| Purpose:Different groups on the observation of invigorating Qi and dissolving phlegm prescription in plasma,BALF and lung tissue in the expression of TNF alpha, IL-17, ts mechanism may be through MAPK 1 and the nf-kappa B signal transduction pathway activation and the influence of Muc5ac and AQP5 mRNA gene expression.Bidirectional intervention in the airway inflammation of this study based on the traditional Chinese medicine Qi and phlegm, for the Chinese medicine "lung main gas", "provide the experimental basis for the scientific connotation of lung water passage" theory and Yiqi Huatan therapy, and provide further experimental basis and guiding clinical treatment of COPD.Methods:1 healthy male C57BL 60 mice, randomly divided into 6 groups:they are normal control group, COPD model group, low dose of Chinese medicine group, middle dose of Chinese medicine group, high dose group, dexamethasone group. Each group of 10 only. The use of cigarettes smoked instillation of lipopolysaccharide combined modeling method to establish a mouse model of chronic obstructive pulmonary disease aerated pipe, from the lung tissue of mice in general, pathological morphology (naked eye observation, HE staining of lung tissue pathological section) observation and BALF cells and total cells consisting of three indexes to evaluate the effects of chronic obstructive pulmonary disease mouse model, Qi and phlegm traditional Chinese medicine for the change of chronic obstructive pulmonary disease mice inflammatory cell level and pathology.2 using WBP whole body volume recorder in the rats of twentieth weeks of mice were PIF, PEF and MV without pulmonary function test record. Understand the animal model of airway resistance and lung compliance, reliability of experimental animal model.3 expression by immunohistochemical method to detect TNF-in lung tissue of mouse alpha, IL-17, Muc5ac. Through the analysis and comparison of effects of Chinese medicine Yiqi Huatan on cytokine expression in lung tissue, to study the therapeutic effect of Yiqi Huatan Decoction on COPD mice.4 Application of double antibody sandwich enzyme linked immunosorbent assay (ELISA) the content of Muc5ac TNF-alpha, IL-17 content and BALF in mice were measured in the plasma and BALF. Discussion of Yiqi Huatan Decoction on anti inflammation, inhibition of sticky mouse model of mechanism of protein secretion.5. The expression of Muc5ac and AQP5 of mRNA gene in lung tissue of mice were detected by using RT-PCR. Observation of Chinese medicine Yiqi Huatan effect on the expression of its efficacy evaluation, and targets of different doses from inflammation/ immune reaction medium angle.6 Protein content using the Western blot method for detection of lung tissue p38-MARK, Erk, JNK and NF-kappa B phosphorylation. To observe the effect of Yiqi Huatan Decoction on lung tissue in mice with COPD MAPK and NF-B signaling pathway, further prove that the traditional Chinese medicine Qi and phlegm is through the IL-17/MAPK, the NF-B signaling pathway regulation of airway inflammation, airway epithelial MUC5AC change mRNA and AQP5 expression.7 Experimental data are expressed by X± s, packet statistics analysis by SPSS16.0 statistical software. The different groups using one-way ANOVA analysis of variance, with P< 0.05, the difference was statistically significant, with P< 0.01 for statistical significance.Results1 Effect of Yiqi Huatan Decoction on indexes of COPD mic1.1 Histological observation:HE staining and observed under light microscope, the results showed:normal control mice in the lungs, bronchial epithelium and alveolar structural integrity, bronchial lumen cilia arranged in neat rows, there is no obvious exudation, tube wall is not thickened submucosa, no obvious inflammatory cell infiltration in the alveolar cavities, no the obvious expansion. The mice in model group in the lungs, bronchial epithelial cells arranged in disorder, columnar epithelial cells shedding serious, the number of goblet cells were increased, a small amount of lodging cilia shedding, arranged irregularly, bronchial wall thickening, lumen