Induction Of Alveolar Epithelial Cell Apoptosis Via Endoplasmic Reticulum Stress In COPD And The Anti-apoptosis Effect Of GRP78

Background The apoptosis of alveolar epithelial cell plays an important role in the pathogenesis of chronic obstructive pulmonary disease(COPD). In recent years, it has been reported that endoplasmic reticulum(ER) may be a new location which participates in intracellular pathway of cell apoptosis. That is called ER stress-mediated apoptosis pathway. GRP78, a 78kDa glucose regulated protein, is a major ER chaperone and plays a critical role in regulating ER homeostasis. Previous studies revealed that p38 mitogen-activated protein kinase (MAPK) may be involved in the induction of GRP78. Cigarette smoke, has been regarded as the main risk factor that triggers development of COPD. Some components in cigarette smoke including ROS, nicotine, heavy metals, and aldehyde may have the potential to induce ER stress. It has been reported that oxidative stress can activate p38MAPK pathway, which play an important role in the inflammation of airway in COPD. Based on these results, we speculate that cigarette smoke can cause ER stress and thereby contributes to apoptosis of lung epithelial cells. Induction of GRP78 protects cells from apoptosis, and p38 MAPK may involved in the up-regulating of GRP78. In present study, we use smoke-induced COPD rat and cigarette smoke extract (CSE) treated human alveolar epithelial cells (A549) as models, to explore the mechanisms of ER stress in COPD, the anti-apoptosis role of GRP78 and the regulative effect of MAPK pathway in this process.Chapter One The ER stress and ER stress-mediated apoptosis in the lung tissues of COPD ratObjective To study the ER stress and the apoptosis of alveolar epithelial cell in COPD rat model.Methods 24 Wister rats were divided into two groups at random: control group and COPD group. COPD rat model was established by intratracheal instillation of lipopolysaccharide (LPS) twice and exposure to cigarette smoke daily. The spirometry was conducted and the pathological changes were observed after the model was established. The mRNA of GRP78 and CHOP were detected by reverse transcription-polymerase chain reaction (RT-PCR).The expression of GRP78 in the lung tissues was examined by immunohistochemistry and the protein expression of GRP78, CHOP and caspase-12 was detected by Western blot.TUNEL was used to analyze alveolar epithelial cell apoptosis.Results Significant decrease of FEV0.3/FVC, Cdyn and increase of RI were found in the COPD group compared with the control group. Immunoperoxidase staining showed GRP78 was localized in the cytoplasm of bronchial epithelial cells, endothelial cells, especially in alveolar epithelial cells, and the GRP78 expression significantly increased in the COPD group. The levels of GRP78 and CHOP mRNA were higher in the COPD group than in the control group. The expression of GRP78, CHOP and active caspase-12 protein were increased in the COPD group. Apoptosis was observed with an in situ TUNEL assay, more apoptotic alveolar epithelial cells were found in the COPD group.Conclusion The COPD rat models was successfully established by intratracheal instillation of LPS and exposure to cigarette smoke; ER stress was triggered in the lung tissues of COPD rat. Alveolar epithelial cell apoptosis was increased in the COPD group. The ER stress-mediated apoptosis pathway may participate in the alveolar epithelial cell apoptosis in COPD.Chapter Two GRP78 induction by cigarette smoke extract and its anti-apoptosis effectObjective To investigate the induction of GRP78 mediated by cigarette smoke extract(CSE), and the anti-apoptosis effect of GRP78.Methods In part one, cultured A549 cells were exposed to CSE at various concentrations and for defferent durations. GRP78 expression was detected by RT-PCR and Western blot. In part two, A549 cells were divided into 4 groups:control group,5%CSE group, GRP78siRNA+5% CSE group, control siRNA+5% CSE group, measured the GRP78 expression by RT-PCR and Western blot, detected the active caspase-3 expression by Western blot,and TUNEL assay was used to detect apoptosis.Results Part one, the expression of GRP78 was gradually increased as raising the CSE concentration or prolonging the treated time. When A549 cells were treated with 5% CSE for 12 hours, the GRP78 expression level reach the peak value. When continue to raise the CSE concentration or prolong the durations, the GRP78 expression was decreased. Part two, pretreated with GRP78 siRNA and then stimulated with CSE significantly reducing the GRP78 expression, indicated that GRP78 gene expression was successfully inhibited by GRP78 siRNA, accompany with the increase of active caspase-3 expression and A549 cells apoptosis.Conclusion In A549 cells, low CSE concentration or short stimulation time up-regulate GRP78 expression via unfolded protein response. But high CSE concentration or long duration could down-regulate GRP78 expression because of the broken compensation of protection mechanism; GRP78 counteract A549 cells apoptosis induced by cigarette smoke.Chapter Three CSE up-regulates GRP78 expression in A549 cells via p38MAPK pathwayObjective To investigate the regulative effect of p38MAPK pathway in GRP78 expression induced by CSE.Methods In part one,24 Wister rats were divided into two groups at random:control group and COPD group. COPD rat model was established by intratracheal instillation of LPS twice and exposure to cigarette smoke daily. After the model was established, P-p38 protein expression was detected by Western blot. The correlation analysis was used to evaluate the association between P-p38 protein level and GRP78 expression. In part two, A549 cells were divided into 3 groups:control group,5%CSE group, SB203580+5%CSE group. P-p38 expression was measured by Western blot, and GRP78 expression was detected by RT-PCR and western blot.Results Part one, the expression of P-p38 was significantly increased in the COPD group compared with the control group, and the correlation analyses showed that P-p38 protein level was positively correlated with GRP78 expression (r=0.848, P<0.01). Part two, P-p38 expression was significantly decreased in SB203580 pretreated group, demonstrated that p38MAPK pathway was successfully blocked by SB203580. The expression of GRP78 which was induced by CSE was inhibited by SB203580.Conclusion p38MAPK pathway was activated in COPD; CSE up-regulate GRP78,which was attributed to p38 pathway.