Tobacco Smoke Extract Down-regulate Lysosomal ClC-3 Protein Through Akt Signaling Pathway And Induce Autophagy Flux Blockage In Tracheal Epithelial HBE Cells

Objective:To observe the effects of tobacco smoke extract on AKT signaling pathway and lysosomal ClC-3 chloride channel protein expression in tracheal epithelial HBE cells and investigate the underlying mechanism of autophagy flux blockage in HBE cells induced by down-regulation of ClC-3 expression by tobacco smoke.Methods: 1.Tobacco smoke extract was obtained by a modified syringe-drivencigarette burning device.2.Western blotting was used to detect the expression of ClC-3 proteinand Akt protein phosphorylation in the tracheal epithelial cells treatedwith tobacco smoke extract.3.The effect of tobacco smoke extract on the expression of Cl C-3protein and phosphorylation of Akt protein in tracheal epithelial cellswas detected by immunofluorescence assay.4.The effect of tobacco smoke extract on the expression of CREB1protein in tracheal epithelial cells was detected byimmunofluorescence assay.5.The effect of ClC-3 expression changes on the lysosomal acidicenvironment was detected by the lysosomal red fluorescent probe.6.The immunofluorescent double staining for autophagosome markerprotein LC3 B and lysosomal marker protein Lamp1 were used toobserve the effects of tobacco smoke extract on autophagosome andautophagosome lysosome fusion in autophagic flow.Result: 1.Tobacco smoke extract down-regulated ClC-3 protein expression intracheal epithelial HBE cells while inhibiting phosphorylation of Aktprotein.2.The Akt signaling pathway inhibitor piperidoline inhibited Aktphosphorylation of HBE cells while down-regulating the expressionof ClC-3 protein.3.Akt signaling pathway activator SC79 promoted Akt phosphorylationin HBE cells while antagonizing the effect of tobacco smoke extracton Cl C-3 expression.4.Tobacco smoke extract down-regulated the expression of thetranscription factor CREB.Perfosine inhibited the expression of thetranscription factor CREB.5.SC79 up-regulated the expression of CREB and antagonized theeffect of tobacco smoke on CREB expression.6.Tobacco smoke extract down-regulated the expression of lysosomalmembrane ClC-3 in HBE cells.7.Tobacco smoke extract reduced the acidity of HBE cell lysosomes.8.Tobacco smoke extract inhibits fusion of lysosome andautophagosome in autophagy flux of HBE cells Conclusion: 1.Tobacco smoke extract down-regulated the expression of ClC-3 byinhibiting phosphorylation of Akt protein and down-regulatingtranscription factor CREB expression.2.Tobacco smoke extract down-regulated the expression of lysosomalmembrane ClC-3 protein,which decreased the acidity of lysosomeand induced autophagic flux blockade due to dysfusion betweenautophagy and lysosome.