Department of Periodontology, The Affiliated Hospital of Qingdao University, Qingdao, Shandong, China; School of Stomatology, Qingdao University, Qingdao, Shandong, China.
Laboratory of Oral Microbiology, Shanghai Research Institute of Stomatology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Int Immunopharmacol. 2021 May;94:107456. doi: 10.1016/j.intimp.2021.107456. Epub 2021 Feb 12.
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).
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.
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.
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.
探讨缺氧和牙龈卟啉单胞菌脂多糖（P. gingivalis-LPS）对人牙龈成纤维细胞（HGFs）中 NACHT 富含亮氨酸重复蛋白 3（NLRP3）炎性小体激活的影响。
采用 1%的缺氧浓度来最佳模拟牙周炎。分别在常氧和缺氧条件下用 P. gingivalis-LPS（1.0μg/ml）刺激 HGFs3 h 和 6 h。用实时定量聚合酶链反应和蛋白质印迹法检测缺氧诱导因子-1α（HIF-1α）、白细胞介素-1β、gasdermin D（GSDMD）和 NLRP3 炎性小体的基因和蛋白表达水平，包括 NLRP3、凋亡相关斑点样蛋白含有 CARD（ASC）、半胱天冬酶-1 和其活化形式。ELISA 用于检测和确定细胞上清液中炎症因子白细胞介素-1β的水平。用乳酸脱氢酶（LDH）释放试验、半胱天冬酶-1 活性试验和 Hoechst 33342/碘化丙啶（PI）染色进一步验证细胞发生了细胞焦亡。
单独使用 P. gingivalis-LPS 或缺氧刺激不会影响 NLRP3 炎性小体（即 NLRP3、ASC、caspase-1）。然而，缺氧和 P. gingivalis-LPS 刺激的联合作用显著增强了炎性小体的激活，并促进了白细胞介素-1β、gasdermin D 和 HIF-1α在基因和蛋白水平上的表达；PI 阳性细胞和 LDH 的释放也升高。
缺氧和 P. gingivalis-LPS 协同诱导 HGFs 中 NLRP3 炎性小体的激活，随后高水平的白细胞介素-1β和 GSDMD 介导的细胞焦亡可引起 HGF 炎症反应，这在牙周炎的发病机制中起重要作用。
