The Effect Of MiR-200s And ZEB1/2 Regulatory System In Early Pulmonary Fibrosis Caused By Lps-induced Acute Lung Injury

Objective: To detect the effect of miR-200b/c or ZEB1/2 on LPS-induced early pulmonary fibrosis and investigate the underlying mechanism.Methods: 1.Early pulmonary fibrosis caused by acute lung injury was built via a lipopolysaccharide three-hit regimen.The lung tissue sections were stained with HE and Masson staining and the lung injury score and fibrosis score were assessed.The expression profiles of miR-200 b,miR-200 c,ZEB1 mRNA and ZEB2 mRNA were detected by real-time PCR.Western blot was utilized to detect the expression levels of ZEB1,ZEB2,E-cadherin,Vimentin,?-SMA proteins in lung tissue.2.Lentiviral packaged miR-200 b and miR-200 c cDNA or ZEB1 and ZEB2 shRNA was intratracheally administrated into the lungs of C57bl/6 mouse.One day after genetransfer,animals received LPS injection according to the foregoing method.3.LPS(0.1mg/ml)or TGF-?(0.5ng/ml),or LPS combined with TGF-? stimulated RLE-6TN cells for 24 h and p38/MAPKs and TGF-?/Smad signaling pathways were further detected by Western-blot.Results: The collagen fibers deposited after LPS treatment and pulmonary fibrosis worsened gradually.With the aggravation of pulmonary fibrosis,miR-200 b and miR-200 c levels were declined.ZEB1/2 mRNA and protein levels were gradually increased in the process of pulmonary fibrosis.Besides,the expression of E-cadherin protein decreased,while Vimentin and ?-SMA protein levels increased gradually.After overexpressing miR-200 b or miR-200 c,their targets ZEB1 and ZEB2 mRNA and protein levels decreased significantly,while,ZEB1/2 shRNA increased miR-200 b and miR-200 c expression markedly.Both overexpression of miR-200b/c and down-regulation of ZEB1/2 could effectively alleviate lung injury and pulmonary fibrosis,reduced Vimentin and ?-SMA expressions as well as increased E-cadherin protein level significantly.LPS or TGF-? stimulation induced an increase of ZEB1 and ZEB2 protein expressions accompanied by upregulating mesenchymal markers Vimentin and ?-SMA as well as downregulating epithelial mark E-cadherin in RLE-6TN cells.Besides,phosph-p38 and Smad3 increased.LPS combined with TGF-? produced additive or synergistic effects on the EMT of RLE-6TN.Conclusion: The imbalance of miR-200b/c and ZEB1/2 expressions in local lung tissue,accompanied with the changes of epithelial marker E-cadherin as well as mesenchymal markers Vimentin and ?-SMA,is very important in the process of early pulmonary fibrosis caused by acute lung injury via LPS.miR-200b/c upregulation or ZEB1/2 downregulation exert a protective effect against LPS-induced epithelial mesenchymal transition mainly through inhibiting p38/MAPKs and TGF-?/Smad pathway activation.