Isoliquiritigenin Alleviates P. Gingivalis-LPS/ATP-induced Pyroptosis By Inhibiting NF-κB/NLRP3/GSDMD Signals In HGFs

Objective: Periodontitis is a common clinical problem and exhibits a high rate of clinical incidence,but its specific mechanism remains unclear and conventional antibiotic treatment has limited effectiveness.Recent studies have shown that pyroptosis plays a pivotal role in the initiation and development of periodontitis.Therefore,pyroptosis signaling pathway may be a novel entry point to explore the pathogenesis of periodontitis,and finding a non-antibiotic drug that specifically inhibits this pathway may be a breakthrough in the treatment of periodontitis.Hence,we aimed to investigate whether pyroptosis is induced by Porphyromonas gingivalis-lipopolysaccharide(P.gingivalisLPS)/ adenosine triphosphate(ATP)through NF-κB/NLRP3/GSDMD signaling in human gingival fibroblasts(HGFs)and whether isoliquiritigenin(ISL)alleviates pyroptosis by inhibition of NF-κB/NLRP3/GSDMD signals.Methods:1.Primary culture and identification of HGFs: the primary HGFs were cultured by enzyme digestion culture method.The cells were identified by optical microscopy and immunofluorescence method.2.Screening of appropriate concentrations of drugs and experimental grouping processing: HGFs were treated with DMSO or ISL at concentrations of 0,0.1%,0.5%,1.0%,2.5%,5.0%,10.0% or 0,2.5,5,7.5,10,20 μM for 24 hours,respectively.CCK-8method was used to detect the effect of DMSO or ISL on HGFs viability,so as to screen out the appropriate concentration for subsequent experiments.The experimental cells were divided into 4 groups: control group,ISL group,LPS+ATP group,and LPS+ATP+ISL group.3.Construction and suppression of pyroptosis model: pyroptosis was optimally simulated using a combination of P.gingivalis-LPS and ATP,and using ISL regulated cell model.The expression levels of genes and proteins of NF-κB,NLRP3 inflammasome,GSDMD,and IL-1β was characterized by q RT-PCR,western blotting and ELISA.The2?,7??dichlorodihydrofluorescein diacetate fluorescence probe was used to determine the intracellular ROS level.Hoechst 33342 and PI double staining,cytotoxicity assay,and caspase-1 activity assay were used to confirm the influence of ISL on pyroptosis in P.gingivalis-LPS/ATP-treated HGFs.Results:1.Primary culture and identification of HGFs: The primary cell morphology observed under the microscope was the most typical morphologies of gingival fibroblasts.Immunofluorescence results also demonstrated that the primary cells were HGFs.2.Screening of appropriate concentrations of drugs: CCK-8 assay data showed that cell viability after 24 h of culture was unaffected by 0,0.1%,0.5%,and 1.0% DMSO.Under0.1% DMSO,when the concentration of ISL was >5 μM,cell viability was decreased with the increase of the ISL concentration.Therefore,0.1% DMSO and 5 μM ISL was selected for subsequent experiments.3.Construction and suppression of pyroptosis model: the results of q RT-PCR,western blot and ELISA showed that the expression levels of gene and protein of NF-κB,the NLRP3 inflammasome,GSDMD,and IL-1β were increased after P.gingivalis-LPS/ATP stimulation,and decreased after treatment with ISL.Similarly,the intracellular ROS level,the proportion of membrane-damaged cells,caspase-1 activity,and the release of LDH were also elevated after P.gingivalis-LPS/ATP stimulation.However,treatment with ISL observably suppressed these effects.Conclusions:P.gingivalis-LPS/ATP induced pyroptosis in HGFs by activating NF-κB/NLRP3/GSDMD signals and ISL attenuated P.gingivalis-LPS/ATP-induced pyroptosis by inhibiting these signals.Our study provides a new idea for the pathogenesis of periodontitis,which may provide a powerful therapeutic target for the treatment of periodontitis.ISL may be a potential drug candidate for the treatment of periodontitis.