Objective

Objective 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.

Methods

3D-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.

Results

GC/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.

Significance

Despite 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.

中文翻译：

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樟脑醌改变口腔黏膜3D共培养模型中细胞外蛋白酶的表达

目的

我们的研究目的是确定CQ对口腔粘膜3D共培养模型（3DCCM）中抗氧化剂蛋白和细胞外蛋白酶表达的影响，并分析CQ在3D-CCM中的分布和稳定性。

方法

3D-CCM由胶原蛋白在人牙龈成纤维细胞（HGF）顶部的细胞培养插入物上的融合角质形成细胞（OKF6 / TERT2）组成。通过在浓度下降的两个时间点向细胞培养插入物中添加CQ来进行处理。质谱用于分析OKF6 / TERT2层上方和下方的CQ浓度。通过qRT-PCR和western blot分析抗氧化剂基因的表达。通过qRT-PCR和Proteome Profiler阵列分析了来自不同家族的细胞外蛋白酶的调控。

结果

GC / MS分析表明，CQ在模型中均匀分布。在OKF6 / TERT2细胞的mRNA和蛋白质水平上诱导了血红素加氧酶-1，NAD（P）H醌脱氢酶1（NQO1）和超氧化物歧化酶1。在HGF中，仅诱导NQO1的转录。初始处理后72小时，细胞外蛋白酶的转录主要在OKF6 / TERT2细胞中增加。CQ调节角质形成细胞层上方和下方的细胞培养上清液中25种分析的细胞外蛋白酶中的10种。

意义

尽管CQ具有高反应活性，但它能够穿透密集的角质形成细胞层，大概穿过质膜。CQ最初诱导细胞抵御氧化应激的防御机制，并改变了细胞外蛋白酶的表达。我们假设这两个过程之间存在关系。