Shuo Xu1, Qihui Zhou2, Chun Fan3, Hongmei Zhao4, Yanwen Wang5, Xiaohui Qiu1, Kai Yang1, Qiuxia Ji6. 1. Department of Periodontology, The Affiliated Hospital of Qingdao University, Qingdao, 266003, China; School of Stomatology of Qingdao University, Qingdao, 266003, China. 2. Institute for Translational Medicine, State Key Laboratory of Bio-Fibers and Eco-Textiles, Qingdao University, Qingdao, 266021, China. 3. Department of Periodontology, The Affiliated Hospital of Qingdao University, Qingdao, 266003, China. 4. Department of Prosthodontics, The Affiliated Hospital of Qingdao University, Qingdao, 266003, China. 5. Stuart Country Day School of the Sacred Heart, 1200 Stuart Road, Princeton, NJ, 08628, United States. 6. Department of Periodontology, The Affiliated Hospital of Qingdao University, Qingdao, 266003, China. Electronic address: jqx_1@163.com.
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.
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.
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