The Role Of Macrophage-Specific Act1 In Periodontitis

BackgroundPeriodontitis is one of the most common oral inflammatory diseases.The global prevalence of severe periodontitis is about 10%,which is one of the main reasons for patients’ missing teeth.At present,non-surgical treatments such as periodontal cleaning and root flattening are regularly performed with the assistance of antibiotics,but there is a problem of unstable treatment effects.Immunomodulation has very important significance in the pathogenesis and targeted therapy of periodontitis.However,the immune mechanism of periodontitis is still unclear,and there is no mature immunotherapy method for clinical application.Therefore,in-depth study of the immune regulation mechanism of periodontitis and exploration of new immune targets in order to treat periodontitis are current scientific frontier issues.Act1 is an NF-κB activating protein,which is highly expressed in macrophages and participates in immune regulation,but its role in periodontitis has not been reported.ObjectiveIn this project,an in vivo study was performed using an anti-Act1 gene-modified mouse periodontitis model that specifically constructed to inhibit Act1 expression in macrophages.To explore the role of macrophage-specific Act1 in periodontitis.Material and MethodsFirst,the PCR method was used to identify anti-Act1 mice.Twenty 8-week-old male anti-Act1 mice and 20 C57BL/6 mice were divided into 4 groups(C57BL/6normal group and periodontitis group;anti-Act1 normal group and periodontitis group).The model of periodontitis was established by ligating the left maxillary second molar with silk thread for 7 days.Micro-CT scan analysis,HE staining and TRAP staining were used to observe the periodontal bone resorption and inflammation severity.The expression of inflammatory factors(IL-1β,IL-6,and TNFα)and chemokines(MCP-1 and MIP-1α/β)in periodontal tissues of mice was observed by RT-q PCR and other methods.The infiltration of macrophages in the periodontal tissues of mice was observed by immunohistochemistry.The number of macrophages and M1/M2 polarization in the periodontal tissues of mice were detected by flow cytometry.Mouse macrophages were cultured in vitro,and LPS was used to induce macrophages as an in vitro inflammation model.The expression level of Act1 in macrophages was detected by RT-q PCR.The migration experiment was used to detect the migration ability of macrophages.RANKL and M-CSF induced macrophages to differentiate into osteoclasts and tested their ability to differentiate into osteoclasts.The secretion of inflammatory factors(IL-1β,IL-6,and TNFα)and MCP-1 in macrophage culture supernatant was detected by ELISA.Results(1)Silk thread ligation can cause obvious pathological changes of periodontitis in mice.Act1 is expressed in mouse periodontal tissues.(2)The specific inhibition of Actl expression of macrophages makes periodontal tissue inflammation increase,bone resorption increases,bone density decreases,the number of osteoclasts increases,and the expression of inflammatory factors increases.(3)Specific inhibition of Act1 expression of macrophages increases the number of macrophages and M1-type polarization in local tissues of periodontitis.(4)Inhibiting macrophage-specific Act1 expression in vitro causes functional changes in macrophages,such as enhancing the migration ability of macrophages and enhancing the differentiation ability of macrophages into osteoclasts.Macrophage secretion of inflammatory factors and MCP-1 increased.ConclusionSilk ligation can effectively and stably induce periodontitis in mice.Act1 is expressed in mouse periodontal tissues and is upregulated during periodontitis.In periodontitis,specifically inhibiting the expression of Act1 in macrophages may promote macrophage migration,M1 polarization,osteoclast differentiation through up-regulation of inflammatory factors and MCP-1 expression,promote inflammatory response and bone resorption,and then accelerate the pathological process of periodontitis.