Camphorquinone alters the expression of extracellular proteases in a 3D co-culture model of the oral mucosa.
Camphorquinone alters the expression of extracellular proteases in a 3D co-culture model of the oral mucosa.
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DOI：
10.1016/j.dental.2020.11.005
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发表时间：
2020-11
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通讯作者：
Renke Perduns;J. Volk;M. Plum;André Jochums;F. Gutzki;V. Kaever;W. Geurtsen
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文献类型：
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作者：
Renke Perduns;J. Volk;M. Plum;André Jochums;F. Gutzki;V. Kaever;W. Geurtsen
我们的研究目的是确定CQ对口腔粘膜3D共培养模型（3DCCM）中抗氧化蛋白和细胞外蛋白酶表达的影响，并分析CQ在3D-CCMs. Methods 3D-CCMs中的分布和稳定性，3D-CCMs由融合的角质形成细胞（OKF 6/TERT 2）在胶原蛋白中的人牙龈成纤维细胞（HGFs）顶部的细胞培养插入物上组成。通过在两个时间点向细胞培养插入物中加入CQ进行处理，浓度下降。使用质谱法分析OKF 6/TERT 2层上方和下方的CQ浓度。通过qRT-PCR和western blot分析抗氧化基因的表达。结果GC/MS分析表明，CQ在模型中分布均匀。血红素氧合酶1、NAD（P）H醌脱氢酶1（NQO 1）和超氧化物歧化酶1在mRNA和蛋白水平上被诱导。在HGFs中，只有NQO 1的转录被诱导。初步处理后72 h，OKF 6/TERT 2细胞中胞外蛋白酶的转录主要增加。在角质形成细胞层上方的细胞培养上清液中，25种分析的细胞外蛋白酶中有10种的量和6种在角质形成细胞层下方的细胞外蛋白酶的量被CQ调节。CQ最初诱导细胞防御机制对抗氧化应激，并改变细胞外蛋白酶的表达。我们假设这两个过程之间存在关系。
ObjectiveObjective of our investigation was to determine the influence of CQ on the expression of antioxidant proteins and extracellular proteases in a 3D co-culture model (3DCCM) of the oral mucosa and to analyze the distribution and stability of CQ within 3D-CCMs.Methods3D-CCMs consist of confluent keratinocytes (OKF6/TERT2) on cell culture inserts on top of human gingival fibroblasts (HGFs) in collagen. The treatment was carried out by adding CQ to the cell culture inserts at two time points with declining concentrations. Mass spectrometry was used to analyze the CQ concentration above and underneath the OKF6/TERT2-layer. The expression of antioxidant genes was analyzed by qRT-PCR and western blot. The regulation of extracellular proteases from different families was analyzed by qRT-PCR and Proteome Profiler arrays.ResultsGC/MS analysis showed that CQ was evenly distributed within the model. Heme oxygenase-1, NAD(P)H quinone dehydrogenase 1 (NQO1), and superoxide dismutase 1 were induced on the mRNA and protein level in OKF6/TERT2 cells. In HGFs, only the transcription of NQO1 was induced. The transcription of extracellular proteases was increased mainly in OKF6/TERT2 cells 72 h after the initial treatment. The quantity of ten out of 25 analyzed extracellular proteases in the cell culture supernatant above and six underneath the keratinocyte-layer were modulated by CQ.SignificanceDespite its high reactivity, CQ is able to penetrate a dense keratinocyte-layer, presumably across plasma membranes. CQ initially induced the cellular defense machinery against oxidative stress and altered the expression of extracellular proteases. We assume a relationship between both processes.