Kai Yang1, Shuo Xu2, Hongmei Zhao3, Lingshuang Liu4, Xiaofang Lv1, Fang Hu1, Lei Wang1, Qiuxia Ji5. 1. Department of Periodontology, The Affiliated Hospital of Qingdao University, Qingdao, Shandong, China; School of Stomatology, Qingdao University, Qingdao, Shandong, China. 2. Laboratory of Oral Microbiology, Shanghai Research Institute of Stomatology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. 3. Department of Prosthodontics, The Affiliated Hospital of Qingdao University, Qingdao, Shandong, China. 4. Department of Endodontics, The Affiliated Hospital of Qingdao University, Qingdao, Shandong, China. 5. Department of Periodontology, The Affiliated Hospital of Qingdao University, Qingdao, Shandong, China; School of Stomatology, Qingdao University, Qingdao, Shandong, China. Electronic address: jqx_1@163.com.
Abstract
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.
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.