The Study On Inflammatory Effects And Mechanism Of COPD Rats

Objective: cigarette smoke is an important part of the sources of indoor air pollution,and it is also the main source of indoor fine particulate matter,which seriously threatens human health.At present,there were three methods,namely Ames test,micronucleus analysis and neutral erythrocyte toxicity test recommended by Cooperation Centre for Scientific Research Relative to Tobacco(CORESTA)to evaluate the toxicity of cigarette smoke in vitro.However,these methods can only initially evaluate the relative toxicity of some of the chemical constituents of cigarette smoke and do not reflect the biological effects of their overall composition.In recent years,foreign researchers have proposed that we can use "the specific disease test",that is,Chronic obstructive pulmonary disease(COPD)animal model for toxicology evaluation of cigarette smoke in vivo,but the relevant research has not been reported.Clinical researches have shown that COPD is mainly associated with increased inflammatory response to harmful gases or particulates,as well as a systemic disease that accumulates extrapulmonary tissue,which is the fourth leading cause of death and the fifth economic burden of disease worldwide currently.Previous studies have shown that COPD rats induced by cigarette smoke showed local(airway and lung tissue)and systemic oxidative stress and injury,as well as a extrapulmonary organ damage(liver and spleen)effect.But the inflammatory response to COPD rats(both pulmonary and systemic)is unclear.Based on this,we used the flue-cured tobacco from three genotypes of tobacco as the material,and used sub-chronic(56 days)smoking test to construct the COPD model of SD rats,and to explore the pathological damage of lung in COPD rats,pulmonary inflammatory response,and systemic inflammatory response,including changes in the degree of pathological changes and some inflammatory markers concentration levels,to assess the toxic effects of different cigarette smoke in vivo.This study aims at providing a theoretical basis of the toxic mechanism and health hazards of complex air pollutants.Methods: 72 male SD rats had divided into control group,CS-1,CS-2 and CS-3group.The three experimental groups of rats were exposed to chronic smoking respectively,to construct the COPD model.After 14,28 and 56 days,lung tissues were obtained for H.E.,Masson and PAS staining to observe the pathological changes and score;The changes of levels of TNF-?,IL-8,IL-6,IFN-? and IL-4 in bronchoalveolar lavage fluid and serum were determined by ELISA method;The changes of levels of NF-?B p65 and Nrf2 in lung tissue were determined by the same method;The expression level of NF-?B p65 in lung tissue was determined by immunohistochemical assay.Result:(1)After 56 days of being exposed to cigarette smoked,CS-1,CS-2 and CS-3 groups rats performed typical COPD pathological features.Including inflammatory cell infiltration around the small airway and pulmonary vascular,mucosal epithelial cell exfoliation erosion,mucous hyperplasia,airway bronchial epithelial cells goblet metaplasia.Moreover,the fibres of bronchial wall hyperplasia,smooth muscle layer thickening,increased mucosal folds caused bronchial lumen stenosis,and alveolar structure disorder.When compared with the control group,pathological score showed that the changes of lung tissue were predominant in experimental group rats(P<0.05).The severity of lesions was CS-2 > CS-1 > CS-3,and compared with CS-2 group,the CS-1 and CS-3 groups had significant difference(P<0.05).(2)With the extension of exposure time,the content of TNF-?,IL-8,IL-6,IFN-?,NF-?B p65,Nrf2,and ratio of IFN-?/IL-4 in BALF or lung tissue of experimental group rats were higher than the control group,but the concentration of IL-4 was lower than the control group counterpart.Simultaneously,at the end of the infection,the levels of TNF-?,IL-8,IL-6,IL-4,NF-?B p65 and Nrf2 in CS-1 and CS-3 groups were significantly lower than those in CS-2 group(P<0.05).However,the ratio of IFN-?/IL-4was not significantly different(P>0.05).And the changes of all indicators were CS-2>CS-1>CS-3.The results showed that different tobacco smoke could activate NF-?B p65 protein and up-regulate the expression of inflammatory factors in different degrees.(3)With the extension of exposure time,the content of CRP,TNF-?,IL-8,IL-6,IFN-?,and ratio of IFN-?/IL-4 in serum of experimental group rats were higher than the control group,but the concentration level of IL-4 was lower than that of the control group.Simultaneously,at the end of the infection,the levels of CRP,TNF-? and IL-8 in CS-1 and CS-3 groups were significantly lower than CS-2 group counterpart(P <0.05).However,the ratio of IFN-?/IL-4 was not significantly different(P>0.05).And the changes of other indicators were CS-2>CS-1>CS-3,except that CRP was CS-2>CS-3>CS-1.The results showed that different genotypes of tobacco smoke induced different degrees of systemic inflammatory response.Conclusion: Exposure to cigarette smoke can construct COPD disease model of SD rat,experimental animals showing typical pathological features like inflammatory and emphysema,as well as local(lung tissue)and systemic(serum)inflammation.And there was a significant difference between the pathologic score and the concentration of inflammation-related factors of the three experimental groups,which may be related to the different genotype and chemical composition of cigarette smoke.At the same time,the difference in toxic effects of the three tobacco species in vitro in this study was basically the same as the previous study(CS-2>CS-1>CS-3).The results suggested that pathological scores of lung tissue and CRP,TNF-?,IL-8 and IL-6 in BALF and/or serum can be used to detect the difference of biological effects among different samples.Moreover,the difference of abnormal inflammatory effect in COPD rats may be related to the expression of NF-?B p65.