Shin Jae-Min, Kang Ju-Hyung, Lee Seoung-Ae, Park Il-Ho, Lee Heung-Man
Department of Otorhinolaryngology-Head and Neck Surgery, Seoul, South Korea.
Am J Rhinol Allergy. 2017 Mar 1;31(2):71-77. doi: 10.2500/ajra.2017.31.4410.
Doxycycline has antibacterial and anti-inflammatory effects, and it also suppresses collagen biosynthesis. This study aimed to confirm the effects and mechanism of doxycycline on transforming growth factor (TGF) beta 1 induced epithelial-mesenchymal transition and cell migration in A549 and primary nasal epithelial cells.
A 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide assay and phalloidin-fluorescein isothiocyanate staining were used to evaluate cytotoxicity and cellular morphologic changes. Western blot and immunofluorescence staining were used to determine the expression levels of E-cadherin, vimentin, alpha-smooth muscle actin, fibronectin, phosphorylated Smad2/3, and mitogen-activated protein kinases. Scratch and transwell migration assays were used to assess cellular migration ability.
Doxycycline (0-10 μg/mL) had no significant cytotoxic effects in A549 and primary nasal epithelial cells. Increased expression of mesenchymal markers, including vimentin, alpha-smooth muscle actin, and fibronectin in TGF beta 1 induced A549 cells were downregulated by doxycycline treatment. In contrast, E-cadherin expression was upregulated in TGF beta 1 induced A549 cells. An in vitro cell migration assay showed that doxycycline also inhibited the ability of TGF beta 1 induced migration. Doxycycline treatment suppressed the activation of Smad2/3 and p38, whereas its inhibitory effects were similar to each element-specific inhibitor in A549 and primary nasal epithelial cells.
Doxycycline inhibited TGF beta 1 induced epithelial-to-mesenchymal transition and migration by targeting Smad2/3 and p38 signal pathways in respiratory epithelial cells.
强力霉素具有抗菌和抗炎作用，还能抑制胶原蛋白的生物合成。本研究旨在证实强力霉素对转化生长因子（TGF）β1诱导的A549细胞和原代鼻上皮细胞上皮-间质转化及细胞迁移的作用和机制。
采用3-（4,5-二甲基噻唑-2-基）-2,5-二苯基溴化四氮唑蓝法和鬼笔环肽-异硫氰酸荧光素染色评估细胞毒性和细胞形态变化。采用蛋白质免疫印迹法和免疫荧光染色法测定E-钙黏蛋白、波形蛋白、α-平滑肌肌动蛋白、纤连蛋白、磷酸化Smad2/3和丝裂原活化蛋白激酶的表达水平。采用划痕实验和Transwell迁移实验评估细胞迁移能力。
强力霉素（0-10μg/mL）对A549细胞和原代鼻上皮细胞无明显细胞毒性作用。强力霉素处理下调了TGFβ1诱导的A549细胞中波形蛋白、α-平滑肌肌动蛋白和纤连蛋白等间充质标志物的表达增加。相反，TGFβ1诱导的A549细胞中E-钙黏蛋白表达上调。体外细胞迁移实验表明，强力霉素也抑制了TGFβ1诱导的迁移能力。强力霉素处理抑制了Smad2/3和p38的激活，而其抑制作用与A549细胞和原代鼻上皮细胞中各元素特异性抑制剂相似。
强力霉素通过靶向呼吸道上皮细胞中的Smad2/3和p38信号通路，抑制TGFβ1诱导的上皮-间质转化和迁移。
