| ObjectiveAlveolar epithelial cell senescence is one of the important mechanisms in the pathogenesis of idiopathic pulmonary fibrosis.It has been shown that alveolar epithelial cells in lung tissue of patients with pulmonary fibrosis have high expression of PAI-1 and loss of PTEN expression.PAI-1 inhibitors have also been shown to play a protective role in bleomycin-induced senescence in rat alveolar epithelial cells.This study intends to study the role of PAI-1 inhibitors in senile alveolar epithelial cells through cytological experiments and initially explore its mechanism,providing preliminary clues for the clinical application of PAI-1 inhibitors in the treatment of idiopathic pulmonary fibrosisMaterials and methodsPart ?:Expression of PTEN and PAI-1 in the lung tissues of IPF patients and two senile models of alveolar epithelial cellsIt is planned to verify the expression level and distribution of PTEN and PAI-1 in lung tissue of patients with IPF by Western blot and immunofluorescence detection of lung tissue samples from patients with IPF transplantation.The senescence models of type ? alveolar epithelial cells(A549 cells)were induced with bleomycin and H2O2,and the expressions of PTEN,PAI-1,P21WAF1 and P16INK4A were detected by Western blot method.The co-immunoprecipitation method was used to verify the interaction between PTEN and PAI-1 in A549 cellsPart ?:Protective effects of PAI-1 inhibitors and knockdown of PAI-1 on senescent A549 cellsSerum-free A549 cells were pretreated with different concentrations of PAI-1 inhibitors for 24 hours.Cells were treated with bleomycin and H2O2,respectively SA-?-gal staining was used to detect the positive rate of A549 cells.Western blot was used to detect A549 cells.The expression of PTEN,PAI-1,P21WAF1 and P16INK4A in cells.Then shPAI-1 lentivirus was used to transfect A549 cells to knock down PAI-1 gene.Western blot was used to detect the expression of PTEN,PAI-1,P21WAF1 and P16INK4A in A549 cells after knocking down PAI-1Part ?:Protective effect of PAI-1 inhibitors on alveolar cell senescence caused by knocking down PTENA549 cells were transfected with shPTEN lentivirus to knock down the PTEN and pretreated with 5uM PAI-1 inhibitor for 24 hours.The cells were treated with bleomycin and H2O2 respectively.Expression of PAI-1,P21wAF1 and P16INK4AResultPart ?(1)The expression of PTEN in the lung tissue of patients with IPF decreased,and the expression of PAI-1,P21WAF1,and P16INK4A increased(P<0.05)Immunofluorescence results suggested that the changes in PTEN and PAI-1 expression mainly occurred in alveolar epithelial cells(2)With the increasing concentration of bleomycin and H2O2,the expression of PTEN in A549 cells gradually decreased,and the expression of PAI-1,P21wAF1 and P16INK4A gradually increased(P<0.05)(3)The results of co-immunoprecipitation suggest that there is protein interaction between PTEN and PAI-1 in A549 cellsPart ?(1)After treatment with bleomycin and H2O2,the expression of PTEN in A549 cells decreased significantly,and the expression of PAI-1,P21WAF1,and P16INK4A increased significantly(P<0.05).With the increase of inhibitor concentration,the expression of PTEN gradually increased.When the PAI-1 inhibitor reached 5uM,PTEN was significantly up-regulated(P<0.05),and the expression of PAI-1 and P21WAF1 was significantly decreased(P<0.05)(2)The results of SA-?-gal staining showed that the positive rates of SA-?-gal staining in the cells without drug treatment were 14?±0.5774%and 5.333?±0.4055%,respectively.When A549 cells were treated with bleomycin and H2O2,the SA-?-gal staining positive rate increased to 70.33%±3.756%and 83.48%±3.127%,SA-?-gal positive staining rate decreased to 44.33%±2.906%and 58%±after intervention with 5uM PAI-1 inhibitor 1.528%(P=0.0054),the positive rate of SA-?-gal staining after intervention with 10M PAI-1 inhibitor was 58%±1.528%and 61.33%± 4.096%(P<0.05)(3)After knocking down PAI-1,the expression of PTEN in A549 cells was up-regulated(P<0.05),and P21WAF1 and P16INK4A had a downward trend.When the cells treated with bleomycin and H2O2,the expression of PAI-1 and P21WAF1 in the knockdown PAI-1 group was significantly reduced(P<0.05),the expression of PTEN showed a tendency to rise,but the expression of P16INK4A did not change significantlyPart ?(1)The expression of PAI-1,P21WAF1 and P16INK4A in A549 cells was significantly increased after the PTEN gene was knocked down(P<0.05).PAI-1 inhibitors could reduce the expression of PAI-1,P21WAF1 and P16INK4A,and increase the expression of PTEN(P<0.05).When A549 cells that knocked down the PTEN gene were induced with bleomycin and H2O2,PAI-1 inhibitors significantly reduced the expression of PAI-1,P21WAF1,and P16INK4A(P<0.05),but the up-regulation of PTEN was not significantConclusions(1)IPF is an aging-related disease.The expression of PTEN in the lung tissue of patients with IPF decreased,and the expression of PAI-1,P2IWAF1,and P16INK4A increased significantly(2)The changes in the expression of PTEN and PAI-1 mainly occured in alveolar epithelial cells.The changes in PTEN and PAI-1 may be related to the senescence of alveolar epithelial cells during the pathological process of IPF(3)Both bleomycin and H2O2 can induce the senescence of A549 cells,and the expression of PTEN decreases and the expression of PAI-1 increases in senescent A549 cells.PAI-1 inhibitors in a certain concentration range can reduce the senescence of A549 cells induced by bleomycin and H2O2(4)Knocking down PAI-1 can up-regulate PTEN and reduce the senescence of A549 cells induced by bleomycin and H2O2.PTEN knockdown can induce senescence of A549 cells,and PAI-1 inhibitors can reduce the senescence of A549 cells caused by PTEN knockdown.(5)There was protein interaction between PTEN and PAI-1 in A549 cells. |
PDF Full Text Request