Preliminary Study Of NOTCH Signaling Pathway In The Process Of Paraquat Induced Interstitial Fibrosis Of Lung Epithelial Cells

Objective:To investigate the changes of key signaling molecules of Notch cell signaling pathway in lung epithelial interstitial fibrosis induced by low dose of PQ(paraquat).Methods:The NICD-expressing lentiviral vector pCDH-nicd had been constructed and proved to up-regulate the NICD level in A549 cells.Normal A549 cells were randomly divided into 4 groups,A.control group(cells without any treatment),B.PQ group(cells treated with paraquat only),C.pCDH-nicd group(cells infected with lentivirus pCDH-nicd),and D.PQ+pCDH-nicd group(cells infected with lentivirus pCDH-nicd and treated with paraquat).Cell morphological changes were observed with phase contrast microscope.Cell viability was measured by MTT method,and the protein expression levels of E-cadherin and alpha-SMA,two key signaling molecules downstream of Notch pathway,were detected by Western-Blot.The difference between groups was analyzed by one-way analysis of variance.Differences were considered statistically significant when the P value was < 0.05.Results:The expression of NICD protein was increased significantly in A549 cells infected with lentivirus pCDH-nicd.Phase contrast microscope observation showed that cells in group A were mainly spindle shaped mixed with some apoptotic cells.In group B:the shape changed from spindle shape to irregular shape with significantly decreased cell survival rate.The cells in group C were similar with group B without any significantly difference.Cells in group D were similar to cells in group B and group C in morphology,and the cell survival rate was obviously lower than both group B and C,while they were all lower than that of group A.The results of Western-Blot showed that compared with in group A,the expression of E-cadherin in group B and group C were significantly decreased(P< 0.05).The expression of E-cadherin protein was significantly decreased in group D compared with group B and group C(P< 0.05),and there was no significant defference between group B and group C(P> 0.05).Compared with group A,the expression of α-SMA protein increased in group B and group C(P< 0.05),still the difference between the latter two groups was not significant(P > 0.05),but the expression of α-SMA protein further increased in group D(P>0.05).Conclusion:Both chronic exposure to PQ and up-regulated expression level of NICD in A549 cells can lead to the obvious morphological changes of A549 cells in EMT.Meanwhile,the expression level of E-cadherin and α-SMA,which are the key signaling molecules in the downstream of NOTCH pathway were changed.Both of them have synergistic effects in promoting the occurrence of EMT in A549,which indicates that Notch signaling pathway is involved in the process of PQ induced EMT in lung epithelial cells.