| Objective:The aim of this study is to provide scientific and reliable theoretical basis for the treatment of COPD with compound mangiferin, and provide the theory and experimental basis for the development of compound mangiferin as new, safe and effective drug for the prevention and treatment of COPD, by study on the efficacy and mechanism of compound mangiferin to the model mice with COPD induced by BLM and LPS.Methods:1Establish the COPD model with the syndrome of qi deficiency and phlegm statisis Healthy male mice were randomly divided into control group, group B1, group L1, group B3, group L3and group B+L,48mice each group. Group B1:Mice were given BLM,3.75mg-mL-1, at day zero by nasal drip. Group L1:Mice were given LPS,5mg·mL-1, at day zero by nasal drip. Group B3:Mice were given BLM,1.25mg·mL-1, respectively at day zero, day seven and day fourteen by nasal drip. Group L3:Mice were given LPS,1.5mg·mL-1, respectively at day zero, day seven and day fouteen by nasal drip. Group B+L:Mice were given BLM,3.75mg·mL-1and given LPS,1.5mg·mL-1,at day zero, then given LPS,1.5mg·mL-1at day seven, and day founteen.16mice of each group were sacrificed at28thd,42thd and56thd, measured the relevant index in lung tissue, icluding observing TCM characterization: claws and tongue skin color changes, locomotor activity, weight changes, observing following changes in the lung tissue by HE staining. Observing the changes in collagen deposition in the lung tissue of mice by Masson staining.Determining cytokines content of IL-1β, IL-4, IL-8, TNF-a and TGF-(31in lung tissue by ELISA. Testing protein expression of MUC5AC and AQP5, which were lung mucus secretion high index, and Nrf2, Bach land y-GCS, which were oxidant/antioxidant index by immunohistochemistry. Testing the content of GSH, MDA and T-AOC, and activity of SOD of lung tissue by using UV spectrophotometry.2. Study on the efficacy and mechanism of compound mangiferin to the model mice According the above research results, the methods of establish model were using the combination of BLM and LPS. Model mice were randomly divided into4groups, model control group(given NS), compound mangiferin high dose group(given compound mangiferin), compound mangiferin high dose group(given compound mangiferin), positive control group(given dexamethasone); and the blank control group (given NS)was normal mice,10mice per group. Mice were given corresponding drugs and NS,20mL·kg-1, by gavage at the first day after establish model. Mice were sacrificed at second day after the last administration, and the relevant index in lung tissue were measured, icluding observing TCM characterization: claws and tongue skin color changes, locomotor activity, weight changes, observing following changes in the lung tissue by HE staining, Observing the changes in collagen deposition in the lung tissue of mice by Masson staining, determining cytokines content of IL-1β, IL-4, IL-8, TNF-a and TGF-β1in lung tissue by ELISA, testing protein expression of MUC5AC and AQP5, which were lung mucus secretion high index, and Nrf2, Bachl and y-GCS, which were oxidant/antioxidant index by immunohistochemistry, testing the content of GSH, MDA, and T-AOC, and activity of SOD of lung tissue by using UV spectrophotometry; testing the mRNA expression of TNF-α, TGF-β1, CXCL8and MUC5AC in lung tissue by using real-time quantitative PCR.Results:1Establish the COPD model (with the syndrome of qi deficiency and phlegm statisis)①TCM characterization:compared with the blank control group, the increasing of weight of model control group were more slow, the group B+L were most slow(P<0.01), group B+L and group Bl’s number of independent activity of were significantly reduced, hind paw skin and tongue color were change into dark purple, group B+L were most obviously(P<0.01).②Lung tissue of model mice appeard significant pathological changes at day twenty-eight, and showed a progressive increase. The group B+L were most obvious than other group, bronchial submucosa and muscular mice showed obvious infiltration of inflammatory cells, there were a lot of exudate within the lumen, partial alveolar wall destructed the and formed emphysema, part of the bronchial ciliated epithelium, vessel wall were infiltrated by inflammatory cell, local alveolar septa became thick, bronchial smooth muscle were significantly became thick, bronchus became narrowing, interstitial fibrous tissue proliferated. The group B+L had the more obvious pathological changes of pulmonary bronchitis, emphysema, lung and bronchial inflammation around than other group, which showed the combination of BLM and LPS, was better than other inducing methods.