Department of Periodontics, Institute of Stomatology, School of Dentistry, Hospital of Stomatology, Tianjin Medical University, Tianjin, 300070, China,
Inflamm Res. 2015 Jun;64(6):441-51. doi: 10.1007/s00011-015-0824-y. Epub 2015 May 7.
Toll-like receptors (TLRs) pathway has been demonstrated to play an important role in periodontitis. However, the regulatory mechanism of microRNAs (miRNAs) on TLRs pathway is still unclear. Hence, this study is to explore the function of miRNA-146a in inflammatory reaction induced by Porphyromonas gingivalis lipopolysaccharide (LPS) in human periodontal ligament cells (hPDLCs).
Cells were treated with 1 or 10 μg/ml P. gingivalis LPS. The expression of TLR2, TLR4 and miRNA-146a were measured by real-time polymerase chain reaction (PCR). Enzyme-linked immunosorbent assay (ELISA) was applied to detect nuclear factor (NF)-κ B p65 nuclear activity, interleukin-1β (IL-1β), IL-6, IL-8 and tumor necrosis factor-α (TNF-α). To examine the underlying mechanisms, cells were exposed to anti-TLR2/4 mAb or miRNA-146a inhibitor/mimic and evaluated by real-time PCR and ELISA.
10 μg/ml P. gingivalis LPS increased the expressions of TLR2 (3.79 ± 0.31), TLR4 (2.21 ± 0.31), and miRNA-146a (4.91 ± 0.87), NF-κ B p65 nuclear activity (6.51 ± 0.77 fold) (p < 0.05). 1 μg/ml P. gingivalis LPS induced TLR2 (3.05 ± 0.23), miRNA-146a (3.66 ± 0.83) and NF-κ B p65 nuclear activity (4.06 ± 0.78 fold) (p < 0.05), except TLR4 (1.11 ± 0.30, p > 0.05). Also, cytokines production increased (p < 0.05). The up-regulation of miRNA-146a could be blocked by anti-TLR2/4 mAb (p < 0.05). After the blockage of miRNA-146a, TLR2, TLR4, NF-κ B p65 nuclear activity and proinflammatory cytokines increased. However, after application of miRNA-146a mimic, the levels of these indexes decreased obviously (p < 0.05).
MiRNA-146a functions as a negative feedback regulator via down-regulating proinflammatory cytokine secretion and blocking TLRs signaling pathway in hPDLCs after P. gingivalis LPS stimulation.
Toll 样受体（TLR）途径已被证明在牙周炎中发挥重要作用。然而，miRNAs（miRNA）对 TLR 途径的调节机制尚不清楚。因此，本研究旨在探讨 miRNA-146a 在牙龈卟啉单胞菌脂多糖（LPS）诱导的人牙周韧带细胞（hPDLC）炎症反应中的作用。
用 1 或 10μg/ml 牙龈卟啉单胞菌 LPS 处理细胞。实时聚合酶链反应（PCR）检测 TLR2、TLR4 和 miRNA-146a 的表达。酶联免疫吸附试验（ELISA）检测核因子（NF）-κB p65 核活性、白细胞介素-1β（IL-1β）、白细胞介素-6（IL-6）、白细胞介素-8（IL-8）和肿瘤坏死因子-α（TNF-α）。为了研究潜在的机制，用抗 TLR2/4 mAb 或 miRNA-146a 抑制剂/模拟物处理细胞，并通过实时 PCR 和 ELISA 进行评估。
10μg/ml 牙龈卟啉单胞菌 LPS 增加 TLR2（3.79±0.31）、TLR4（2.21±0.31）和 miRNA-146a（4.91±0.87）的表达，NF-κB p65 核活性（6.51±0.77 倍）（p<0.05）。1μg/ml 牙龈卟啉单胞菌 LPS 诱导 TLR2（3.05±0.23）、miRNA-146a（3.66±0.83）和 NF-κB p65 核活性（4.06±0.78 倍）（p<0.05），但 TLR4（1.11±0.30，p>0.05）。此外，细胞因子的产生也增加了（p<0.05）。miRNA-146a 的上调可被抗 TLR2/4 mAb 阻断（p<0.05）。阻断 miRNA-146a 后，TLR2、TLR4、NF-κB p65 核活性和促炎细胞因子增加。然而，应用 miRNA-146a 模拟物后，这些指标水平明显下降（p<0.05）。
在牙龈卟啉单胞菌 LPS 刺激后，miRNA-146a 通过下调促炎细胞因子的分泌并阻断 TLRs 信号通路，在 hPDLCs 中作为负反馈调节剂发挥作用。