stenosis, deformation, submucosal glands hyperplasia, accompanied by massive infiltration of inflammatory cells, neutrophils, monocytes to predominantly macrophages and lymphocytes. Alveolar wall thinning or fracture, alveolar cavity is enlarged, partial fusion of pulmonary alveoli into pulmonary bulla, leading to alveolar number decreased, pulmonary interstitial and alveolar cavity shows a large number of inflammatory cell infiltration1.2 Observe the lung tissue sections:in lung tissue, PAS staining can be dyed purple red slime. The normal control group, the lungs of mice bronchial mucus less, PAS positive cells dyeing ratio is relatively low, and lung tissue of model group mice bronchial mucus in PAS staining cells increased significantly, the number increased significantly.1.3 The changes of pulmonary respiratory function index:using WBP whole body volume recorder in the rats of twentieth weeks of mice without pulmonary function test record. Results show that, compared with the normal control mice, mice in the model group PIF, PEF and MV values decreased significantly (p<0.01), the clinical features are consistent with clinical COPD. High reaction condition, high, low dose of Chinese medicine Yiqi Huatan Decoction can obviously improve the airway of mice with COPD (p<0.05), improve the effect showed a dose dependent relationship with some traditional Chinese medicine Yiqi Huatan Decoction, high dose of improve the strongest effect, but the data of each index is still lower than that of normal control mice, traditional Chinese medicine Yiqi Huatan Decoction dose the improvement of the function, improve the effect of Chinese medicine Yiqi Huatan Decoction for weak low doses of dexamethasone, and no obvious improvement in COPD airway resistance (p>0.05).1.4 Change in total BALF cell components and cell:the study found that the total white blood cell count was highest in COPD model group, there was significant difference compared with the normal group (P> 0.05), Chinese medicine Yiqi Huatan high, low dose group and model group were significant difference (P< 0.05 or P< 0.01), conclusion:the traditional Chinese medicine Yiqi Huatan has obvious inhibiting effect on inflammatory cells in COPD rats; In the cell counting and classification, the groups are dominated by mononuclear macrophage, compared with normal control group, mononuclear macrophage percentage of model group decreased significantly (P＜0.01), and lymphocyte, neutrophil percentage were significantly increased (P<0.01), compared with the model group, Chinese medicine Yiqi Huatan high, low dose group of mononuclear macrophage increased significantly (P<0.01 or P＜0.05), lymphocyte, neutrophil percentage, significantly reduced ((P<0.01 or P<0.05), suggesting that cigarette smoke with tracheal instillation of lipopolysaccharide molding can induce rat mononuclear macrophage percentage decreased, increased lymphocyte, neutrophil percentage, mononuclear macrophage percentage and Yi Qi phlegm medicine can obviously the increase of COPD in mice, the percentage of reduced lymphocyte, neutrophil, could obviously inhibit the secretion of inflammatory cells in mice with COPD; Effects of different dose of Yiqi Huatan Decoction on inflammatory cells in mice with obvious dose effect relationship. High, low dose of Yiqi Huatan Decoction has inhibitory effects on inflammatory reaction mediated by IL-17, including the number of dexamethasone group in BALF mice inflammatory cells decreased most significantly, high dose of Chinese medicine group, followed by the middle dose of Chinese medicine group, low dose of Chinese medicine group in BALF mice inflammatory cells to reduce the number of the most obvious.2 Effect of Yiqi Huatan Decoction on the expression of TNF- alpha, IL-17, Muc5ac2.1 Changes in the immunohistochemical results of COPD expression in lung tissue of mice of TNF- alpha, IL-17, Muc5ac display, IL-17, TNF-alpha, Muc5ac weakly positive expression in lung tissue of mice in normal. In the lung tissue of mice in COPD model group, IL-17, TNF- alpha, Muc5ac was strong positive expression. Tip IL-17 can