The Mechanism Of Chronic Obstructive Pulmonary Disease (COPD) Induced By Cigarette Smoke

Chronic obstructive pulmonary disease (COPD) is one of the major causes of chronic morbidity and mortality throughout the world. It is currently the fourth leading cause of death in the world and is projected to rank the fifth as a worldwide burden of disease by 2020. It is characterized by chronic inflammation throughout the airways and parenchyma. No currently available treatments reduce the progression of COPD or suppress the inflammation in small airways and lung parenchyma.Although COPD is a major public health problem in the world, but it receives relatively little attention compared with diseases with a similar impact such as coronary heart disease and cancer. One of the main reasons is not only complex pathogenesis of COPD but also lack of reliable model of COPD.Reliable model of COPD will not only increase our understanding about pathogenesis, but will also facilitate the development and introduction of new therapeutic strategies. Therefore, more research is needed to develop reliable model of COPD and to understand the cellular and molecular mechanisms of COPD to aid the development of new therapies.Cigarette smoking is a risk factor for the development of airway hyperresponsiveness and chronic obstructive pulmonary disease. In this study, we examined the possibility of developing an animal model of chronic obstructive pulmonary disease (COPD) induced by cigarette smoke and to study the role of inflammatory cell, cytokine in the chronic airway inflammation of COPD.Study objective(1) The present study was conducted to determine whether chronic whole-body exposure of rats to sidestream cigarette smoke could produce significant, time-related increases in the incidence of COPD, and to establish an experimental model for smoke-induced COPD. (2)To investigate the character of airway inflammation in chronic obstructive pulmonary disease (COPD). (3)To study the role of cytokine in the chronic airway inflammation of COPD.Methods(1) 132 rats were randomly divided into normal control or cigarette smoke (CS) rats. The rat models of COPD were established by exposure to cigarette smoke daily for 36 weeks (2 h/day, 7 days/week). For each CS group, air control rats were studied at the same time (all n = 7, respectively). (2) At the end of 2, 4, 6, 8, 10, 12, 24 and 36 week of smoke treatment, a forced oscillation (FO) technique independently estimated the lung function. (3) Rats were sacrificed and the levels of interleukin(IL)-2, IL-4, IL-6, IL-10, IL-13, IL-18, interferon (IFN)-γ,endothelin-1 (ET-1), monocyte chemotactic protein -1 (MCP-1) in bronchoalveolar lavage fluid (BALF) or serum were measured by enzyme-linked immunosorbent assay (ELISA). (4) Total and differential cell counts in BALF and blood were done. (5) Enzyme-linked immunosorbent assays (ELISA) for mucin were performed on culture medium after tracheas were incubated. (6) Tissue was fixed by infiating with formalin from right lung. Sagittal seetions were stained with hematoxylin-eosin or AB-PAS or V.G. (7) The pathomorphological changes, including epithelial cell mucus occupying ratio (MOR),the ratio of the wall thickness to the external diameter in the bronchus,the ratio of collagen area per unit of length of basilemma in the bronchus,mean linear intercept (MLI),mean alveolar airspace (MAA) and inflammatory cell counts (ICC) in rat lung tissues were measured with image-analysis system. (8) The expression of CD3, CD4, CD8 and CD68 in pulmonary tissue was observed by immunohistochemistry technique.Results1. The development of weight in CS rat were significantly inhibited compared with normal control.2. The increase of Raw and Ers in group CS 36wk were significantly differences than those in group normal control (P<0.05).3. The CS rats shared specific pathological features in trachea,bronchi and lung tissues with that of human chronic bronchitis and emphysema. It shows that the proliferation epithelial cell and fibroblast, epithelial layer rising into the lumens goblet cell and mucus gland significantly increased, mucus accumulation in lumens. An extensive macrophages and lymphocytes in filtrate around the pulmonary blood vessels and airways was seen in the lung interstitium of CS rats compared with normal control animals. From 10 wk CS exposure, irregular cilia arrangement of bronchial epithelium appeared in the rat bronchus. The evidence for mucus hypersecretion was also observed. The detachment of bronchial epithelium and merged cilia was found in the bronchus of CS rats after 12 wk exposure. From 12 to 24 wk CS exposure, air spaces were much enlarged in an irregular manner, some alveoli were merged and bullae were formed. Following 24 wk CS exposure, the pathological evidence of bronchitis became more apparent with hypersecretion of mucus, detachment of bronchial epithelium, merged cilia, irregular arrangement of bronchial smooth muscles and fibrosis on basement membrane. At 36 wk CS exposure, air spaces were markedly enlarged, the