Department of Oral Microbiology, Faculty of Dentistry, Mahidol University, Bangkok, 10400, Thailand.
Department of Microbiology, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand.
Inflamm Res. 2018 Sep;67(9):723-726. doi: 10.1007/s00011-018-1168-1. Epub 2018 Jul 6.
The aim of this study is to investigate the involvement of TLR9 in the regulation of iNOS expression and nitric oxide (NO) production in Porphyromonas gingivalis LPS-treated mouse macrophages.
Mouse macrophage cell line (RAW264.7) was transfected with siRNAs against TLR9 and then stimulated with P. gingivalis LPS. At indicated time points, the activated cells were lysed. Gene and protein expression of iNOS were determined by RT-PCR and immunoblotting, respectively. The level of nitric oxide (NO) production in the supernatant of the activated cells was determined by Griess reaction assay.
Depletion of TLR9 in mouse macrophages demonstrated the markedly decreased iNOS gene and protein expression by P. gingivalis LPS compared to those of the wild-type or control siRNA transfected cells. In consistent with these results, the level of NO secretion was also significantly diminished in TLR9-depleted cells after challenged with P. gingivalis LPS. These results indicate that TLR9 involves in the regulation of the iNOS expression and the NO secretion in P. gingivalis LPS-treated macrophages.
本研究旨在探讨 TLR9 是否参与调节牙龈卟啉单胞菌脂多糖（LPS）诱导的小鼠巨噬细胞中诱导型一氧化氮合酶（iNOS）的表达和一氧化氮（NO）的产生。
用 TLR9 的 siRNA 转染小鼠巨噬细胞系（RAW264.7），然后用 P. gingivalis LPS 刺激。在指定的时间点，裂解活化的细胞。通过 RT-PCR 和免疫印迹分别检测 iNOS 的基因和蛋白表达。通过 Gries 反应测定活化细胞上清液中 NO 产生的水平。
与野生型或对照 siRNA 转染细胞相比，用 P. gingivalis LPS 处理 TLR9 耗尽的小鼠巨噬细胞后，iNOS 基因和蛋白表达明显降低。与这些结果一致的是，在 TLR9 耗尽的细胞中用 P. gingivalis LPS 刺激后，NO 的分泌水平也显著降低。这些结果表明 TLR9 参与了 P. gingivalis LPS 处理的巨噬细胞中 iNOS 的表达和 NO 分泌的调节。
