Effect Of NLRP3/Caspase-1/IL-1? On Orthodontically Induced Inflammatory Root Resorption

Orthodontically induced inflammatory root resorption(OIIRR)is the most common complication in the correction of maxillofacial deformity.According to statistics,the incidence of OIIRR is about 90%,and the incidence of severe root resorption exceeding 4 mm or reaching a third of the length of the root is about 5%.Severe OIIRR can cause tooth mobility or even falling off,which has become an important potential factor to cause doctor-patient disputes.In recent years,with the wide application of Cone beam computed tomography(CBCT)in clinical,the rate of detection of OIIRR is increasing.However,the pathogenesis of OIIRR is not clear,and there are no effective measures to prevent or interfere with OIIRR.In the context of orthodontic tooth movement,sterile necrosis in periodontal tissues occur in response to localized orthodontic load-induced compression of the vasculature and subsequent ischaemia in the periodontal ligament,the secretion of inflammatory cytokines such as IL-1?,IL-6,TNF-?,chemotactic factor and transforming growth factor increased and a sterile inflammatory reaction formed in the periodontal tissues.Subsequently,removal of necrotic tissues in the compressive zone of the periodontium by osteoclasts,cementoclasts/odontoclasts and inflammasome,and they have the collateral damaging effect of root resorption.Early in the orthodontic tooth movement,the secretion of IL-1? increases,which maintains a local proinflammatory environment by enhancing the recruitment and activation of peripheral blood mononuclear cells.IL-1? is an important proinflammatory cytokine in the early period of root resorption.It has been reported that in the periodontal tissue of OIIRR,the secretion of IL-1? and the number of M1 macrophages increased,while M1 macrophages were the main cells that produced IL-1?.NLRP3 inflammasome is a potential sensor of aberrant metabolites or extracellular force signals,the activation of which leads to matured IL-1? secretion through pro-caspase-1 activation.It has reported that NLRP3 mediated the progression of diabetes,gouty arthritis,atherosclerosis and other diseases through secretion IL-1?.In consideration of the effects of NLRP3 on IL-1? production and inflammatory reaction,we speculate that NLRP3 inflammasome regulates the production of IL-1? in M1 macrophages,promoting the occurrence of OIIRR.Therefore,the expression of NLRP3,caspase-1,IL-1? and the distribution of M1 macrophages in the root resorption region were examined in this study to explore the role and mechanism of NLRP3 inflammasome-mediated OIIRR.The aim of the study was to provide new therapeutic ideas and methods for clinical prevention and treatment of OIIRR.The study included the following two parts: Part 1 Changes of expression of NLRP3/caspase-1/IL-1? and distribution of M1 macrophages in root resorption area during orthodontic in rat OIIRR model Objective: To study the expression of NLRP3,caspase-1 and IL-1? and the distribution of M1 macrophages in periodontal tissue of OIIRR in rat.Methods: To establish a rat OIIRR model,36 healthy male Sprague-Dawley(SD)rats were randomly divided into six groups: 0-day group,1-day group,4-day group,7-day group,14-day group and 28-day group,six rats per group,rats were sacrificed after the end of each group,removed maxilla.Through H&E staining,the root resorption of rats was observed and the root resorption area of each group was calculated.Expression of NLRP3,caspase-1 and IL-1? around root resorption in different groups were detected by immunohistochemical staining,the expression and distribution of M1 macrophages in root resorption region were detected by immunofluorescence double staining.Results: H&E staining results showed that the cementum in pressure side began to absorb in the 4-day group,and the number and area of root resorption fossa increased gradually with increasing force-loading time.Immunohistochemical staining showed that NLRP3,caspase-1 and IL-1? were mainly distributed in the root resorption area and the edge of alveolar bone.The expression of IL-1? was increased gradually in the periodontal tissue and peaked on the fourth day,then the expression of IL-1? decreased,there were significant differences in the expression between the 1-day group and the 4-day group(P<0.05).The expression of NLRP3 decreased slightly after the fourth day,the difference was not statistically significant,there was no significant difference between 7 days,14 days and 28 days(P>0.05).Through immunofluorescence double staining,we found that the number of CD68+CD11b+ M1 macrophages increased gradually with the increasing force-loading time,and reached its peak on day 28,and mainly distributed in the pressure side of the root.Conclusion: In pressure-side periodontal tissues of rat OIIRR model,the expression of NLRP3,caspase-1,IL-1? increased,and the number of CD68+CD11b+ M1 macrophages increased.Part 2 Study of force-pretreated hPDLCs on NLRP3 inflammasome activation and IL-1? production in macrophages Objective: To investigate the effect of force-pretreated hPDLCs on macrophage polarization,NLRP3 inflammasome activation and IL-1? production.Methods: A co-culture system of hPDLCs and macrophages was established in vitro.1g/cm2 compressive stress were applied to hPDLCs with 12 hours,then cultured with macrophages for 72 hours.The effect of force-pretreated hPDLCs on expression of NLRP3,pro-caspase-1,caspase-1 and IL-1? in M1 macrophages was determined by Western Blot and immunofluorescence.The expression of NLRP3,pro-caspase-1,caspase-1 and IL-1? in M1 macrophages were detected by adding MCC950,a specific inhibitor of NLRP3.Results: Western Blot results showed increased expression of NLRP3,pro-caspase-1,caspase-1 and IL-1? in macrophages after co-culture with force-pretreated hPDLCs.Immunofluorescence double staining showed increased expression of CD68+IL-1?+ M1 macrophages in the co-culture group of force-pretreated hPDLCs and macrophages.After adding MCC950,the expression of NLRP3,pro-caspase-1,caspase-1 and IL-1? in the co-culture system decreased,changes in CD68+IL-1?+ M1 macrophages were also decreasing.Conclusion: Force-pretreated hPDLCs promote the polarization of M1 macrophages and the expression of NLRP3,caspase-1 and IL-1? in M1 macrophages.MCC950 inhibited CD68+IL-1?+ M1 macrophage polarization and NLRP3 activation and then reduced expression of IL-1?.