Drp1 Competitive Binding To HK1 Induces MPTP Over-Opening To Regulate The Inflammatory Response Of Periodontitis Macrophages

Objective: Imbalance of the host immune response and excessive inflammation play an important role in periodontal disease,leading to periodontal tissue damage and thus triggering the onset of periodontitis.Porphyromonas gingivalis(P.g),present in the oral biofilm,is one of the most important causative agents of periodontitis,and its cell wall lipopolysaccharide(LPS)is the main causative agent of inflammation,directly damaging tissues and stimulating the immune system.As a mitochondrial kinetics-related protein,Drp1 plays an important role in periodontitis,but the specific mechanism is not clear.This study aimed to reveal the specific mechanism of Drp1 regulating the inflammatory response of periodontitis macrophages through protein interactions in the P.g.LPSperiodontitis model,providing an experimental reference and theoretical basis for exploring the pathogenesis of periodontitis.Methods: Inflamed gingival tissues from periodontitis patients and healthy gingival tissues from volunteers were extracted separately,and Drp1 tetramer formation and phosphorylation were detected by Western blot.P.g.LPS(1μg/m L)was applied to RAW264.7 mouse macrophages to establish a periodontitis cell model,and the formation of Drp1 tetramer and phosphorylation of Drp1 at different time points(0 h,12 h,24 h,48h)were detected by Western blot.Mitochondrial fragmentation and membrane potential alterations were assessed by immunofluorescence staining,and NLRP3 inflammasome expression and inflammatory factor IL-1βsecretion were detected by Western blot and ELISA.The expression of Drp1 in macrophages was silenced using lentivirus to detect changes in the expression of inflammasome,the secretion of inflammatory factors and mitochondrial fragmentation in the P.g.LPS-periodontitis macrophage model.The protein interactions between hexokinase 1(HK1)and Drp1 were clarified by proteomic analysis and verified by immunoprecipitation,and the opening of the mitochondrial membrane permeability transition pore(mPTP)was detected using immunofluorescence staining in the P.g.LPS-periodontitis macrophage model.The above experiments clarify the role of Drp1 competitive binding to HK1 to induce mPTP over-opening in the pathogenesis of periodontitis.Results: 1.Increased Drp1 tetramer formation and increased phosphorylation of the Drp1Ser616 site in gingival tissue with periodontitis patients and P.g.LPS(1μg/m L)induced periodontitis macrophage models(P<0.05).2.Immunofluorescence staining experiments showed fragmentation of mitochondria and altered membrane potential in the P.g.LPSperiodontitis macrophage model(P<0.05).3.NLRP3 inflammasome were activated and IL-1βinflammatory factor was released,and silencing of Drp1 resulted in a corresponding decrease in NLRP3 expression levels and IL-1βrelease was correspondingly decreased(P<0.05).4.Drp1 competitive binding to HK1 in the P.g.LPS-periodontitis macrophage model to induce mPTP over-opening,and silencing Drp1 inhibits mPTP over-opening(P<0.05).Conclusion: 1.Drp1 hyperactivation was observed in gingival tissue from patients with periodontitis and in the P.g.LPS-induced periodontitis cell model.2.Mitochondria damage in the P.g.LPS-periodontitis macrophage model and silencing Drp1 reduces mitochondrial damage.3.The P.g.LPS-periodontitis macrophage model undergoes inflammatory injury,NLRP3 inflammasome activation,and IL-1β release,and silencing Drp1 inhibits these processes.4.Drp1 hyperactivation may regulates periodontitis by competitively binding to HK1 to induce excessive mPTP opening macrophage inflammatory response.