Lyn Kinase Represses Mucus Hypersecretion By Regulating IL-13-Induced Endoplasmic Reticulum Stress In Airway Epithelial Cells

bjective:To study the mechanism of Lyn kinase in interleukin-13(IL-13)-induced endoplasmic reticulum stress(ER stress)and MUC5 AC in airway epithelial cell.Methods : 1.Lyn high expression cell lines and Lyn low expression cell lines were constructed by human bronchial epithelial cells(16HBE cells):(1)Lyn high expression vector constructed in our laboratory were transfected into 293 T cells through Lipofectamine 2000 to produce infectious lentiviral vector-containing particles,and 16 HBE cells were transfected with Lyn overexpression lentivirus to create Lyn Highly expressed cell lines(Lyn+/+-16 HBE cell lines);At the same time,16 HBE cells were transfected with lentivirus containing only GFP and the control cell lines(CTRL-16 HBE cell lines)were constructed.(2)Lyn low expression cell lines(Lyn-siRNA-16 HBE cell lines)were prepared by Lyn siRNA via Lipofectamine 2000 and transfected with 16 HBE cells.At the same time,negative control cell lines(siCTRL-16 HBE cell lines)were constructed using non-silent siRNA transfected cells.2.Cell grouping:(1)Lyn+/+-16 HBE cells and CTRL-16 HBE cells were stimulated with IL-13,and PBS stimulated as control.(2)Lyn-siRNA-16 HBE cells and siCTRL-16 HBE cells were stimulated with PI-103 or 4-PBA,and PBS was stimulated as a control,followed by the addition of IL-13 or PBS to stimulate the cells.3.Detection method:(1)Western blot was used to detect the expression of Lyn,p-Lyn,NF?B p65,p-NF?B p65,Akt1,p-Akt1,PI3 K p85?,p-PI3 K p85?,CHOP,Bi P,histone H3,?-actin.(2)MUC 5AC,BiP,CHOP,NF ?B p65 were detected by immunofluorescence and the results were observed with SP5 Leica confocal microscope with Leica Application Suite Software.4.All statistical analyses were done using the SPSS 17.0 software(Chicago,IL,USA),and all data were presented as the mean ± standard deviation().Differences among multiple groups were analyzed using a one-way ANOVA.Significant differences between two groups were analyzed by using the Tukey-Kramer post-test or Dunnett's T3.Statistical significance was defined as a P value<0.05.Results: 1.Successfully constructed the target cells of Lyn+/+-16 HBE cell lines,CTRL-16 HBE cell lines,Lyn-siRNA-16 HBE cell lines and siCTRL-16 HBE cell lines.2.in CTRL-16 HBE cell group,Immunofluorescence analysis showed that IL-13 could induce the protein levels of MUC5 AC,BiP and CHOP,and the difference was statistically significant(P <0.05),Western blot analysis showed that IL-13 induced the increase in the relative phosphorylation levels of PI3 K p85,Akt1,NF?B p65(P <0.05);In the IL-13-induced Lyn+/+-16 HBE cell group and CTRL-16 HBE cell group,Immunofluorescence showed that Lyn overexpression decreased the expression of IL-13-induced MUC5 AC,BiP,CHOP(P<0.05),and Western blot showed that the relative phosphorylation levels of PI3 K p85,Akt1 and NF?B p65 in Lyn+/+ cell group was significantly lower than that in CTRL-16 HBE cell group(P <0.05).3.Western blot was used to detect the expression of NF?B in the nucleus or cytoplasm by Western blot,In CTRL-16 HBE cell group,IL-13 reduced the level of NF?B p65 in cytoplasmic extract(P <0.05),but IL-13 increased the level of NF?B p65 in the nucleus extract(P <0.05);In the IL-13-induced Lyn+/+-16 HBE cell group and CTRL-16 HBE cell group,Lyn overexpression reduced the level of NF?B p65 in the nucleus extract(P <0.05).4.Western blot analysis showed that the relative phosphorylation levels of PI3 K p85,Akt1,NF?B p65 in Lyn-siRNA-16 HBE cells was significantly higher than that in siCTRL-16 HBE cells stimulated by IL-13 cells(P <0.05),the protein levels of BiP and CHOP in Lyn-siRNA-16 HBE cells were higher than those in siCTRL-16 HBE cells,and 4-PBA could decrease the expression of BiP and CHOP in siCTRL-16 HBE cells and Lyn-siRNA-16 HBE cells;Immunofluorescence showed that the fluorescence intensity of MUC5 AC,BIP and CHOP was significantly increased by IL-13 stimulated Lyn-siRNA-16 HBE cells compared with siCTRL-16 HBE cells(P<0.05).In the IL-13 stimulated Lyn-siRNA-16 HBE cell group,Immunofluorescence revealed that 4-PBA or PI-103 significantly reduced the fluorescence intensity of MUC5 AC,BiP,CHOP in siRNA-16 HBE cells(P <0.05);In addition,PI-103 decreased the phosphorylation levels of Akt1 and NF?B p65 in Lyn-siRNA-16 HBE cells by Western blot(P <0.05),However,PI-103 did not inhibit the phosphorylation level of PI3 K p85,and 4-PBA significantly reduced the level of p-NF?Bp65/NF?Bp65 in Lyn-siRNA-16 HBE cells(P <0.05),but 4-PBA did not significantly inhibit the levels of p-PI3 K p85/PI3 K p85 and p-Akt/Akt.Conclusions: 1.In Lyn overexpressing bronchial epithelial cells,Lyn kinase inhibits IL-13-induced endoplasmic reticulum stress and MUC5 AC,which involves PI3 K p85/Akt/NF?B pathway.2.In Lyn knockdown bronchial epithelial cells,endoplasmic reticulum stress modulates NF?B activity,rather than modulates PI3 K p85/Akt activity.3.PI3 K p85/Akt pathway plays a key role in IL-13-induced endoplasmic reticulum stress and mucin hypersecretion in Lyn knockdown bronchial epithelial cells.4.Lyn kinase inhibits IL-13-induced endoplasmic reticulum stress through the PI3 K p85/Akt/NF?B signaling pathway to reduce mucus hypersecretion of airway epithelial cells.