Nanjing Stomatological Hospital, Medical School of Nanjing University, Nanjing, China.
Central Laboratory of Stomatology, Nanjing Stomatological Hospital, Medical School of Nanjing University, Nanjing, China.
J Periodontal Res. 2021 Jun;56(3):523-534. doi: 10.1111/jre.12852. Epub 2021 Feb 3.
BACKGROUND/OBJECTIVES: Iron homeostasis plays a crucial role in the combat against pathogen invasion. Ferrous iron can trigger generous production of reactive oxygen species (ROS) by Fenton reaction. Nuclear receptor coactivator 4 (NCOA4), a selective cargo receptor to deliver ferritin to lysosome, may trigger release of ferritin-bound iron into the cytosol. The aim of the present study was to explore whether NCOA4-mediated ferritinophagy participated in the pathogenesis of periodontitis, and its role in promoting the periodontal inflammation.
Inflamed and healthy periodontal tissues were harvested for immunobiological staining of ferritinophagy-related genes in the periodontal tissues, while real-time quantitative PCR (qPCR) was utilized to detect mRNA transcription. Periodontal ligament fibroblasts (PDLFs) were isolated and infected with Porphyromonas gingivalis. The mRNA transcription and protein expression of genes involved in the iron metabolism, including NCOA4, transferrin receptor 1 (TFR1), and ferroportin (SLC40A1) were detected by qPCR and western blot. Levels of labile iron pool and ROS production were detected by flow cytometry and confocal endoscopy. Small interference RNA was utilized to knock down NCOA4.
Elevated expression of NCOA4, ferritin heavy chain, and light chain were observed in the diseased periodontal tissues. P. gingivalis infection promoted expression of TFR1, NCOA4, and microtubule-associated protein 1-light chain 3 B (LC3B), enhanced levels of intracellular labile iron pool and ROS production. NCOA4 knockdown reduced ROS generation in PDLFs in response to P. gingivalis and mitigated production of pro-inflammatory monocyte chemoattractant protein-1 and interleukin 6. P. gingivalis triggered activation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase signaling pathway. In addition, inhibitors of JNK, SP600125, and inhibitors of p38, SB203580 blocked NCOA4 transcription.
NCOA4-ferritinophagy participated in the progress of periodontitis progression. P. gingvalis-triggered ferritinophagy aggravated production of ROS and inflammatory responses in PDLFS. These findings suggest iron homeostasis plays an important role in the pathogenesis of periodontitis.
背景/目的：铁稳态在抵御病原体入侵中起着至关重要的作用。亚铁离子可以通过芬顿反应引发大量活性氧（ROS）的产生。核受体共激活因子 4（NCOA4）是一种将铁蛋白递送至溶酶体的选择性货物受体，可能会触发铁蛋白结合的铁向细胞质内释放。本研究旨在探讨 NCOA4 介导的铁蛋白自噬是否参与了牙周炎的发病机制，以及其在促进牙周炎症中的作用。
采集炎性和健康牙周组织进行牙周组织中铁蛋白自噬相关基因的免疫生物学染色，同时利用实时定量 PCR（qPCR）检测 mRNA 转录。分离牙周韧带成纤维细胞（PDLFs）并感染牙龈卟啉单胞菌。通过 qPCR 和 Western blot 检测参与铁代谢的基因，包括 NCOA4、转铁蛋白受体 1（TFR1）和铁输出蛋白（SLC40A1）的 mRNA 转录和蛋白表达。通过流式细胞术和共聚焦内镜检测不稳定铁池和 ROS 产生水平。利用小干扰 RNA 敲低 NCOA4。
在患病的牙周组织中观察到 NCOA4、铁蛋白重链和轻链表达升高。牙龈卟啉单胞菌感染促进了 TFR1、NCOA4 和微管相关蛋白 1 轻链 3B（LC3B）的表达，增加了细胞内不稳定铁池的水平并促进了 ROS 的产生。NCOA4 敲低降低了 PDLFs 对牙龈卟啉单胞菌的 ROS 生成，并减轻了促炎单核细胞趋化蛋白-1 和白细胞介素 6 的产生。牙龈卟啉单胞菌触发了 c-Jun N 末端激酶（JNK）和 p38 丝裂原活化蛋白激酶信号通路的激活。此外，JNK 抑制剂 SP600125 和 p38 抑制剂 SB203580 阻断了 NCOA4 的转录。
NCOA4-铁蛋白自噬参与了牙周炎的进展。牙龈卟啉单胞菌触发的铁蛋白自噬加剧了 PDLFS 中 ROS 的产生和炎症反应。这些发现表明铁稳态在牙周炎的发病机制中起着重要作用。
