Study of the common activating mechanism of apoptosis and epithelial-to-mesenchymal transition in alveolar type II epithelial cells.

Infection and severe trauma can result in acute lung injury/acute respiratory distress syndrome (ALI/ARDS) and eventually pulmonary fibrosis. Epithelial-to-mesenchymal transition (EMT) is related to pulmonary fibrosis. Our study found that pyocyanin (PCN) could promote apoptosis and EMT in alveolar type II epithelial A549 cells. We hypothesized that there might be a common mechanism related to both apoptosis and EMT in A549 cells. The aim of this study was to determine whether reactive oxygen species (ROS) induced by PCN is the common stimulus upstream of apoptosis and EMT as well as the relevant signalling pathways. A549 cells were challenged with PCN; ROS was then detected by immunofluorescence, and apoptosis was measured by flow cytometry. Caspases, EMT markers and the TGF-β/Smad pathway were assessed by Western blot, qPCR or ELISA. The results showed that PCN promoted ROS production, and the apoptosis rate was clearly increased. E-cadherin downregulation, vimentin and α-SMA upregulation in A549 cells, cleaved caspase-9 and caspase-3, TGF-β1 and activated Smad2/3 were also detected. Interestingly, the protein expression of cleaved caspase-3 and vimentin was highly positively correlated. Inhibition of ROS could partially reverse PCN-induced EMT and apoptosis in A549 cells, and EMT could also be reversed by TGF-β1 inhibitors. In conclusion, ROS may be a common activating mechanism of apoptosis and EMT in alveolar epithelial cells, during which the degree of apoptosis is positively related to EMT. ROS may induce alveolar epithelial cell apoptosis through the mitochondrial pathway or endoplasmic reticulum pathway. ROS activates TGF-β1, followed by SMADs, eventually inducing EMT.