Effect of autophagy on EMT in alveolar epithelial cells induced by pyocyanin

Abstract

Objective Epithelial-Mesenchymal Transition (EMT) plays an important role in the occurrence and development of pulmonary fibrosis, which can cause severe cell damage. Autophagy is a process of maintaining cell balance through degradation and reuse of damaged organelles, proteins, invading pathogens and other substances. Autophagy can protect cells to a certain extent, while uncontrolled and defective autophagy will further aggravate cell damage. At present, it has been reported that autophagy can reduce the level of apoptosis and mesenchymal transformation caused by certain pathogenic factors. Therefore, the aim of this study was to investigate the effect of autophagy on EMT in alveolar type II epithelial cells stimulated by pyocyanin (PCN). Methods After stimulating human alveolar type II epithelial cell line A549 with different concentrations of PCN in vitro, EMT changes were detected by Western blot and Real-time PCR, and autophagy levels were detected by immunofluorescence and Western blot. Then autophagy was inhibited and EMT marker protein levels and nucleic acid levels were detected. Finally, the changes of TGF-β/Smad pathway markers were detected after the addition of autophagy inhibitor 3-MA. Result After stimulating A549 cells with PCN (5ug/ml, 10ug/ml, 25ug/ml, 50ug/ml) for 24h, The expression levels of epithelial marker E-cadherin protein and mRNA were significantly decreased compared with the control group, and the expression levels of mesenchymal marker α-SMA protein and mRNA were increased compared with the control group (p < 0.05), suggesting that EMT phenomenon occurred after PCN stimulated A549 cells. At the same time, the expression of autophagy marker LC3 in protein level and immunofluorescence level was significantly higher than that in control group (p < 0.05), suggesting that PCN induced autophagy in A549 cells. After inhibition of autophagy with 3MA, the protein and nucleic acid expression levels of autophagy marker LC3 and epithelial marker E-cadherin were significantly decreased compared with control group, while the protein and nucleic acid expression levels of mesenchymal marker α-SMA were increased compared with control group (p < 0.05), indicating that the EMT phenomenon was enhanced after inhibition of autophagy. Further study showed that TGF-β1 nucleic acid level and p-Smad2/3 protein expression level in the addition of autophagy inhibitor 3MA group were significantly increased compared with the control group and PCN group (p < 0.05), indicating that inhibition of autophagy may enhance EMT by affecting TGF-β/Smad pathway. Conclusion PCN can induce EMT and autophagy in alveolar epithelial cells, and autophagy can inhibit the further development of EMT, which may inhibit the occurrence of EMT by reducing the activity of TGF-β/Smad pathway. These results suggest that autophagy may prevent pulmonary fibrosis.