Department of Periodontology and Endodontology, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, Japan.
Department of Oral Microbiology, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, Japan.
J Periodontal Res. 2020 Aug;55(4):539-550. doi: 10.1111/jre.12741. Epub 2020 Mar 13.
Diabetes mellitus (DM), a risk factor of periodontal diseases, exacerbates the pathological condition of periodontitis. A major factor for DM complications is advanced glycation end-products (AGEs) that accumulate in periodontal tissues and cause inflammatory events. Lipocalin 2 (LCN2) is an antimicrobial peptide and inflammation-related factor, and LCN2 levels increase in DM. In this study, the effects of AGEs and lipopolysaccharide of Porphyromonas gingivalis (P g-LPS) on LCN2 expression in human oral epithelial cells (TR146 cells) and the role of secreted LCN2 in periodontitis with DM were investigated.
TR146 cells were cultured with AGEs (AGE2) and control BSA and cell viability was estimated, or with P g-LPS. Conditioned medium and cell lysates were prepared from cultures of epithelial cells and used for Western blotting and ELISA to analyze LCN2, RAGE, IL-6, MAPK, and NF-κB. RNA was isolated from AGE-treated TR146 cells and differentiated HL-60 (D-HL-60) cells and used for quantitative real-time PCR to examine the expression of LCN2 and interleukin-6 (IL-6) mRNAs. RAGE- and LCN2-siRNAs (siRAGE, siLCN2) were transfected into epithelial cells, and AGE-induced LCN2 expression was investigated. D-HL-60 cells were co-cultured with TR146 cells that were transfected with siLCN2 and treated with AGEs, and IL-6 mRNA expression in D-HL-60 cells and cell migration was investigated.
AGEs increased the expression levels of LCN2 and IL-6 in oral epithelial cells. siRAGE and a neutralizing antibody for RAGE inhibited AGE-induced LCN2 expression. AGEs stimulated the phosphorylation of ERK, p38, and NF-κB in epithelial cells, and their inhibitors suppressed AGE-induced LCN2 expression. In contrast, P g-LPS did not show a significant increase in LCN2 level in TR146 cells that expressed Toll-like receptor 2. In co-culture experiments, AGE-induced LCN2 inhibited IL-6 mRNA expression in D-HL-60 cells, and LCN2 knockdown in epithelial cells suppressed HL-60 cell migration.
These results suggested that AGEs increase LCN2 expression via RAGE, MAPK, and NF-κB signaling pathways in oral epithelial cells, and secreted LCN2 may influence the pathological condition of periodontitis with DM.
糖尿病（DM）是牙周病的一个危险因素，会使牙周炎的病理状况恶化。DM 并发症的一个主要因素是糖基化终产物（AGEs），其在牙周组织中积累并引起炎症事件。脂钙蛋白 2（LCN2）是一种抗菌肽和炎症相关因子，且其在 DM 中水平升高。在本研究中，我们研究了 AGEs 和牙龈卟啉单胞菌脂多糖（P g-LPS）对人口腔上皮细胞（TR146 细胞）中 LCN2 表达的影响，以及分泌的 LCN2 在 DM 相关牙周炎中的作用。
用 AGE2 和对照 BSA 培养 TR146 细胞并估计细胞活力，或用 P g-LPS 培养。从上皮细胞培养物中制备条件培养基和细胞裂解物，并用于 Western blot 和 ELISA 分析 LCN2、RAGE、IL-6、MAPK 和 NF-κB。用 AGE 处理的 TR146 细胞和分化的 HL-60（D-HL-60）细胞分离 RNA，并用于定量实时 PCR 检测 LCN2 和白细胞介素 6（IL-6）mRNA 的表达。将 RAGE 和 LCN2-siRNA（siRAGE、siLCN2）转染至上皮细胞，并研究 AGE 诱导的 LCN2 表达。将 siLCN2 转染的 D-HL-60 细胞与转染 siLCN2 的 TR146 细胞共培养，并研究 D-HL-60 细胞中 IL-6 mRNA 的表达和细胞迁移。
AGEs 增加了口腔上皮细胞中 LCN2 和 IL-6 的表达水平。siRAGE 和 RAGE 的中和抗体抑制了 AGE 诱导的 LCN2 表达。AGEs 刺激上皮细胞中 ERK、p38 和 NF-κB 的磷酸化，其抑制剂抑制了 AGE 诱导的 LCN2 表达。相比之下，表达 Toll 样受体 2 的 TR146 细胞中，P g-LPS 并未显著增加 LCN2 水平。在共培养实验中，AGE 诱导的 LCN2 抑制了 D-HL-60 细胞中 IL-6 mRNA 的表达，上皮细胞中 LCN2 的敲低抑制了 HL-60 细胞的迁移。
这些结果表明，AGEs 通过口腔上皮细胞中的 RAGE、MAPK 和 NF-κB 信号通路增加 LCN2 的表达，分泌的 LCN2 可能影响 DM 相关牙周炎的病理状况。
