodontitis,which is recognized as an important periodontal pathogen at present.The host’s defense ability interferes P.gingivalis through various mechanisms,which leads to the destruction of periodontal connective tissue and the absorption of alveolar bone.Normal alveolar bone metabolism is active,and alveolar bone function depends on the dynamic balance between osteogenesis of osteoblasts and bone resorption of osteoclasts.When periodontitis occurs,this coupling balance is broken,the bone formation of osteoblasts is inhibited,and the bone resorption function of osteoclasts is continuously enhanced,which eventually leads to the continuous absorption and destruction of alveolar bone.Erythropoietin producing hepatocyte kinases(Eph)receptor is a member of tyrosine kinase receptor family,it can combine with its ligand Erythropoiesis producing hepatocyte kinases receptor interacting protein(Ephrin)to generate bidirectional signal transduction and participate in regulating various cellular biological processes.It is found that EphB4 receptor in osteoblasts and EphrinB2 ligand in osteoclasts are important coupling regulators in bone formation and bone resorption,EphB4 receptor in osteoblasts is activated by EphrinB2 ligand in osteoclasts as a forward conduction signaling,osteoblast’s bone formation activity is promoted,and EphrinB2 ligand in osteoclasts is activated by EphB4 receptor in osteoblasts as a reverse conduction signaling,osteoclast’s bone resorption activity is inhibited,when EphB4 / EphrinB2 signal is blocked,the activity of osteoblasts is reduced,osteoclasts differentiation is promoted,and finally which affects the expression of transcription factors and proteins relating of osteoblast and osteoclast differentiation.EphB4 / EphrinB2 bidirectional signaling plays an important role in maintaining bone balance and regulating bone remodeling.Therefore,based on the above research background,in this study,a rat model of periodontitis was built with P.gingivalis,and the existence of EphB4 / EphrinB2 in periodontitis and normal tissues were verified.By establishing a model of osteoblasts and osteoclasts with P.gingivalis,the role of P.gingivalis on EphB4 / EphrinB2 signaling pathway and its possible molecular mechanisms were explored.Methods: 1.The rat maxillary first molar was ligated with P.gingivalis,which was applied around the gum to establish the periodontitis infection model.By scanning Micro-CT,the distance from enamel cementum boundary to the crest of alveolar ridge(CEJ-ABC)was measured,the alveolar bone absorption was observed,the inflammation around alveolar bone was observed by hematoxylin eosin staining,and the expressions of EphB4 and EphrinB2 were detected by immunohistochemistry.2.MC3T3-E1 cells were cultured into osteoblasts by inducing solution.The protein expressions of EphB4,osteoblast active factors Runx2,ALP and OPN were detected by Western-blot and immunofluorescence after different time with MOI=100 P.gingivalis.3.RAW264.7 cells were induced to be osteoclasts,and the expressions of EphrinB2,osteoclast active factors NFATc1,C-fos,and Cts K proteins were detected by Western-blot and immunofluorescence after different time with MOI=100 P.gingivalis.4.To clarify the role of EphB4 in osteoblasts,a simulated co-culture system of EphrinB2-Fc expressing osteoblasts was established.The expression of EphB4,osteogenic active factor Runx2 and ALP protein were detected in co-culture system after siRNA silencing or plasmid overexpression of EphB4.5.To clarify the role of EphrinB2 in osteoclasts,a simulated co-culture system of EphB4-Fc expressing osteoclasts was established.The expression of EphrinB2,osteoclast active factor C-fos,NFATc1 and Cts K were detected in co-culture system after siRNA silencing or plasmid overexpression of EphrinB2.Results: 1.The results of animal experiments showed that with the increase of infection time(4 w,6 w and 10 w),the alveolar bone resorption of the periodontitis group was higher than the control group(P< 0.05).The results of HE showed that the inflammation of periodontal tissue was gradually increased.Immunohistochemical results showed that the expression of EphB4 and EphrinB2 in the periodontitis group is higher than the control group(P< 0.05),but with the extension of infection time of P.gingivalis,the expressions of EphB4 and EphrinB2 gradually were decreased.2.When osteoblasts or osteoclasts were infected with MOI=100 P.gingivalis,With the increase of time,the expressions of EphB4 and osteogenic active factors were decreased(P< 0.05).Compared with the control group,the expression of EphrinB2 was increased at6 h,12 h and 24 h,while the expression of EphrinB2 was decreased at 48 h,and the expression of osteoclast activity factor were increased,they were most obvious at 24 h(P<0.05).3.In the imitated co-culture system consisted of osteoblasts expressing EphrinB2-Fc,q PCR and Western-blot showed that the expression of osteogenic active factors was decreased after siRNA transfection of EphB4(P< 0.05).and after overexpressing EphB4,the expression of osteogenic active factors was increased(P< 0.05).the expression of EphB4 was inhibited with P.gingivalis.4.In the imitated co-culture system consisted of osteoclasts expressing EphB4-Fc,q PCR and Western-blot showed that the expression of osteoclasts active factors was increased after siRNA transfection of EphrinB2(P< 0.05).and after overexpressing EphrinB2,the expression of osteoclasts active factors was decreased(P< 0.05).the expression of EphrinB2 was inhibited with P.gingivalis.Conclusion: 1.During the development of periodontitis in rats,with the increase of infection time,the alveolar bone absorption with the periodontitis group was increased,and the periodontal inflammation was gradually aggravated.The expressions of EphB4 and EphrinB2 were higher than the control group.At the bone resorption edge,EphB4 was mainly expressed in the cytoplasm of osteoblasts and a few osteoclasts,and EphrinB2 was mainly expressed in the cytoplasm of osteoclasts and a few osteoblasts at the bone resorption edge.With the prolongation of infection time,the expressions of EphB4 and EphrinB2 were gradually decreased.osteoblasts function was inhibited and osteoclasts function was promoted with P.gingivalis by regulating EphB4 and EphrinB2.EphB4 and EphrinB2 may be involved in alveolar bone remodeling during the development of periodontitis.2.Osteoblasts and osteoclasts were infected with P.gingivalis respectively,with the increase of time,the expression of EphB4 and EphrinB2 protein were regulated in osteoblasts and osteoclasts,The expression of osteogenic factors Runx2,ALP and OPN were decreased and the activity of osteoblasts were weakened.On the contrary,the expression of osteoclastic factors NFATc1,C-fos and Cts K were increased,and the activity of osteoclasts were strengthened.3.By establishing the imitated co-culture system of EphrinB2-Fc expressing osteoblasts or EphB4-Fc expressing osteoclasts,EphB4 and EphrinB2 were inhibited by P.gingivalis,the activity of osteoblasts was inhibited and the activity of osteoclasts was promoted.The expressions of EphB4 and EphrinB2 were regulated between osteoclasts and osteoblasts,which may play a regulatory role through EphB4 / EphrinB2 bidirectional signaling transduction and affect the imbalance of bone homeostasis. |
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