The Regulation Of Cigarette-induced Expression Of PDK1 On Airway Mucus Hypersecretion

Objective:Airway mucus hypersecretion is an important pathophysiological feature of chronic airway inflammatory diseases such as chronic obstructive pulmonary disease,bronchial asthma,bronchiectasis and pulmonary cystic fibrosis.Inflammatory response and oxidative stress induced by cigarette smoke and other irritants can damage airway epithelial cells,cause ciliated cell exfoliation,goblet cells proliferate and submucous hypertrophy,and eventually lead to excessive secretion of airway mucus.Persistent mucus hypersecretion can obstruct the airway lumen,induce bronchopulmonary infection,and lead to the destruction of bronchial and pulmonary parenchyma structure,resulting in sustained irreversible lung function damage.The clinical manifestations include cough,expectoration,repeated acute exacerbations,which is closely related to the poor clinical outcome of the patients.Recent researchs have shown that chronic airway inflammation,oxidative stress,protease imbalance,cholinergic nerve dysfunction and other pathophysiological mechanisms can induce or aggravate airway mucus hypersecretion,in which chronic airway inflammation is an critical factor.Therefore,to study the mechanism of airway mucus hypersecretion in order to explore effective intervent approaches has become an important issue in the study of chronic airway inflammatory diseases.PGE2 participates in the inflammatory process of COPD.The level of PGE2 in induced sputum can be used as an index to evaluate the severity of COPD,but it is not clear whether PGE2 is involved in the regulation of goblet cell metaplasia and mucus hypersecretion in airway remodeling.PDK1 is an important protein kinase in vivo,and PDK1 signaling pathway plays an important role in cell growth,survival and angiogenesis.It was also found that the secretion of PGE2 level in bronchoalveolar lavage fluid and mucin secretion of airway epithelium and the expression of PDK1 in airway epithelium of smoking rats were increased,and PDK1 inhibitor could reduce the airway mucus hypersecretion caused by smoking.In previous studies,it has been found that PKC and AKT/PKB signaling pathways were involved in cigarette-induced airway mucus hypersecretion.As the upstream regulator of AKT signal,PDK1 participates in the regulation of PKC and AKT/PKB signal pathways,thus affecting airway mucus hypersecretion.So,does cigarette induce airway mucus hypersecretion by increasing the expression of PDK1 regulated by PGE2? At present,there is no related research on the above problems at home and abroad.We hypothesized that cigarette smoke induced increased PGE2 secretion in airway epithelial cells,then bound to its receptor EP2 or EP4.c-jun or AP-2 located in the PDK1 promoter sequence could up-regulate the expression of intracellular PDK1,and then activate downstream signals(such as AKT,MAPK,etc.)which may involve in the regulation of airway mucus hypersecretion.This study attempt to explore the molecular mechanism of PDK1 in cigarette-induced airway mucus hypersecretion and airway remodeling in vivo and in vitro,so as to find a new target for the treatment of airway mucus hypersecretion.Materials and Methods:1.Cell experiment in vitro: the most suitable concentration and time of CSE for stimulating BEAS2 B cells were detected by CCK8.After stimulating BEAS-2B cells with different concentrations of CSE(0%,1%,2.5%,5%,10%),proteins were extracted,and the expression of PDK1 was detected by Western-blot to further verify the optimal concentration of CSE.Using 5%CSE to stimulate BEAS-2B in different time points(0h,6h,12 h,24h,48h),Western-Blot method was used to detect the expression and activity changes of MUC5 AC,PDK1,AKT,c-jun,AP-2 and so on in PDK1 signal pathway.EP2-siRNAsiRNA and EP4-siRNAsiRNA were used to transfect cells to inhibit the gene expression of EP2 or EP4.CSE was used to interfere with transfected cells.After protein extraction,Western Blot was used to detect the expression of MUC5 AC,PDK1,AKT,c-jun,AP-2 and so on in PDK1 signal pathway.The cells were transfected with PDK1-siRNA,c-jun-siRNAsiRNA and AP-2-siRNAsiRNA,and treated with PDK1 inhibitor.After the cells were stimulate with CSE,the expression of MUC5 AC,PDK1,AKT,etc.in the signal pathway were detected by Western Blot.2.In vivo experiment: 50 SPF grade C57BL/6 male mice,aged 8-10 weeks and weighing