Objective: Soluble triggering receptors expressed by myeloid cells-1 (sTREM-1) and inflammatory cytokine tumor necrosis factor-α (TNF-α) in macrophage cells were stimulated by Porphyromonas gingivalis-lipopolysaccharide (Pg-LPS) to investigate the expression of triggering receptors expressed by myeloid cells-1 (TREM-1) and further explore the correlation between TREM-1 and the pathogenesis of periodontitis.
Methods: THP-1 cells (a human monocytic cell line derived from an acute monocytic leukemia patient) were induced to differentiate THP-1 macrophages by phorbol-12-myristate-13-acetate and were injected with 0 (blank control), 0.5, or 1.0 μg·mL⁻¹ Pg-LPS. The THP-1 cells were then grouped in accordance with incubation time, and each group was incubated for 4, 6, 12, or 24 h. The expression of the TREM-1 mRNA in macrophages was detected by real-time quantitative polymerase chain reaction, while the expression of TREM-1 protein was detected by Western blot; the site where TREM-1 protein expression was observed in macrophages was detected by immunofluorescence staining, and the expression of soluble sTREM-1 and TNF-α in cell culture medium was detected by enzyme-linked immunosorbent assay.
Results: Compared with the blank control group, the expression of TREM-1 mRNA, TREM-1 protein, and sTREM-1 in Pg-LPS-stimulated macrophages was significantly upregulated (P<0.05). The expression of TREM-1 mRNA, TREM-1 protein, and sTREM-1 in the supernatant of cell culture was higher in the 1.0 μg·mL⁻¹ Pg-LPS group than in the 0.5 μg·mL⁻¹ group; this expression was statistically significant since the 6, 4, and 4 h time point (P<0.05). Cell immunofluorescence staining showed that TREM-1 protein was positive when the THP-1 macrophages was stimulated by Pg-LPS (1.0 μg·mL⁻¹) for 24 h, and the staining sites of TREM-1 were mainly located in the cell membrane of the macrophages (P<0.05). The expression level of TNF-α increased in groups stimulated by Pg-LPS, and the expression level of TNF-α was significantly higher in 1.0 μg·mL⁻¹ Pg-LPS stimulated groups than in 0.5 μg·mL⁻¹ Pg-LPS-stimulated groups since the 6 h time point (P<0.05). The expressions of TREM-1 mRNA, TREM-1 protein, and sTREM-1 in 0.5 μg·mL⁻¹ Pg-LPS-stimulated macrophages were positively correlated with one another (r=1, P<0.05), but no statistically significant correlation was found in the expression of TNF-α. The positive correlation between sTREM-1 and TNF-α expressions was detected when macrophages were stimulated by 1.0 μg·mL⁻¹ Pg-LPS (r=1, P<0.05).
Conclusions: The expression of TREM-1 mRNA, TREM-1 protein, and sTREM-1 in the culture supernatant in Pg-LPS-stimulated macrophages was significantly upregulated on the basis of the concentration of Pg-LPS; moreover, their upregulation was positively correlated with one another. The expression of TNF-α in the supernatant of cell culture was also upregulated and was positively correlated with the expression of sTREM-1 at the group of high Pg-LPS concentration (1.0 μg·mL⁻¹). Results reveal that TREM-1, which has been realized as a proinflammatory receptor protein, can promote the development of periodontitis by regulating the expression of TNF-α in macrophages.
目的 探索牙龈卟啉单胞菌脂多糖（Pg-LPS）刺激的巨噬细胞中髓样细胞触发受体-1（TREM-1）、可溶性TREM-1（sTREM-1）及肿瘤坏死因子-α（TNF-α）的表达，探讨TREM-1与牙周炎发病机制的可能关联。方法 使用豆蔻酰佛波醇乙酯诱导人单核细胞系细胞THP-1分化为巨噬细胞，分别予以0（空白对照）、0.5、1.0 μg·mL⁻¹的Pg-LPS刺激，并根据不同时间分组：孵育4、6、12、24 h组。实时定量聚合酶链反应检测巨噬细胞中TREM-1 mRNA的表达，Western blot分析TREM-1蛋白表达的差异，细胞免疫荧光染色检测TREM-1在巨噬细胞中的表达部位，并通过酶联免疫吸附试验对细胞培养液中的sTREM-1及TNF-α进行检测。结果 与空白对照组相比，Pg-LPS刺激巨噬细胞后TREM-1 mRNA、TREM-1蛋白及细胞培养上清液中的sTREM-1表达均显著增强（P<0.05）；1.0 μg·mL⁻¹ Pg-LPS组TREM-1 mRNA、TREM-1蛋白及细胞培养上清液中的sTREM-1表达量高于0.5 μg·mL⁻¹组，且分别从6、4、4 h时间点开始差异具有统计学意义（P<0.05）；细胞免疫荧光染色结果显示，Pg-LPS（1.0 μg·mL⁻¹）刺激巨噬细胞24 h后，可见TREM-1蛋白呈阳性染色，且TREM-1的染色部位主要位于巨噬细胞的细胞膜区域；Pg-LPS刺激巨噬细胞后TNF-α的分泌水平升高（P<0.05），其中1.0 μg·mL⁻¹ Pg-LPS组表达量高于0.5 μg·mL⁻¹组，且从12 h开始差异具有统计学意义（P<0.05）；0.5 μg·mL⁻¹ Pg-LPS刺激巨噬细胞后TREM-1 mRNA、TREM-1蛋白、sTREM-1间表达呈正相关（r=1，P<0.05），但与TNF-α表达不存在相关性；在1.0 μg·mL⁻¹ Pg-LPS刺激巨噬细胞后检测到了具有统计学意义的sTREM-1与TNF-α表达的正相关性（r=1，P<0.05）。结论 巨噬细胞受到Pg-LPS刺激后可出现Pg-LPS浓度依赖性的TREM-1 mRNA、TREM-1蛋白及细胞培养上清液中的sTREM-1表达上调，且能观测到三者之间表达的正相关性；同时细胞培养上清液中的TNF-α也出现Pg-LPS浓度依赖性的表达上调，在高浓度Pg-LPS（1.0 μg·mL⁻¹）刺激下与sTREM-1表达量呈正相关。该结果提示，TREM-1作为促炎受体蛋白，可能通过调节巨噬细胞TNF-α的表达来实现牙周炎发展的促进作用。.
Keywords: Porphyromonas gingivalis; lipopolysaccharide; macrophage; periodontitis; triggering receptors expressed by myeloid cells-1; tumor necrosis factor-α.