ABSTRACT
Objective:To investigate the effect of protein tyrosine kinase 6(PTK6) on TNF-α induced human airway epithelial barrier dysfunction and mechamism.Methods: After cultivating 16HBE cells in vitro,the recombined PTK6 and PTK6 siRNA was respectively transfected into the cells,with empty vector and scramble siRNA as control.The cells were incubated with exogenous TNF-α.Cell viability was detected by MTT assay.Cells TER and permeability were detected.ZO-1 and Occludin mRNA were analyzed by RT-PCR.PTK6,ZO-1,Occludin,p-ERK1/2,p-JNK1/2 and p-p38MAPK protein were assayed by Western blot.Results: TNF-α remarkably decreased ZO-1 and Occludin mRNA and protein and TER;increased cells permeability,as well as p-ERK1/2,p-JNK1/2 and p-p38 protein(P<0.05).Upregulation of PTK6 further decreased ZO-1 and Occludin mRNA and protein and TER,enhanced cells permeability and p-JNK1/2 and p-p38 protein(P<0.05),and downregulated PTK6 ,the changes of these indices were opposite(P<0.05).Whereas upregulation or downregulation of PTK6 had no effect on p-ERK1/2.Conclusion: Downregulation of PTK6 inhibits the phosphorylation of JNK1/2 and p38MAPK,thus improving TNF-α-induced human airway epithelial barrier dysfunction.
ABSTRACT
Objective: To explore the effect of caveolin-1 ( Cav-1 ) on lipopolysaccharide ( LPS )-induced airway mucous hypersecretion.Methods:16HBE human airway epithelial cells with Toll-like receptor 4(TLR4) inhibitor,nuclear factor-kappa B(NF-κB) inhibitor,Cav-1 siRNA or plasmid pr-treated,further stimulated with LPS.The cells were divided into 8 groups:the control group, the LPS group,the LPS+Cav-1 expression group,the LPS+Cav-1 siRNA group,the LPS +negative siRNA group,the LPS +empty vector group,the LPS +E5564 group, the LPS +PDTC group.Cell survival rate was detected by MTT assay.Transcription level of mucin(MUC)5AC was evaluated with RT-PCR.The level of MUC5AC protein was measured by ELISA.The expression of TLR4,Cav-1 and phosphorylated IκBα( p-IκBα) were measured by Western blot.MUC5AC protein changes were observed by immunofluorescence and confocal laser technology.Results:LPS remarkably increased MUC5AC,as well as TLR4,p-IκBα(P<0.05).These effects were prevented by E5564 and PDTC.We found that the overexpression of Cav-1 further enhanced the expression of TLR4, p-IκBαand MUC5AC.However,downregulation of Cav-1 inhibited the expression of TLR4,p-IκBα,MUC5AC.Conclusion: Cav-1 enhances LPS-induced MUC5AC hypersecretion through TLR4/NF-κB signaling pathway.