The Mechanism Of NLRP3 Inflammasome Activation In Gingival Fibroblasts Cells

OBJECTIVES : Observing the gene expressions of NLRP3 inflammasome and inflammatory factors IL-1β secretion in P.gingivalis LPS,P.gingivalis or heat-killed P.gingivalis(HP.gingivalis)-induced human gingival fibroblasts(h GFs)cultured in vitro.Moreover,further investigating the effects of exogenous ATP on the activation of NLRP3 inflammasome and interleukin-1β(IL-1β)secretion under inflammatory microenvironment.METHODS: HGFs were cultured with tissue block mehthod to obtain primary cells in vitro.P4-P5 h GFs were selected to be simulated by 1ug/ml P.gingivalis LPS,100 MOI live P.gingivalis or 100 MOI HP.gingivalis in vitro then treated with 5mmol/L ATP.Real-time PCR was carried out to dectect genes expression of NLRP3,ASC,Caspase-1 and IL-1β.The intracellular protein level of NLRP3,Caspase-1 and IL-1β were measured by western blot.Finally,IL-1β secretion were assessed by ELISA technology.RESULTS : Comparing with control group.P.gingivalis could decrease NLRP3,ASC genes whlie upregulate IL-1β gene expression.Furthermore,the intracellular protein expressions of NLRP3 and IL-1β were downregulated.The gene and intracellular protein expressions of NLRP3,ASC and IL-1β could be increased by P.gingivalis LPS or HP.gingivalis induction,while Caspase-1m RNA and IL-1β secretion were unaffected with P.gingivalis LPS,P.gingivalis or HP.gingivalis stimulus.The increase of NLRP3 inflammasome and IL-1β genes as well as intracellular protein NLRP3,Caspase-1expressions and IL-1β secretion level were remarkable potentiating with ATP/P.gingivalis LPS,ATP/P.gingivalis or ATP/HP.gingivalis stimuli in h GFs.CONCLUSIONS : P.gingivalis LPS,P.gingivalis or HP.gingivalis could serve as first signal to regulate the gene expression of NLRP3 inflammasome and the intracellular protein level of NLRP3 and IL-1β.The exogenous ATP could serve as a second stimulating signal in NLRP3 inflammasome activation of h GFs and adjust inflammatory factor IL-1β secretion.