③The content of IL-1β, IL-4, IL-8, TNF-α and TGF-β1in lung tissue of model group were obviouly increased(P<0.01-0.05). The protein expression of MUC5AC of model mice were obviously increased(P<0.01), the protein expression of AQP5of model mice were obviously decreased at day forty-two(P<0.05), the protein expression ofy-GCS and Nrf2of model mice were obviously increased(P<0.01-0.05), the protein expression of Bachlof model mice were obviously decreased(P<0.01-0.05).⑤The content of GSH, SOD and T-AOC of model mice were obviously decreased(P<0.01-0.05), the content of MDA of model mice were obviously increased(P<0.01-0.05).2. Study on the efficacy and mechanism of compound mangiferin to the model mice①Compared with the model control group, compound mangiferin could obviously recoverd the increasing of mice weight with the COPD, improving the condition of hind paw skin and tongue color, recovered the autonomic activity in mice(P<0.01-0.05).②Compound mangiferin could obviously improve the pathological changes in the lungs, bronchial submucosa and muscular, vascular wall had only mild inflammatory cell infiltration, small amount of exudate within the lumen, destroied a small part of the alveolar walls, mild bronchial stenosis, a small amount of bronchial ciliated epithelium, bronchial smooth muscle thickness increased slightly, slight thickening of alveolar septa, mild interstitial fibrous tissue hyperplasia. Compound mangiferin could improve the pathological changes of lung tissue at different degrees, reduce pulmonary bronchitis, bronchial inflammation around the lungs(P<0.01-0.05), inhibited the formation of emphysema(P<0.01-0.05).③Compound mangiferin could decrease the content of IL-1β, IL-4, IL-8, TNF-α and TGF-β1in the lung tissue of model mice(P<0.01-0.05).④Compound mangiferin could obviously decrease the protein expression of MUC5AC in lung tissue(P<0.01-0.05), and obviously increase the expression of AQP5in lung tissue(P<0.01-0.05).⑤Compound mangiferin could obviously increase the content of GSH, SOD and T-AOC(P<0.01-0.05), obviously decrease the content of MDA.⑥Compound mangiferin could obviously inhibit the gene expression of TNF-α, CXCL8, TGF-β1and MUC5AC mRNA in lung tissue of model mice.Conclusions:1Establish the COPD model (with the syndrome of qi deficiency and phlegm statisis):The combination of the BML and LPS can successfully used in the establishing of the COPD mice model (with the syndrome of qi deficiency and phlegm statisis), which not only reflect the signs and symptoms of TCM(Qi deficiency, phlegm stasis, and blood stasis), but also reflect the pathological changes of COPD. 2Study on the efficacy of compound mangiferin:Compound mangiferin can restore the growth of mouse weight, restore the spontaneous activity of mice, reduce the ability of skin blood stasis tongue and jaw performance, further confirm that compound mangiferin has effects of nourishing qi, eliminating phlegm and activating blood.3Study on the mechanism of compound mangiferin:Compound mangiferin can protect the COPD mice model by the multiple mechanisms, including inhibition of lung tissue and airway inflammation, reducing mucus hypersecretion, improving the imbalance between oxidant and antioxidant. The mechanism may be related with the effects of mangiferin, including reducing the contents of1L-1β, IL-4, IL-8, TNF-α and TGF-β1in lung tissue, inhibiting the mRNA expression of CXCL8, TNF-α and TGF-β1, inhibiting the protein expression of MUC5AC, decreasing the content of TNF-α, inhibiting the mRNA expression of MUC5AC, enhancing the level of GSH, SOD and T-AOC in tissue of model mice, decreasing the level of MDA, down-regulating the protein expression of Nrf2, up-regulating the protein expression of Bachl, down-regulating the protein expression of γ-GCS.
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