| Objective:Through medication experiment to stable SD rat model of chronic obstructive pulmonary disease, to study the changes of general condition, lung function, pathology and intestinal flora of chronic obstructive pulmonary disease model rats after treatment by using the recuperating lung decoction,and clarify the rat model of chronic obstructive pulmonary disease in the gut microflora imbalance, recuperating lung decoction can regulate the structure of intestinal flora in patients with chronic obstructive pulmonary disease. The electron spin magnetic resonance technology and other means of detection was used to test the oxygen free radicals, endotoxin and cytokine levels of COPD rats, consummate the mechanism of recuperating lung decoction adjusting the structure of intestinal flora through oxidative stress damage and airway inflammation in the treatment of chronic obstructive pulmonary disease.Method:1.Thirty six healthy male SD rats were randomly divided into model control group (n=18), blank control group (n=18). The oxidative stress model of rats with chronic obstructive pulmonary disease was established by using the method of smoke plus lipopolysaccharide infusion combined with ozone exposure. The general state of rats was observed, and the lung function of rats in each group was determined. After the materials were drawn, the bronchoalveolar lavage fluid was prepared and the number of inflammatory cells was counted. The degree of lung tissue damage was judged by HE staining of lung tissue. By bacterial culture, we study on microbial ecology of intestinal flora by observing the number of flora and culture of intestinal Escherichia coli, enterococci, bifidobacteria and lactic acid bacteria. Using the PCR method of molecular biology, the16S rDNA gene sequence of the intestinal flora of rats was analyzed, and the changes of the intestinal flora structure were observed.2. Seventy healthy male SD rats were randomly divided into blank control group, model control group, dexamethasone group, probiotics group, recuperating lung decoction high, middle, low dose,10 rats in each group. Establishment of rat model of chronic obstructive pulmonary disease in addition to the blank control group. The glucocorticoid group and the probiotics group were treated with glucocorticoid and probiotics respectively, and the high, medium and low doses group of traditional Chinese medicine were administered to the rats with different doses of recuperating lung decoction.After drug intervention, the general condition of rats were observed, and the lung function of rats in each group were determined. Lung and bowel tissues stained by he were observed under microscope. By bacterial culture, we study on microbial ecology of intestinal flora by observing the number of flora and culture of intestinal Escherichia coli, enterococci, bifidobacteria and lactic acid bacteria. Using the PCR and Qualcomm sequencing method of molecular biology, the 16S rDNA gene sequence of the intestinal flora of rats was analyzed, and the changes of the intestinal flora structure were observed. The superoxide anion free radical, nitric oxide free radical, hydroxyl radical and superoxide dismutase in serum and lung homogenate of rats were detected by electron paramagnetic technique. IL-1,4,5,13,16,17, CRP and TNF-in serum were detected by ELISA method. Observing the plasma endotoxin levels by matrix LAL method. The method of direct count of eosinophils was used to count the serum eosinophils.Result:1.Structural analysis of intestinal flora in rats with chronic obstructive pulmonary disease Model control group rats in the modeling process have a certain degree of shortness of breath, cough frequently, spirit is dispirited, yellow teeth, messy and dry hair, restlessness, reduced food intake. The rats in control group have no these features. Lung function test result, Compared with blank control group, the FVC, FEV1, FEV1/FVC% in COPD model control group was lower. Lung tissue biopsy, stained with hematoxylin and eosin visible:In COPD model control group, it’s can be seen that bronchioles narrow deformation and respiratory bronchiolar lumen narrow or occlusion, wall thickening, centrilobular emphysema and panlobular emphysema; proliferation of epithelial cells, goblet cells were obviously proliferated. There are numbers of inflammatory cell infiltration around mucosa and peribronchiolar, which mainly are lymphocytes. Besides, airway smooth muscle thickening, alveolar wall thinning and expansion of the alveolar space, part of alveolar rupture is formed by the fusion of the bullae of lung can be seen. In the blank control group, the bronchial wall was smooth and the fold was clear. Bronchoalveolar lavage fluid (BALF) results, the numbers of macrophagocyte and lymphocytes in BALF of model control group was more than that in the blank control group, there were statistically significant differences (P<0.05). The ratio of GSH/GSSG in the serum was shown that in the model control group, the level of GSH decreased, the level of GSSG increased, the ratio of the two decreased, and the difference was statistically significant (P< 0.05).Intestinal tissue pathological sections showed that there were eosinophils between the colon and the mucosa