Department of Periodontology, Institute of Dentistry, University of Turku, Turku, Finland.
Department of Chemistry, Purdue Institute for Drug Discovery, Purdue University, West Lafayette, IN, USA.
J Periodontal Res. 2018 Jun;53(3):414-421. doi: 10.1111/jre.12528. Epub 2018 Jan 17.
Quorum-sensing molecules regulate the behavior of bacteria within biofilms and at the same time elicit an immune response in host tissues. Our aim was to investigate the regulatory role of dihydroxy-2,3-pentanedione (DPD), the precursor of universal autoinducer-2 (AI-2), and its analogs (ethyl-DPD, butyl-DPD and isobutyl-DPD) in the integrity of gingival epithelial cells.
Human gingival keratinocytes were incubated with four concentrations (10 μmol L , 1 μmol L , 100 nmol L and 10 nmol L ) of DPD and its analogs for 24 hours. The numbers of viable cells were determined using a proliferation kit, matrix metalloproteinase (MMP)-2 and -9 activities were determined by gelatin zymography, and expression of occludin protein and occludin mRNA were determined by western blotting and RT-qPCR, respectively.
Increased cell proliferation was observed in gingival keratinocytes incubated with 100 nmol L of butyl-DPD. MMP-9 activity was elevated in cells incubated with 10 μmol L of ethyl-DPD. On the other hand, MMP-2 activity did not show any significant change when gingival keratinocytes were incubated with or without DPD or analogs. Western blot analyses demonstrated five forms (105, 61, 52.2, 44 and 37 kDa) of occludin. Incubation with 1 μmol L and 100 nmol L of DPD and with 10 nmol L of ethyl-DPD increased dimeric (105 kDa) forms of occludin, while incubation with 100 nmol L of isobutyl-DPD increased monomeric (61 kDa) forms. DPD and ethyl-DPD decreased, and 100 nmol L of isobutyl-DPD and 10 nmol L of butyl-DPD increased, the monomeric (52.2 kDa and 44 kDa) forms of occludin, whereas ethyl-DPD decreased and isobutyl-DPD increased, the low-molecular-weight (37 kDa) forms. According to RT-qPCR analysis, the exposure of gingival keratinocytes to 10 μmol L of isobutyl-DPD up-regulated expression of occludin.
The results indicate that isobutyl-DPD has the potential to enhance the integrity of the epithelium by stimulating the formation of occluding, without affecting the proliferation or gelatinolytic enzyme activities of the exposed cells. The modulatory effect of an AI-2 analog on the epithelial cell response is shown for the first time.
群体感应分子调节生物膜内细菌的行为，同时在宿主组织中引发免疫反应。我们的目的是研究二羟丙酮（DPD），即通用自诱导物-2（AI-2）的前体及其类似物（乙基-DPD、丁基-DPD 和异丁基-DPD）在牙龈上皮细胞完整性中的调节作用。
将人牙龈角质形成细胞在四种浓度（10 μmol L、1 μmol L、100 nmol L 和 10 nmol L）的 DPD 及其类似物中孵育 24 小时。使用增殖试剂盒测定活细胞数量，通过明胶酶谱法测定基质金属蛋白酶（MMP）-2 和 -9 的活性，通过 Western blot 和 RT-qPCR 分别测定封闭蛋白和封闭蛋白 mRNA 的表达。
在孵育 100 nmol L 丁基-DPD 的牙龈角质形成细胞中观察到细胞增殖增加。在孵育 10 μmol L 乙基-DPD 的细胞中 MMP-9 活性升高。另一方面，当牙龈角质形成细胞孵育有或没有 DPD 或类似物时，MMP-2 活性没有显示出任何显著变化。Western blot 分析显示封闭蛋白有五种形式（105、61、52.2、44 和 37 kDa）。孵育 1 μmol L 和 100 nmol L 的 DPD 以及 10 nmol L 的乙基-DPD 增加了二聚体（105 kDa）形式的封闭蛋白，而孵育 100 nmol L 的异丁基-DPD 增加了单体（61 kDa）形式。DPD 和乙基-DPD 减少，100 nmol L 的异丁基-DPD 和 10 nmol L 的丁基-DPD 增加了单体（52.2 kDa 和 44 kDa）形式的封闭蛋白，而乙基-DPD 减少，异丁基-DPD 增加了低分子量（37 kDa）形式。根据 RT-qPCR 分析，10 μmol L 的异丁基-DPD 暴露于牙龈角质形成细胞中，上调了封闭蛋白的表达。
结果表明，异丁基-DPD 通过刺激封闭蛋白的形成，有可能增强上皮的完整性，而不会影响暴露细胞的增殖或明胶酶活性。首次显示了 AI-2 类似物对上皮细胞反应的调节作用。