between 19-23 g,were randomly divided into five groups: normal control group(n = 10),smoking group(n = 10),low-dose PDK1 inhibitor group(n = 10),medium-dose PDK1 inhibitor group(n = 10)and high-dose PDK1 inhibitor group(n= 10).After 12 weeks of smoking,the blood samples,BALF samples and lung tissue samples of mice were collected to detect the total number of cells and the proportion of diffrent cells in BALF.The pathological changes of lungs of each group were observed by HE staining,and the airway mucus secretion was observed by ABPAS staining.The expression of MUC5 AC in airway was observed by immunohistochemical method.Results:1.CSE decreased the viability of BESA-2B cells and increased the concentration of PGE2 in a time-dependent manner(P<0.05).The optimal concentration of CSE was 5%CSE(P<0.05),and the duration was 48 h.CSE induced the expression of PDK1,c-jun,p-c-jun,p-AKT,AP-2 and MUC5 AC compared with the control group,with statistically significantly difference(all P <0.05).CSE had little effect on the expression of AKT(P =0.21),but promote phosphorylation of AKT.2.Both EP2 siRNA and EP4 siRNA inhibited the expression of c-jun,AP-2,PDK1,p-PDK1,p-AKT and MUC5 AC induced by CSE compared with the control group with statistically significantly difference(P <0.05),and the inhibition effect of EP4 siRNA group was more obvious(P <0.05).These results suggest that both EP2 and EP4 may be involved in CSE-mediated activation of PDK1/ AKT signaling pathway and regulation of MUC5 AC expression through binding with PGE2,and EP4 maybe the main receptor.3.PDK1 siRNA and PDK1 inhibitor OSU-03012 could down-regulate the protein expressions of PDK1,p-PDK1,p-AKT and MUC5 AC compared with the control group with statistically significantly difference(P <0.01),but had little effect on the expression of AKT(P=0.144).4.Both c-jun siRNA and AP-2 siRNA could down-regulate the protein expression of PDK1,p-AKT and MUC5 AC compared with the control group with statistically significantly difference(P <0.01),but had little effect on the expression of AKT(P=0.07),indicating that c-jun and AP-2,as nuclear transcription factors located in the PDK1 promoter region,could affect the expression of MUC5 AC by upregulating the expression of PDK1 and activating the PDK1/AKT pathway,which were blocked by c-jun siRNA and AP-2 siRNA.5.Smoking established the model of airway mucus hypersecretion in mice successfully.The body weight of mice in the smoking group decreased and the total number of inflammatory cells,neutrophils,macrophages and lymphocytes in BALF increased,compared with the control group with statistically significantly difference(P<0.01).PDK1 inhibitor OSU-03012 could decrease the above changes in low-dose,middle-dose and high-dose groups,and there were significant differences compared with smoking group(P<0.01).It was more obvious in the high dose group(P<0.01).6.Submucosal gland hypertrophy,goblet cell hyperplasia and mucus obstruction were observed in the central airway in fumigating mice.Inflammatory cell infiltration and mucus obstruction were observed in the airway,bronchioles and alveolar sacs.Airway cilia lodging,shortening and shedding;Alveolar dilatation,thinning of alveolar walls,formation of large alveoli and significant reduction in the number of alveoli were also observed.Concurrently,AB-PAS staining showed that the mucus secretion was lower than that in the smoking group,and the expression of MUC5 AC was decreased showed by immunohistochemistry(all P <0.05).Compared with the smoking group,the airway mucus obstruction,inflammatory cell infiltration,alveolar wall thinning and alveolar dilatation were alleviated in the low,middle and high dose OSU-03012 groups.AB-PAS staining showed that the mucus secretion was lower than that in the smoking group,and immunohistochemistry showed that the expression of MUC5 AC was lower than that in the smoking group,and the greater the dose,the more obvious improvement.Conclusion:This experiment confirmed that cigarette stimulated the secretion of PGE2 in airway epithelium,then bound to its receptor EP2 or EP4,in which EP4 may be the main receptor.Up-regulation of nuclear transcription factor c-jun or AP-2 located in the PDK1 promoter sequence increased the expression of intracellular PDK1,and then activated the downstream signal Pmurac AKT,up-regulates the expression of MU5 AC,resulting in increased airway mucus secretion.