The Role Of PTEN/TLR4/NF-kB Pathway In PM Induced Inflammatory Injury Of Alveolar Epithelial Cells

ObjectiveThe epidemiological investigation found that PM2.5 exposure during pregnancy was related to the lung function damage and asthma of offspring.The previous animal experiments of our research group also revealed that PM2.5 exposure during pregnancy caused the decrease of lung function and the increase of lung inflammatory factor exudation,but the specific molecular mechanism was not clear.In this study,the rat lung epithelial cells and the differentiation system of human embryonic stem cells into lung progenitor cells was used to study the effect of PM exposure on them and the possible pathogenesis of their inflammatory damage.Methods1.Rat alveolar epithelial cells(RLE-6TN)were exposed to PM at doses of 50,100,200 and 400ug/ml respectively.The morphological changes of the cells were observed and the proliferation of the cells was detected by CCK8 method.2.Detection of IL-1β expression by real-time fluorescence quantitative PCR in alveolar epithelial cells after PM exposure.3.To detect the role of PTEN/TLR4/NF-κB pathway in the inflammatory injury of pulmonary epithelial cells induced by PM exposure: the expression levels of PTEN,TLR4 and NF-κB were detected by real-time fluorescence quantitative PCR and western blotting;after inhibition of NF-κB activation by bms-345541,the expression level of IL-1β was detected by q PCR.4.To establish an in-vitro model of human embryonic stem cells(hESCs)differentiation into lung precursor phase,and to carry out PM exposure in different phase.The exposure concentration in the stage of D1-D3 was5,50,100 ug/ml,the expression levels of c-kit and CXCR4 were detected by flow cytometry,the expression levels of NF-κB were detected by western blotting;The exposure concentration of stage of D10-D15 was 5,50ug/ml,TTF1,a differentiation index of lung progenitor,was detected by western blotting after PM exposure.Results1.After PM exposure,the cell morphology of RLE-6TN in 100,200 and400ug/ml concentration groups was changed.With the increase of PM concentration,the cell morphology changed from cluster like and paving stone like to cell shrinkage,cell gap widening,irregular shape and cell surface covered by PM particles.The higher the PM concentration,the greater the morphological change and cell death occurred in the high concentration group.Compared with the control group,the proliferation ability of RLE-6TN cells in each PM treatment group decreased and showed a downward trend with the increase of PM concentration.2.The results of q PCR showed that compared with the control group,the expression level of IL-1β increased with the increase of PM exposure(P<0.01).3.The results of q PCR showed that the expression levels of TLR4,PTEN and NF-κB decreased with the increase of the concentration compared with the control group;WB showed that TLR4 increased significantly at the concentration of 50ug/ml(P<0.01),PTEN decreased significantly at the concentration of 100,200 and 400ug/ml(P<0.05),P-NF-κB/NF-κB increased with the increase of the exposure concentration,and increased significantly at the concentration of 200ug/ml(P<0.001);The mRNA expression level of IL-1β decreased significantly with the addition of NF-κB pathway activation inhibitor.4.HESCs was exposed to PM during induction to the definitive endoderm and the results of flow cytometry showed that the expression level of c-kit was decreased(p<0.05),while the expression level of CXCR4 was no significant difference.The results of Western blotting showed that the expression level of P-NF-κB/NF-κB increased with the increase of exposure concentration,compared with the control group.TTF1 was expressed in all groups of cells when they were exposed to PM from Day10 to day15,and there was no significant difference between the concentration groups.Conclusion1.PM exposure may cause lung injury by reducing the proliferation of rat pulmonary epithelial cells.2.PM exposure may induce the inflammatory injury of alveolar epithelial cells through the activation of PTEN/TLR4/NF-κB pathway,and initiate the occurrence of pulmonary inflammation,thus leading lung injury.3.By using the in vitro hESCs induced culture model,it is clarified that PM exposure may affect the differentiation efficiency of the definitive endoderm through NF-κB pathway,thus affecting the early development of human embryos.