thickness of air walls was greatly increased,and large number of accumulated inflammatory cells was observed. The clear characteristics of chronic bronchitis including hyperplasia of bronchial epithelial cells, hypersecretion of mucus and development of peribronchial fibrosis were found at 36 wk CS exposure.4. Using quantitative histomorphology techniques, it was found that Lm and AIA of CS rats increased respectively by 24.6% and 22% after 24 wk inhalation, and by 44.8% and 43.7% after 36 wk inhalation.5. It appeared that up to approximately 29 fold increase in the number of inflammatory cells being observed in the lungs of CS rats.6. Morphometric Analysis of the aiways the thickness of each category airway walls in CS group were significantly greater than in normal control group. There was a progressive trend to the ratio of the wall thickness to the external diameter in the bronchus of CS exposed and normal control rats.7. Significant increase in thickening of collagen and the ratio of collagen area per unit of length of basilemma in the bronchus were found in CS group (P<0.05 or P<0.01) than that of normal control group.8. Stained mucin area on trachea epithelium was faint in color and small in size in normal group. Stained mucin area of CS group on trachea had the apparent increased optical density and the epithelial cell mucus occupying ratio (MOR) after 4, 8, 10, 12, 24 and 36 week inhalation were higher than those of control group respectively.9. Approximately up to 2.5 or 4 fold increase in mucin release was achieved following 1 hr incubation period. Approximately up to 1.5 or 7.5 fold increase in mucin release was achieved following 3 hr incubation period. Approximately up to 1.6 or 11.3 fold increase in mucin release was achieved following 6 hr incubation period. After 16 hr incubation, mucin secretion was elevated by 4 or 41 fold in comparison with the secretion from normal control rats.10. Compared with Sham group rats, up to approximately 8.3, 2.3 and 9.9 fold increase in the number of neutrophils, lymphocytes and dust cells was observed in the BALF of CS rats following inspiring cigarette smoke for 24 or 36 wk. At 12 wk cigarette smoking, however, only the number of dust cells was elevated in the BALF. Cigarette smoking had little effect on the number of macrophages, eosinophils and epithelial cells in the BALF of CS rats. The number of neutrophils, lymphocytes, monocytes, eosinophils and basophils in rat blood was not significantly altered after up to 36 wk cigarette smoking.11. Statistic analysis showed that level of IL-10 in serum of CS rats after 2, 6, 8, 10 and 36 week inhalation were higher than those of control group respectively,while that level of IL-10 in BALF decreased respectively by 56, 38.5 and 59% after 4, 24, and 36 wk inhalation.12. The level of IL-13 in serum and BALF decreased respectively by 75.3 and 54.5% after 36 wk inhalation.13. The level of IL-18 in serum of CS 36 wk rats was increased to 188% of control values. The level of IL-18 in BALF of CS rats were higher than those of control rats after 2, 6, 8, 10 and 36 wk inhalation.14.The level of IFN-γin BALF of CS rats increased respectively by 149 and 103% after 24 and 36 wk inhalation.15.The level of MCP-1 in BALF of CS rats increased respectively by 59 and 85.8% after 24 and 36 wk inhalation.16. It appeared that up to approximately 5.5 and 10.7 fold increase in concentration of ET in serum after after 24 and 36 wk inhalation.17. The level of IL-2 in BALF of CS rats were higher than those of control rats after 10, 24 and 36 wk inhalation.18. The level of IL-4 in BALF of CS rats were higher than those of control rats after 24 and 36 wk inhalation.19. The level of IL-6 in BALF of CS rats were higher than those of control rats after 12, 24 and 36 wk inhalation.20. Statistic analysis showed that the number of CD3+ (CS 2 wk, CS 8 wk, CS 12 wk, CS 24 wk, CS 36 wk),CD8+ (CS 2 wk, CS 8 wk, CS 24 wk, CS 36 wk),CD68+ (CS 2 wk, CS 4 wk, CS 6 wk, CS 12 wk, CS 24 wk, CS 36 wk) were higher than those of control group respectively, while the number of CD4 in lung parenchyma was not significantly altered after up to 36 wk cigarette smoking. Conclusions(1)The COPD models were made by the rats exposed to cigarette smoke for 36 weeks. This experimental model replicates many of the features of human chronic obstructive pulmonary disease and should facilitate studies of pathogenetic mechanisms and of potential therapeutic agents.(2) The results suggest that many inflammatory cells involve in the development and progress of COPD. The infiltration of inflammatory cells involve neutrophils, monocytes and lymphocytes in lumina of airways, and CD3+,CD8+,CD68+ in lung parenchyma.(3) IL-2,4,6,10,18, IFN-γ, MCP-1 and ET-1 might play important roles in the airway inflammation of COPD.(4) Hypersecretion of mucus, increases in inflammatory cells, increases in airway wall thickness and collagen deposition diffusely within the airway wall lead to small airway remodeling and played important role in the airflow obstruction.