Downregulation Of Endoplasmic Reticulum Stress Resist Airway Epithelial Cell Apoptosis In COPD:the Role Of PERK/eIF2α Pathway

Airway epithelial cell apoptosis is an important mechanism of COPD pathogenesis, endoplasmic reticulum stress (ERS) mediated apoptosis is also more and more attented. Tobacco and other harmful substances to stimulate the cells by initiating the unfolded protein response (UPR) decreased protein synthesis and increased degradation of the unfolded protein to reduce cell stress burden. The inner state of the endoplasmic reticulum stress, PERK pathway activated first, and has an extremely important role. PERK by oligomerization and autophosphory lation activation, resulting in its downstream factor of eukaryotic transla tionn initiation factor2a subunit (eIF2a) phosphorylation, phosphory lation of eIF2a direct inhibition of protein synthesis, and the resulting nascent polypeptide outflow endoplasmic reticulum to alleviate its burden of maintaining the balance of the intracellular environment. If the stimulation persists, endoplasmic reticulum stress induced CHOP, caspase-12expression and induced apoptosis. In vitro studies have found that the PERK/eIF2a pathway knockout cell endoplasmic reticulum stress due to increased sensitivity to apoptosis, and enhanced PERK/eIF2a path, reduced apoptosis, prove raised PERK pathway in ERS against apoptosis. In the pathogenesis of COPD, PERK/eIF2a pathway is not clear, the first part of this study build COPD rat model, and detected the PERK pathway signaling molecules and apoptosis signal indicators the PERK pathway activator salubrinal of intervention in COPD rat model to observe the airway epithelial apoptosis. Second part of human bronchial epithelial cells (HBE) study, cigarette smoke extract (CSE) intervention PERK small interference from the cellular level to confirm the PERK pathway in CSE-induced airway epithelial cell apoptosis has important role to provide new theoretical basis and therapeutic targets for the clinical treatment of COPD.Chapter I The role of PERK/eIF2a pathway in COPD lung tissues of rats endoplasmic reticulum stress-induced apoptosisObjective:To observe the PERK pathway in the endoplasmic reticulum stress-induced apoptosis in the COPD lung tissue of rats and the protection of salubrinal.Methods:36Wistar rats were randomly divided into three groups: normal control group, COPD model group, COPD model plus drug group (intervention group). Passive smoking plus intratracheal instillation of lipopolysaccharide rat model of COPD drug group while trachea for infusion of lipopolysaccharide the infusion salubrinal (lmg/kg). Upon completion of the modeling measured the lung function of the rats in each group; pathological changes of the lung tissue; Immunohistochemical detected expression of p-PERK, p-eIF2α, Caspase-12in bronchopulmonary tissue; western blot detected the expression of p-PERK、p-eIF2α、active caspase-12and active caspase-3; The TUNEL assay bronchopulmonary organization epithelial cell apoptosis。Results:the lung function index of model group were significantly lower than normal group, but salubrinal group significantly increased than model group. The expressions of p-PERk, p-eIF2α was higher compared than normal group, while the highest expression level exsited in the intervention group; The apoptosis index of active caspase-12and active caspase-3expression in model group was significantly higher than the normal group, and decreased in the intervention group compared with the model group.Conclusion:(1) The intratracheal instillation of lipopolysaccharide plus smoke successfully build a rat model of COPD;(2) ERS ocuured in COPD rat bronchopulmonary tissue, started PERK-mediated UPR signaling pathway;(3)Salubrinal can protect the rat model of COPD bronchial alveolar epithelial cells from apoptosis, its protective effect may be achieved by regulating the PERK/eIF2a pathway. Chapter II the role of PERK/eIF2a pathway in cigarette smoke extract induced human bronchial epithelial cell apoptosisObjective:To observe the impact of the CSE in HBE cells and PERK pathway related protein expression, explore the role of PERK pathway in the endoplasmic reticulum stress-induced apoptosis.Method:First, cultured HBE cells give5%CSE them at different times (0h-24h) intervention in vitro, the expression of p-PERK, p-eIF2a were detected by western blot, immunofluorescence; Sencond, increase with different concentrations of Salubrinal (0-100uM) intervention the HBE cells stimulated by the CSE, the results show a protective effect on the cells, and the protective effect of increasing concentrations; then divided the HBE into six groups:the control group,5%CSE group, Salubrinal group, Salubrinal (1μM)+5%CSE group; Salubrinal (10μM)+5%CSE group, Salubrinal (100μM)+5%CSE group; The expression of p-PERK, p-eIF2a and caspase4were detected by western blot. AnnexinV-PI double standard flow method to measure the HBE cell apoptosis. Then, after the PERK siRNA, treat the HBE cells with5%CSE and salubrinal, RT-PCR detected the expression of PERK mRNA, the expression of p-PERK、p-eIF2a and active caspase-3、4were measured by western blot, AnnexinV-PI double standard flow method to measure the HBE cell apoptosis.Results:With the CSE the concentration increase or intervention time prolonged the expression of p-PERK and p-eIF2a protein was gradually increased in HBE cells,5%CSE,6h group intervention increased the most obviously, but p-PERK and p-eIF2a expression decreased when the CSE exsited persistely; Salubrinal (0-100uM) intervention the CSE process HBE cells obtained has a protective effect on cells, and its role in increasing concentration increase the maximum concentration of100uM protection, to cells after Salubrinal intervention group p-eIF2a protein expression was significantly higher than other groups; After PERK siRNA, the expression of PERK decreased notably by RT-PCR and western blot, PERK siRNA block the expression of PERK mRNA successfully; With the knock out of PERK, the expression of active caspase-3、caspase-4and cell apoptosis increase greatly compared with simple CSE stimulation group, but decreased if the HBE cell stimulated with salubrinal, and the expression of p-eIF2a increased with p-PERK no statistical significance.Conclusion:(1)PERK/eIF2α pathway negative regulate endoplas mic reticulum stress to resist the CSE-induced HBE apoptosis, the integrity of critical path HBE cell survival; (2) Salubrinal can protect HBE cells from CSE induced apoptosis;(3) Salubrinal might regulating PERK pathway activity to protect the HBE cells against endoplasmic reticulum stress induced apoptosis.