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 by Porphyromonas gingivalis-lipopolysaccharide (P. gingivalis-LPS) stimulation and the anti-inflammatory eff ;ect of doxycycline (Dox) in human gingival fibroblasts (HGFs).

Design: The optimal concentration of P. 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 with P. 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.

Keywords: Adenosine triphosphate; Doxycycline; IL-1β; NLRP3 inflammasome; P. gingivalis-LPS; Periodontitis.