The role of γ-secretase inhibitor in pulmonary fibrosis epithelial-mesenchymal transition

doi:10.11958/20220307

Abstract

Abstract:

Objective To investigate the formation of pulmonary fibrosis epithelial-interstitial transformation (EMT) induced by transforming growth factor TGF-β1 in vitro and to detect the effect of Notch signal specific inhibitor DAPT on this transformation and its possible mechanism. Methods A549 cells were divided into the control group (cultured in RPMI 1640 complete medium), the TGF-β1 group (containing 10 μg/L TGF-β1 in RPMI 1640 medium), the TGF-β1+DAPT group (containing 10 μg/L TGF-β1 and 2 μmol DAPT in RPMI 1640 medium) and the DAPT group (only 2 μmol DAPT was cultured in RPMI 1640 medium). The cell morphology was observed by inverted microscope. The mRNA expression of alveolar epithelial cell specific protein levels of E-cadherin and interstitial cell specific protein α-actin (α-SMA) were detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR), and the expression levels of E-cadherin and α-SMA were detected by Western blot assay. Results The cells in the control group were polygonal, irregular and closely linked, while those in the TGF-β1 group were spindle-shaped with reduced intercellular connections. In the TGF-β1 + DAPT group, only a few cells showed spindle shape, and most cell morphology was similar to that of the control group. The cell morphology of the DAPT group was similar to that of the control group. Compared with the control group, expression levels of α-SMA protein and mRNA increased significantly in the TGF-β1 group (P<0.05), while the expression of E-cadherin and its mRNA decreased significantly (P<0.05). Compared with the TGF-β1, the expression of α-SMA and mRNA decreased significantly in the TGF-β1 and DAPT group, while the expression of E-cadherin and mRNA increased significantly (P<0.05). There were no significant differences in expression levels of α-SMA and E-cadherin protein and mRNA between the DAPT group and the control group (P>0.05). Conclusion TGF-β1 can induce lung epithelial interstitial transformation, while Notch signal inhibitor DAPT can block, partially or completely reverse this transformation process.

Key words: transforming growth factor-β1, pulmonary fibrosis, epithelial-mesenchymal transition, γ-secretase inhibitor

CLC Number:

R563.9

Figures/Tables 5

References 19

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基因名称	引物序列（5′→3′）	产物大小（bp）
β-actin	上游：AGCGAGCATCCCCCAAAGTT	285
β-actin	下游：GGGCACGAAGGCTCATCATT	285
α-SMA	上游：TCATGGTCGGTATGGGTCAG	150
α-SMA	下游：CGTTGTAGAAGGTGTGGTGC	150
E-cadherin	上游：CGTAGCAGTGACGAATGTGG	175
E-cadherin	下游：CTGGGCAGTGTAGGATGT	175

基因名称	引物序列（5′→3′）	产物大小（bp）
β-actin	上游：AGCGAGCATCCCCCAAAGTT	285
β-actin	下游：GGGCACGAAGGCTCATCATT	285
α-SMA	上游：TCATGGTCGGTATGGGTCAG	150
α-SMA	下游：CGTTGTAGAAGGTGTGGTGC	150
E-cadherin	上游：CGTAGCAGTGACGAATGTGG	175
E-cadherin	下游：CTGGGCAGTGTAGGATGT	175

组别	E-钙黏合素	α-SMA
对照组	0.997±0.149	1.045±0.174
TGF-β1组	0.044±0.036^a	1.863±0.095^a
TGF-β1+DAPT组	0.802±0.093^b	1.369±0.149^b
DAPT组	1.026±0.168^b	1.075±0.065^b
F	14.254^＊	26.142^＊

组别	E-钙黏合素	α-SMA
对照组	0.997±0.149	1.045±0.174
TGF-β1组	0.044±0.036^a	1.863±0.095^a
TGF-β1+DAPT组	0.802±0.093^b	1.369±0.149^b
DAPT组	1.026±0.168^b	1.075±0.065^b
F	14.254^＊	26.142^＊

组别	E-钙黏合素	α-SMA
对照组	0.551±0.052	0.357±0.037
TGF-β1组	0.286±0.023^a	0.647±0.042^a
TGF-β1+DAPT组	0.497±0.057^b	0.484±0.049^ab
DAPT组	0.590±0.059^b	0.380±0.040^b
F	22.121^＊	29.753^＊