Department of Endodontics, School of Stomatology, China Medical University, Shenyang, China; Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan.
Comprehensive Dental Clinic, Okayama University Hospital, Okayama University, Okayama, Japan.
Arch Oral Biol. 2020 Oct;118:104841. doi: 10.1016/j.archoralbio.2020.104841. Epub 2020 Jul 18.
Porphyromonas gingivalis (P. gingivalis) is a major bacterium responsible for the progression of periodontitis. P. gingivalis produces small vesicles called outer membrane vesicles (OMVs) containing virulence factors. Increasing evidence suggests a close relationship between periodontitis and respiratory system diseases, such as aspiration pneumonia. However, little is known about whether P. gingivalis OMVs give rise to the impediment of lung epithelial cells. We investigated the effect of the OMVs on cell viability and tight junctions of lung epithelial cells.
Human lung epithelial A549 cells were treated with P. gingivalis OMVs. Cell viability was evaluated, and cell morphology was examined using scanning electron and phase contrast microscopies. To detect apoptosis induced by P. gingivalis OMVs, activation of caspase-3 and poly ADP-ribose polymerase (PARP) cleavage was examined by using Western blotting. Immunocytochemistry was performed to stain tight junction proteins.
P. gingivalis OMVs decreased cell viability in A549 cells in a dose- and time-dependent manner. Microscopic analysis revealed that the OMVs induced morphological changes leading to irregular cell membrane structures. The OMVs caused cell shrinkage, membrane blebbing, and cytoplasmic expulsion in a dose-dependent manner. Western blot analysis showed the OMVs induced caspase-3 activation and PARP cleavage. Treatment with the OMVs disrupted the intact distributions of tight junction proteins.
These results indicate that P. gingivalis OMVs induced cell death by destroying the barrier system in lung epithelial cells. Our present study raises the possibility that P. gingivalis OMVs is an important factor in the engagement of periodontitis with respiratory system diseases.
牙龈卟啉单胞菌（P. gingivalis）是导致牙周炎进展的主要细菌。P. gingivalis 产生含有毒力因子的小泡，称为外膜囊泡（OMVs）。越来越多的证据表明，牙周炎与呼吸系统疾病如吸入性肺炎密切相关。然而，目前尚不清楚 P. gingivalis OMVs 是否会导致肺上皮细胞功能障碍。我们研究了 OMVs 对肺上皮细胞活力和紧密连接的影响。
用人肺上皮 A549 细胞处理 P. gingivalis OMVs。通过扫描电子显微镜和相差显微镜观察细胞活力和形态。通过 Western blot 检测 caspase-3 和聚 ADP-核糖聚合酶（PARP）切割的激活，检测 P. gingivalis OMVs 诱导的细胞凋亡。免疫细胞化学染色检测紧密连接蛋白。
P. gingivalis OMVs 以剂量和时间依赖的方式降低 A549 细胞的活力。显微镜分析显示，OMVs 诱导的形态变化导致不规则的细胞膜结构。OMVs 以剂量依赖的方式引起细胞收缩、细胞膜起泡和细胞质外溢。Western blot 分析显示 OMVs 诱导 caspase-3 激活和 PARP 切割。OMVs 处理破坏了紧密连接蛋白的完整分布。
这些结果表明，P. gingivalis OMVs 通过破坏肺上皮细胞的屏障系统诱导细胞死亡。我们的研究结果提示 P. gingivalis OMVs 可能是牙周炎与呼吸系统疾病相关的重要因素。
