Effects Of PDK1 On PI3K-AKT Signal Pathway And P 16 And LC3B Proteins In Airway Epithelial Cells Of COPD Mice

Background:Chronic obstructive pulmonary disease(COPD)is one of the most common chronic diseases of the respiratory system.Smoking is the biggest risk factor for COPD.Cigarette smoke(CS)exposure can induce chronic airway inflammation,protease / antiprotease imbalance,oxidative stress,etc.,leading to small airway lesions and alveolar destruction,resulting in progressive incomplete reversible airflow limitation.In addition to the classical pathogenesis mentioned above,recent studies have suggested that CS can induce senescence and autophagy of airway epithelial cells,which is closely related to the occurrence and development of COPD,but the mechanism of CS-induced senescence and autophagy of airway epithelial cells in COPD formation has not been fully understood.Previous studies have shown that3-phosphoinositide dependent protein kinase-dependent protein kinase 1(PDK1)is an important component of phosphatidylinositol-3-kinase(PI3K)-serine / threonine kinase(AKT)signal pathway,which regulates cell cycle,proliferation,metabolism,autophagy and senescence by activating AKT.The previous results of this study showed that the expression of PI3K-AKT signal pathway,LC3 B protein(autophagy marker)and p16 protein(aging marker)increased in airway epithelial cells of COPD mice model established by intraperitoneal injection of cigarette smoke extract(CSE)combined with CS exposure,which played a corresponding role in the formation of COPD.However,there are few studies on this signal pathway regulating the senescence and autophagy of COPD airway epithelial cells.Based on the above analysis,our team speculated that CS may increase the expression of LC3 B protein and p16 protein in airway epithelial cells by activating PI3K-AKT signal pathway,and then promote airway inflammation and emphysema-like changes,and participate in the formation of COPD;PDK1 may play a positive regulatory role in this process.Objective:The COPD mouse model was established by intraperitoneal injection of CSE combined with CS exposure to explore the effects of PDK1 on PI3K-AKT signal pathway and p16 and LC3 B protein expression in airway epithelial cells of COPD mice.Methods:C57BL/J mice were randomly divided into three groups: normal control group(NC)(n=8),COPD group(n=9)and COPD+PDK1 inhibitor GSK-2334470 group(COPD+GSK)(n=9).The COPD mouse model was established by intraperitoneal injection of CSE(days 1,12,23)combined with CS exposure(4 weeks).At the beginning of the model construction,the mice in COPD+GSK group were intraperitoneally injected with GSK(80mg/kg/times,three times a week),the other group was given the same frequency and dose of solvent intervention(as the method of the text for details).The general condition of mice was recorded,samples were collected on the 29 th day,and the following corresponding tests were completed:(1)lavage the left lung and collect bronchoalveolar lavage fluid(BALF),detect the concentration of interleukin-6(IL-6)in BALF by enzyme-linked immunosorbent assay(ELISA),calculate the total number of cells in BALF by Nexcelom Cellometer cell counter,and prepare cell liquid-based slices combined with Pap staining to count different classification cells in BALF.(2)the right lung tissue was taken for pathological analysis:(1)Hematoxylin-eosin(HE)staining was used to observe the pathological changes of lung tissue,and the mean alveolar interlining interval(MLI)and alveolar destruction index(DI)were calculated.(2)The expression levels of PDK1,PI3 K and AKT proteins in airway epithelium were analyzed by immunohistochemical(IHC)staining,and the average optical density(AOD)of proteins in airway epithelium was calculated by Image J software;(3)The expression levels of p16 and LC3 B proteins in airway epithelium were analyzed by immunofluorescence(IF)staining,and the mean fluorescence intensity(MFI)of proteins in airway epithelium was calculated by Image J software.Results:1.Comparison of the general conditions of mice in each group: NC group’s mice had normal activity and diet;In COPD and COPD+GSK group,the activity and diet decreased,the oral and nasal secretions increased,but COPD+GSK group’s mice have a lesser extent,and this two group each has one mouse died.2.The comparison of IL-6 concentration,total cell count,neutrophil count and macrophage count in BALF of each group was as follows: COPD group > COPD+GSK group > NC group,the difference was statistically significant(p<0.05).3.The comparison of MLI and DI in each group was as follows: COPD group > COPD+GSK group > NC group,the difference was statistically significant(p<0.05).4.The AOD comparison of PI3 K,PDK1 and AKT protein in each group: PI3K:COPD group and COPD+GSK group >NC group,PDK1 and AKT:COPD group > COPD+GSK group > NC group,the difference was statistically significant(p<0.05).5.The MFI comparison of p16 and LC3 B protein in each group was as follows: COPD group > COPD+GSK group > NC group,the difference was statistically significant(p < 0.05).Conclusion:PDK1,PI3 K,AKT,p16 and LC3 B proteins in airway epithelial cells can participate in mouse COPD formation induced by intraperitoneal injection of CSE combined with exposure to CS.2.PDK1 inhibitor GSK-2334470 may reduce the expression of p16 and LC3 B proteins in airway epithelial cells by regulating PI3K-AKT signal pathway,thus the COPD induced by intraperitoneal injection of CSE combined with exposure to CS in mice was protected..