of the model group. According to the results of bacterial culture, the numbers of Escherichia coli and Enterococcus were increased in model control group, and the number of Bifidobacterium and lactobacillus were significantly reduced (P<0.05).16Sr DNA results showed that the number of Muciniphila, Bacteroides, Prevotella, lactobacillus in model control group were decreased, and the numbers of Qscillospira, Prevotella were increased.2. Structural modulation of gut microbiota in chronic obstructive pulmonary disease rats treated by recuperating lung decoctionModeling rats appear cough and wheezing after Seven days’ smoking. Four months later, the symptoms of wheezing, throat phlegm, listlessness, back arched and curled up, yellow fur were obviously. There were no positive symptom in blank control group, while the rest of the group like dexamethasone group probiotics group, traditional Chinese medicine (TCM) low, middle and high dose group rats appear visible fur yellow, acerbity and no luster as well as shedding, weight loss. Futhermore, the shortness of breath, restlessness, cough and wheezing, increased urination and other symptoms even unresponsive were shown at the same time. To the end of the drug, the symptoms alleviation of model control group and probiotics group was not obvious, the Chinese medicine group and dexamethasone group had a certain degree of reduction. The pulmonary function of the rats in each drug group after different drug treatments were improved to different degree, the pulmonary function alleviation of high dose group and dexamethasone group were close to that of blank control group, middle dose group was next to the former, and probiotic group and low dose group was slightly worse, but still was superior to the control group. Lung tissue HE staining results show that the degree of disease from high to low in order is model control group, low dose group of Chinese medicine, probiotics group, middle dose group of Chinese medicine, high dose group of Chinese medicine, dexamethasone group, blank control group. Bacterial culture results showed in both three Chinese medicine groups and Probiotic group, the Enterococcus and Enterobacter content decreased obviously and the Bifidobacteria as well as lactic acid content increased distinctly. Compared with the blank control group, model control group rats intestinal Enterobacteriaceae and Enterococcus content increased significantly, Bifidobacterium and Lactobacillus content decreased significantly. Compared with dexamethasone group, the difference was statistically significant between the probiotic group and recuperating lung decoction group, and there was no dose effect relationship between different dose Chinese medicine groups.16S rDNA results showed that the intestinal flora of rats with chronic obstructive pulmonary disease after treatment with dexamethasone and the high dose group was more and more close to the blank control group.It was dose related to Lactobacillus species and the Prussian coli in three Chinese medicine dose groups, the abundance of high dose group was significantly higher than that of low dose group. According to the paramagnetic results, the clearance rate of oxygen free radical in serum and lung homogenate of the model control group was lower than that of the control group, and the difference was statistically significant, after the treatment, the clearance rate of oxygen free radical in serum and lung homogenate of rats was higher than that of model control group, the dexamethasone group was better than that of recuperating lung decoction groups and Probiotic group, recuperating lung decoction groups did not show a dose-response relationship. Cytokine detection results show that the IL-1,4,5,13,16,17, CRP, TNF-a, endotoxin levels in the serum of model control group were higher than those in blank control group, the difference was statistically significant, after drug treatment, the IL-1,4,5,13,16,17, CRP, TNF-a, endotoxin levels in the serum were lower than those in model control group, the dexamethasone group was better than that of recuperating lung decoction groups and probiotics group, It’s showed a certain dose-response relationship between different Chinese medicine dose groups, the high dose group is better than the low dose group. The results showed that the serum eosinophils in the dexamethasone group were the lowest, the model control group was the highest, and the dose-response relationship was not found between the three dose groups. Conclusion:1.We have successfully replicated the oxidative stress of chronic obstructive pulmonary diseases rat model, and chronic obstructive pulmonary disease rats model have intestinal microflora Dysbiosis.2.The pathological changes of lung tissue in rats of each groups can reflect the difference of therapeutic effect of different drugs3.In the treatment of chronic obstructive pulmonary disease, the structure of the intestinal flora changed, and the intestinal flora of the high dose group was closer to the normal rats.4.Recuperating lung decoction can regulate COPD rats intestinal flora, and it may be related to the regulation of COPD rats oxidative stress injury, inhibit airway inflammation and release of endotoxin. |
PDF Full Text Request