Abstract:

Objective To explore the mechanism of autophagy in idiopathic pulmonary fibrosis（IPF）and the impact of autophagy modifications on EMT regulation，as well as to offer an experimental foundation for further IPF pathogenesis research. Methods Different concentrations of transforming growth factor⁃β（TGF⁃β1）was used to induce human alveolar epithelial cells A549 cells，and changes of cell morphology were observed by microscope. CCK8 assay detected the effects of different concentrations of TGF ⁃ β1 on A549 cell viability. Western blot mea⁃ sured the effects of different concentrations of TGF⁃β1 on the expression of autophagy and EMT⁃related proteins， and the optimal induction concentration and time were selected to establish the model of IPF in vitro.Transmission electron microscopy（TEM）was used to observe the effects of 5 ng/mL TGF⁃β1 on autophagy of A549 cells. RAPA， an autophagy agonist，was used to study the effect of autophagy on EMT. A549 cells were divided into four groups：control，model（5 ng/mL TGF⁃β1），RAPA（10 nmol/L RAPA），and TGF⁃β1+RAPA（5 ng/mL TGF⁃β1 +10nmol/L RAPA）. Western blot and Immunofluorescence were used to detect the expression of autophagy and EMT⁃ related proteins after the treatment of rapamycin（RAPA），an autophagy agonist. Results Compared with the control group，the cell morphology of the experimental group changed from irregular polygon to spindle shape and the cells were scattered，showing the typical appearance of EMT. TGF⁃β1 had no significant cytotoxic effect on A549 cells in the experimental concentrations range（P > 0.05）. Compared with the control group，the protein expressions of Beclin1 and E ⁃cadherin in the TGF ⁃β1 concentration gradient induced groups were decreased，while the protein expressions of p62 and N⁃cadherin were increased（P < 0.05）. TEM showed that compared with the control group， the cell morphology changed to spindle shape after TGF⁃β1 treatment，and the number of autolysosomes decreased significantly. Western blot and Immunofluorescence data showed that in RAPA group，the expressions of LC3， Beclin1，and E⁃cadherin were up⁃regulated，while the expressions of p62，N⁃cadherin，and Fibronectin were down⁃ regulated（P < 0.05）. Conclusion TGF⁃β1 can suppress the level of autophagy and promote the occurrence of EMT in A549 cells，whereas RAPA enhanced autophagy may alleviate pulmonary fibrosis by inhibiting EMT process.

Key words:

idiopathic pulmonary fibrosis, autophagy, transforming growth factor ? β1, RAPA, epithelial?mesenchymal transition