Study On The Mechanism Of Regulating Nrf2 In Rats With Chronic Obstructive Pulmonary Disease By Lifei Decoction

Objective:To evaluate the therapeutic effect and explore the anti-oxidation mechanism of Recuperating Lung Decoction(RLD)by observing the general condition,lung histopathology,inflammatory factors of the COPD rat model and detecting the indices of oxidative stress,including GST、CAT、GSH、GSSG、Nrf2 and Nrf2mRNA.Methods:Sixty SPF grade SD rats were randomly divided into 6 groups,including normal group,model group,dex group,RLD high-dose,medium-dose and low-dose group,and feeding for one week adaptively.We got the COPD rat medels by giving them passive smoking(5 cigarettes/time,twice a day in fixed time of the morning and afternoon for half an hour,for 12 weeks),sensitizing them 200ug lipopolysaccharide for intratracheal instillation(on the first day and the fifteenth day,a total of two times),and ozone exposure(once a week).After got the COPD rat medels,we gave the rats intragastric administration for 28days.RLD groups were given RLD in concentration of 1.825g/kg,3.65g/kg,7.3g/kg from low dose to high dose.Dex group were given 0.81mg/kg dexamethasone solution.The other group was given the same amount of 0.9%saline.During replication of COPD rats,observed and recorded The general condition of the rats.After modeling,taken the rat lung tissue for HE staining,inflammatory pathology score,measured the levels of IL-6,IL-8,GST,CAT,GSH and GSSG in lung tissue were by ELIS A,detected the expression of Nrf2 protein in lung tissue of COPD rats by immunohistochemical staining,and the expression of Nrf2 mRNA by RT-PCR.Results:1.The general condition of the rats:The normal group of rats breathed regularly and had no obvious positive symptoms.The rest of the rats showed varying degrees of wheezing,shortness of breath,cough,throat phlegm,lips and clawed cyanosis and other performance.During the whole experiment,the body weight of each group increased,among which the dex group had the slowest weight gain and the lightest weight.The dex group rats were irritable in the gavage.2.Histopathological observation of lung tissue by HE staining:In the normal group,the airway epithelium was intact and the cilia were arranged neatly.The number of inflammatory cells infiltrated with lymphocytes was found in the trachea,and the alveolar structure was complete.The model group showed bronchial stenosis deformation,wall thickening,cilia lodging,adhesion,shedding;goblet cell hyperplasia hypertrophy;secretionsubmucosaland bronchial inflammatory cell infiltration;airway and pulmonary vascular smooth muscle hypertrophy;alveolar wall thinning,alveolar cavity,alveolar number decreased,part of alveolar rupture fusion.Compared with model group,the pathological damage of the dex group and the RLD groups was reduced in different degrees,the preliminary judgment of the degree of pathological changes from high to low is:the model group,the RLD low-dose group,the RLD medium-dose group,the RKD high-dose group,the dex group,the normal group.Inflammatory pathology score:In the aspect of inflammation of lung tissue,the scores were from high to low,followed by model group,RLD low-dose group,middle-dose group,high-dose group,dex group and normal group.In the emphysema,mucus secretion and the total score also showed the same trend.3.Determination of Inflammatory Factors in Lung Tissue of RatsCompared with the normal group,the levels of IL-6 and IL-8 in the lung tissue of the model group were significantly increased(P<0.01).Compared with the model group,the levels of IL-6 and IL-8 in the lung tissue of the treatment group decreased significantly(P<0.01),followed by the high-dose group(P<0.05),the decline of IL-6 of the RLD low-dose group was not statistically significant.There was no difference in IL-6 expression among the RLD groups,but the IL-8 of the RLD high-dose group was significantly decreased(P<0.05).4.Detection of oxidative stress in lung tissueCompared with the normal group,the levels of GST,CAT and GSH in the lung tissue of the model group were significantly lower than those in the normal group(P<0.01);Compared with the model group,GST,CAT,GSH and GSSG were decreased(P<0.05)in the RLD low,middle and high dose groups,and the dose-effect relationship was found in the RLD groups.In the treatment groups,GST,CAT,GSH increased,GSSG decreased most significantly in the dex group(P<0.01),followed by high dose group.5.Immunohistochemical staining:Nrf2 protein was strongly expressed in the bronchial,alveolar and alveolar septum of model group and treatment groups,Its strength from high to low is the model group,RLD low-dose group,RLD middle-dose group,RLD high-dose group,the dex group and the normal group.Rat lung tissue Nrf2 protein integral optical density value:The expression of Nrf2 protein in the lung tissue of the model group increased(P<0.01);Compared with the model group,the expression of Nrf2 protein in the RLD low and middle dose group was lower(P<0.05),the RLD high dose group was significantly decreased(P<0.01).There was no significant difference among treatment groups(P>0.05).6.Fluorescence quantitative RT-PCR:Compared with the normal group,the expression of Nrf2 mRNA in the lung tissue of the model group increased(P<0.01).Compared with the model group,the expression of Nrf2 mRNA in the lung tissue of the RLD middle and high dose group was significantly decreased(P<0.01).There was no significant difference among treatment groups(P>0.05).Conclusions:1.This study successfully replicated the rat model of COPD,preliminary evidence of lung powder on COPD model rats have improved antioxidant capacity,and improve the role of gastrointestinal function.2.The RLD decreased the expression of Nrf2 protein and Nrf2 mRNA,suggesting that this may be the mechanism of the treatment of COPD.