Mao Yajun, Feng Hong
Rehabilitation Medicine Department, The First Affiliated Hospital, Zhejiang Chinese Medical University, Hangzhou, Zhejiang 310006, P.R. China.
Respiratory Department, The Fourth Hospital of Baotou City, Baotou, Inner Mongolia Autonomous Region 014030, P.R. China.
Exp Ther Med. 2022 May;23(5):357. doi: 10.3892/etm.2022.11284. Epub 2022 Mar 29.
Vitamin D3 supplementation has been previously reported to inhibit the occurrence and development of chronic obstructive pulmonary disease (COPD). However, the underlying mechanism remains unclear. Epithelial-mesenchymal transition (EMT) and fibrogenesis have been associated with the development of COPD. The aim of the present study was to investigate the potential effects and mechanism of vitamin D3 in an model of cigarette smoke (CS)-induced EMT and fibrosis, with specific focus on the role of club cell protein 16 (CC16). CS extract (CSE) at different concentrations (5, 10 and 20%) was used to treat 16-HBE cells to induce EMT and fibrogenesis following which they were treated with vitamin D3. Subsequently, the 20% CSE group was selected for further experiments, where 16-HBE cells were divided into the following five groups: The control group; the CSE group; the low-dose vitamin D3 group (250 nM); the medium-dose vitamin D3 group (500 nM); and the high-dose vitamin D3 group (1,000 nM). Western blot analysis was used to detect the protein expression levels of the EMT-related proteins E-cadherin, N-cadherin, Slug and α-SMA, fibrogenesis-related proteins collagen Ⅳ and fibronectin 1, proteins involved in the TGF-β1/SMAD3 signaling pathway and CC16. Immunofluorescence was used to measure the protein expression levels of E-cadherin, N-cadherin and collagen Ⅳ. Specific CC16 knockdown was performed using short hairpin RNA transfection to investigate the role of CC16. The results of the present study found that vitamin D3 could increase the protein expression level of CC16 to inhibit the activation of the TGF-β1/SMAD3 signaling pathway; thereby reducing the 20% increase in CSE-induced EMT- and fibrogenesis-related protein expression levels. Following CC16 knockdown, the inhibitory effects of vitamin D3 on EMT- and fibrogenesis-related protein expression were partially reversed. To conclude, these results suggest that vitamin D3 can inhibit the protein expression levels of EMT- and fibrogenesis-related proteins induced by CSE, at least partially through the function of CC16. These findings are expected to provide novel theoretical foundations and ideas for the pathogenesis and treatment of COPD.
先前有报道称补充维生素D3可抑制慢性阻塞性肺疾病（COPD）的发生和发展。然而，其潜在机制仍不清楚。上皮-间质转化（EMT）和纤维化与COPD的发展有关。本研究的目的是在香烟烟雾（CS）诱导的EMT和纤维化模型中研究维生素D3的潜在作用和机制，特别关注俱乐部细胞蛋白16（CC16）的作用。使用不同浓度（5%、10%和20%）的CS提取物（CSE）处理16-HBE细胞以诱导EMT和纤维化，随后用维生素D3处理。随后，选择20% CSE组进行进一步实验，将16-HBE细胞分为以下五组：对照组；CSE组；低剂量维生素D3组（250 nM）；中剂量维生素D3组（500 nM）；高剂量维生素D3组（1000 nM）。采用蛋白质印迹法检测EMT相关蛋白E-钙黏蛋白、N-钙黏蛋白、锌指蛋白Slug和α-平滑肌肌动蛋白（α-SMA）、纤维化相关蛋白Ⅳ型胶原和纤连蛋白1、参与转化生长因子-β1（TGF-β1）/SMAD3信号通路的蛋白以及CC16的蛋白表达水平。采用免疫荧光法检测E-钙黏蛋白、N-钙黏蛋白和Ⅳ型胶原的蛋白表达水平。使用短发夹RNA转染进行特异性CC16敲低，以研究CC16的作用。本研究结果发现，维生素D3可增加CC16的蛋白表达水平，抑制TGF-β1/SMAD3信号通路的激活；从而降低CSE诱导的EMT和纤维化相关蛋白表达水平升高20%。CC16敲低后，维生素D3对EMT和纤维化相关蛋白表达的抑制作用部分逆转。总之，这些结果表明，维生素D3可抑制CSE诱导的EMT和纤维化相关蛋白的表达水平，至少部分是通过CC16的功能实现的。这些发现有望为COPD的发病机制和治疗提供新的理论基础和思路。