synergistically activated macrophages produce TNF-a, promote TNF-a induced a variety of neutrophil chemotaxis, amplification of inflammatory response, so that increased mucus secretion. Hormone can block or inhibit the inflammatory chemokine expression, the experiments also prove that dexamethasone can significantly reduce the expression of TNF-and IL-17 in lung tissue of COPD in mice.2.2 Changes of ELISA results of TNF-alpha, the content of IL-17 in plasma and BALF in COPD mice display, with significant difference in plasma and BALF in plasma of normal mice and BALF and COPD in the model group compared with the content of TNF-alpha (P< 0.01); plasma of normal mice and BALF model and COPD model of centralized secretion of inflammatory mediators by IL-17 raised the neutrophil, a variety of induced chemotactic factor, the inflammatory cells reach the target site. Compared with the COPD mice model group, herbal medicine Yiqi Huatan high, and the content of TNF-alpha, IL-17 in plasma and BALF in low dose group decreased, obvious difference (P< 0.01). The positive control drug dexamethasone group compared with the model group could significantly decrease the content of TNF-alpha and IL-17 in plasma and BALF in COPD mice (P< 0.01). The prompt traditional Chinese medicine Yiqi Huatan can reduce airway inflammation, and its mechanism may be that Th17IL-17 mediated COPD chronic inflammatory and immune protective response related.2.3 Changes of ELISA Muc5ac content of COPD in mice results in the display of BALF, content of BALF OD value of Muc5ac in the normal mice is lower than that of COPD model mice, suggesting that an animal model after IL-17, prompting the upregulation of MUC5AC. Traditional Chinese medicine Yiqi Huatan high, Muc5ac content, low dose of BALF decreased gradually, the content of Muc5ac and BALF in COPD model group but there were no significant differences between the low dose group (P> 0.05), Muc5ac content and COPD in the model group, high dose group and dexamethasone group BALF had statistical significance (P< 0.05), of unknown origin suggest that inhibition of Chinese medicine Yiqi Huatan secretion of Muc5ac in lung tissue of COPD mice, so as to improve the airway remodeling.2.4 Changes of RT-PCR COPD in lung tissue of mice results of Muc5ac and AQP5 mRNA levels showed, compared with the normal mice, the mice in COPD model group Muc5ac mRNA levels increased significantly (P< 0.01), while the AQP5 mRNA level was significantly lowered (P< 0.01). Results suggest that expression of Muc5ac mRNA of hyperthyroidism lung tissue of experimental mice, AQP5 inhibition of mRNA expression, the expression of both negative correlation; traditional Chinese medicine Yiqi Huatan high, medium, low dose groups were significantly lowered the level of Muc5ac mRNA in lung tissue of COPD mice, the up regulation of AQP5 mRNA levels, including high, middle dose group of Muc5ac and AQP5 mRNA expression level and the model group have significant difference (P< 0.01), low dose group, the down-regulation of Muc5ac mRNA expression level and there was significant difference between model group (P< 0.05). The experimental results indicated that the high dose of Yiqi Huatan Decoction reduced the level of Muc5ac mRNA in lung tissue of COPD mice, the up regulation of AQP5 mRNA levels effect is better than the low dose group, and the expression of each dose group of Muc5ac and AQP5 mRNA were dose-dependent.2.5 Lung tissue in mice with COPD Muc5ac and AQP5 mRNA protein level of western with blot results show, compared with the normal mice, the mice in COPD model group Muc5ac mRNA levels increased significantly (P< 0.01), while the AQP5 mRNA level was significantly lowered (P< 0.01). The prompt traditional Chinese medicine Yiqi Huatan to the level of Muc5ac mRNA protein in lung tissue of mice inhibited the upregulation of AQP5, mRNA protein levels; traditional Chinese medicine Yiqi Huatan high, low dose of COPD in lung tissue of mice Muc5ac protein band in mRNA mark colors are shallow, expression intensity was decreased significantly (P<0.01), AQP5 mRNA strap mark color