Abstract
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 in vitro 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.