Doxycycline inhibits NAcht Leucine-rich repeat Protein 3 inflammasome activation and interleukin-1β production induced by Porphyromonas gingivalis-lipopolysaccharide and adenosine triphosphate in human gingival fibroblasts

Abstract Objective To investigate the effect of adenosine triphosphate (ATP) on inflammasome activation byPorphyromonas gingivalis-lipopolysaccharide (P. gingivalis-LPS) stimulation and the anti-inflammatory effect of doxycycline (Dox) in human gingival fibroblasts (HGFs). Design The optimal concentration ofP. gingivalis-LPS (1.0 µg/mL) for cellular viability was determined by observing cell morphology and measuring the amount of formazan and the expression of pro-caspase-1. The expression of genes and proteins related to the NAcht Leucine-rich repeat Protein 3 (NLRP3) inflammasome, including NLRP3, apoptosis-associated speck-like protein containing CARD (ASC), caspase-1 and its activated forms, and the inflammatory factor interleukin-1β (IL-1β) and its activated forms were measured. Results The NLRP3 inflammasome (i.e., NLRP3, ASC, caspase-1) was not affected by stimulation withP. gingivalis-LPS or ATP. However, a combination of P. gingivalis-LPS and ATP significantly enhanced inflammasome activation and IL-1β production at the gene and protein levels as measured by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot, respectively. Furthermore, doxycycline addition markedly inhibited inflammasome activation and IL-1β production induced by a combination of P. gingivalis-LPS and ATP. Conclusions LPS, ATP, and doxycycline play critical roles in regulating host immune responses. This evidence provides guidance for the application of tetracycline drugs for the clinical treatment of periodontal disease.