Department of Otorhinolaryngology-Head and Neck Surgery, Guro Hospital, Korea University College of Medicine, Seoul, South Korea.
Int Arch Allergy Immunol. 2012;159(3):278-86. doi: 10.1159/000337460. Epub 2012 Jun 21.
Myofibroblasts are detected in nasal polyps and are involved in nasal polyp formation by inducing extracellular matrix accumulation. Reactive oxygen species (ROS) are released during the differentiation of fibroblasts to myofibroblasts. The purpose of this study was to investigate ROS production and nicotinamide adenine dinucleotide phosphate oxidase (NOX) expression in nasal polyp-derived fibroblasts (NPDFs) and to evaluate whether ROS from NOX mediates transforming growth factor (TGF)-β1-induced production of alpha smooth-muscle actin (α-SMA) and collagen production.
NPDFs were incubated and treated with TGF-β1. The mRNA expression of NOXs, α-SMA, and collagen type I and IV was determined by reverse transcription-polymerase chain reaction, and the expression of α-SMA protein was determined by immunofluorescence microscopy. The amount of total soluble collagen production was analyzed by the SirCol assay. The ROS generation of cells was investigated using the 2',7'-dichlorfluorescein-diacetate. The fluorescence was captured by fluorescent microscope and measured using a fluorometer.
Stimulation with TGF-β1 increased ROS production by NPDFs compared with NPDFs not treated with TGF-β1. Stimulation with TGF-β1 increased the expression of NOX4 mRNA most potently among various Nox enzymes. siNOX4 was able to decrease the level of ROS production. Myofibroblast differentiation and the production of collagen in NPDFs were prevented by inhibition of ROS generation with diphenyliodonium, N-acetylcysteine, ebselen, and siNox4.
This study showed that NOX4 and ROS have a role in myofibroblast differentiation and collagen production of TGF-β1-induced NPDFs and that these processes are inhibited by the elimination of ROS.
肌成纤维细胞存在于鼻息肉中，并通过诱导细胞外基质积累参与鼻息肉的形成。在成纤维细胞向肌成纤维细胞分化的过程中会释放活性氧（ROS）。本研究旨在探讨鼻息肉衍生成纤维细胞（NPDFs）中 ROS 的产生和烟酰胺腺嘌呤二核苷酸磷酸氧化酶（NOX）的表达，并评估 NOX 产生的 ROS 是否介导转化生长因子（TGF）-β1 诱导的α平滑肌肌动蛋白（α-SMA）和胶原产生。
孵育并处理 NPDFs 使其与 TGF-β1 接触。通过逆转录聚合酶链反应测定 NOXs、α-SMA 和胶原 I 型和 IV 型的 mRNA 表达，通过免疫荧光显微镜测定α-SMA 蛋白的表达。通过 SirCol 分析测定总可溶性胶原产生量。通过 2',7'-二氯二氟荧光素二乙酸酯研究细胞内 ROS 的产生。通过荧光显微镜捕获荧光并用荧光计进行测量。
与未用 TGF-β1 处理的 NPDFs 相比，TGF-β1 刺激可增加 NPDFs 中 ROS 的产生。与各种 Nox 酶相比，TGF-β1 刺激最能增加 NOX4 mRNA 的表达。siNOX4 能够降低 ROS 产生水平。用二苯基碘鎓、N-乙酰半胱氨酸、依布硒啉和 siNOX4 抑制 ROS 生成可防止 NPDFs 中肌成纤维细胞分化和胶原产生。
本研究表明，NOX4 和 ROS 在 TGF-β1 诱导的 NPDFs 中肌成纤维细胞分化和胶原产生中起作用，并且这些过程可通过消除 ROS 而受到抑制。