have deepened, expression strength enhanced significantly (P<0.01 or P＜0.05), including high, low dose group, Muc5ac and AQP5 mRNA expression level and the model group have significant difference (P< 0.01), experiments indicated that high dose of Yiqi Huatan Decoction on the level of Muc5ac and dexamethasone group regulating mRNA protein in lung tissue of COPD mice, downregulation of AQP5 mRNA level is better than low dose effect group, and the expression of each dose group of Muc5ac and AQP5 mRNA protein were dose-dependent.3 Effect of Yiqi Huatan Decoction on lung tissue in mice with COPD MAPK and NF-B signaling pathway3.1 Changes of Western blot results of COPD in lung tissue of mice MAPK pathway shows:compared with the normal group, COPD model group mice in lung tissue Erk, P38 and JNK protein levels were significantly increased, protein phosphorylation ratio was significantly increased in the lung tissue of mice, and Erk, P38 and JNK protein content and phosphorylation in the low level; tip in the lung tissue of mice in COPD model group, MAPK signaling pathway is significantly activated; Qi and phlegm in high, low, can significantly inhibit the activation of COPD in lung of mice with the MAPK signaling pathway, in which the Chinese medicine high dose the strongest inhibition, time in the traditional Chinese medicine agent amount, the weaker effect of low dose of Chinese medicine. The positive control drugs, dexamethasone inhibited the activation of COPD in lung of mice with MAPK signal pathway.3.2 Changes of Western blot results of COPD in lung tissue of mice NF-kappa B signal pathway shows:compared with the normal group, the lung tissue of COPD mouse cytosolic IkappaB content is very high, the NF-kappa B signaling pathway activation in model mice, suggesting that COPD in lung tissue, NF-kappa B signaling pathways play a regulatory role downstream; traditional Chinese medicine Yiqi Huatan high, low dose group, Chinese medicine can significantly inhibit the activation of inhibition of COPD in lung of mice with NF-kappa B signal pathway. Conclusion1 The presence of high expression of IL-17 cytokine mediated by CD4+T/Thl7 cells in the lung tissue of mice COPD model, and the stimulation of proinflammatory cytokines by the main effect factor IL-17 recruitment of neutrophils and other inflammatory factors such as TNF- alpha, prompting Muc5ac was strong positive expression in airway mucus secretion, increase, and further lead to airway remodeling.2 Traditional Chinese medicine Yiqi Huatan can reduce plasma COPD in mice lung tissue, and BALF IL-17, TNF- alpha and other inflammatory cell content, reduce Muc5ac content, significantly improve the pathological injury of lung tissue in mice; mononuclear macrophage percentage of Yiqi Huatan can significantly increase the percentage of COPD mice, reduced lymphocyte, neutrophil, has obvious inhibiting action the COPD mice inflammatory cell secretion.3 Traditional Chinese medicine Yiqi Huatan can reduce numerical pulmonary ventilation function mouse COPD PIF, PEF and MV, significantly improve the state of airway high reaction in COPD mice. But the data of each index is still lower than the normal control group4 Traditional Chinese medicine Yiqi Huatan can the level of Muc5ac mRNA protein in lung tissue of mice raised COPD, downregulation of AQP5 mRNA level, improve mucus hypersecretion state.5 Yiqihuatan prescription by inhibiting the activation of COPD in lung of mice with MAPK signaling pathway and downstream of NF-kappa B signaling pathway, rise to adjust and improve airway remodeling of COPD mice inflammatory cells, mucus hypersecretion, play a key role in these two pathways that regulate IL-17 receptor mediated signal, which may be one of the mechanism of Yiqi Huatan bidirectional immune regulation.6 Different dose of Yiqi Huatan Decoction has a dose effect relationship of inflammatory cells, alveolar experimental rats by affecting cell factor, tissue Muc5ac protein, APQ5 expression of mRNA and MAPK, NF-kappa of the B signaling pathway in the lavage. |
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