PURPOSE

To investigate exogenous ATP-dependent activation of NLRP3 inflammasome and interleukin-1β ( IL-1β) secretion in P.gingivalis infected and heat-killed P.gingivalis induced gingival fibroblasts cells ( hGFs) in vitro.

METHODS

Gingival tissues were obtained from healthy patients and hGFs were cultured in vitro with tissue block method to harvest primary cells. HGFs was simulated by being treated with 100 MOI live P.gingivalis or 100 MOI heat-killed P.gingivalis (HP.gingivalis) after 5 mmol/L ATP pre-treatment. Real-time PCR was carried out to assess mRNA expression of NLRP3, ASC, caspase-1 and IL-1β. The protein level of NLRP3 , caspase-1 and IL-1β was evaluated by Western blot. IL-1β secretion was measured using ELISA. Statistical analysis was performed using Graphpad prism 6 statistical package and the measurement data were analyzed by t test or one-way ANOVA.

RESULTS

Compared with the control group, P.gingivalis downregulated NLRP3 mRNA and ASC mRNA while upregulated IL-1β mRNA. Moreover, the protein expression of NLRP3 and IL-1β was decreased. The gene and protein expression of NLRP3, ASC and IL-1β was induced by HP.gingivalis, while caspase-1mRNA and IL-1βsecretion was free from P.gingivalis or HP.gingivalis stimulus. All those genes as well as intracellular protein expression and IL-1βsecretion were significantly potentiated with ATP/P.gingivalis or ATP/HP.gingivalis stimuli in hGFs.

CONCLUSIONS

Exogenous ATP may be a potential stimulus signal in favour of NLRP3 inflammasome activation of hGFs and mediated inflammatory factor IL-1β secretion.