Hypoxia and Porphyromonas gingivalis-lipopolysaccharide synergistically induce NLRP3 inflammasome activation in human gingival fibroblasts.

OBJECTIVE

To investigate the effects of hypoxia and Porphyromonas gingivalis- lipopolysaccharide (P. gingivalis-LPS) on activation of the NACHT leucine-rich repeat protein 3 (NLRP3) inflammasome in human gingival fibroblasts (HGFs).

DESIGN

Periodontitis was optimally simulated using a hypoxic concentration of 1%. HGFs were stimulated using P. gingivalis-LPS (1.0 μg/ml) in normoxia and hypoxia for 3 h and 6 h, respectively. The expression levels of genes and proteins of hypoxia-inducible factor-1α (HIF-1α), interleukin-1β, gasdermin D (GSDMD) and the NLRP3 inflammasome, including NLRP3, apoptosis-associated speck-like protein containing CARD (ASC), caspase-1 and its activated forms, were measured using quantitative real-time polymerase chain reaction and western blot. ELISA was used to detect and determine levels of the inflammatory factor interleukin-1β in cell supernatants. Lactate dehydrogenase (LDH) release assay, caspase-1 activity assay and Hoechst 33342/Propidium Iodide (PI) staining were performed to further verify the presence of pyroptosis.

RESULTS

The NLRP3 inflammasome (i.e., NLRP3, ASC, caspase-1) was not affected by individual stimulation using P. gingivalis-LPS or hypoxia. However, the combination of both hypoxia and P. gingivalis-LPS stimulation significantly enhanced inflammasome activation and promoted the expression of interleukin-1β, gasdermin D and HIF-1α at gene and protein levels; PI positive cells and the release of LDH were also elevated.

CONCLUSION

Hypoxia and P. gingivalis-LPS synergistically induced NLRP3 inflammasome activation in HGFs, and subsequently high levels of interleukin-1β and GSDMD-mediated pyroptosis can cause an HGF inflammatory response, which plays an important role in the pathogenesis of periodontitis.