| Introduction and ObjectiveBronchial asthma is one of the most common chronic respiratory disease. Persistant allergic airway inflammation is the pathological and physiological basis of recurrent attacks of bronchial asthma. During the proceed of allergic airway inflammation, airway epithelial was damaged by sensitizing, biotoxin and so on. The impaired epithelia interact with dendritic cells, smooth muscle cells, fibroblast and inflammatory cells through the cytokines which become the priming and center piece in the asthma onset. They participate the airway inflammation, mucus hypersecretion, airway remoding and airway hyperresponsiveness. Then identifying the critical signaling molecule which affect the structural integrity or defense function of airway epithelial and dendritic cells, and on the basis of which, setting up effective intervention will be of great significance. It is the hot spot of international respiratory physiological research at present.Since professor Smith discovered AQP1 in 1988, so far 13 different AQPs have been identified in mammals, AQP0-12. At least 6 kinds of AQPs are expressed in airways and lung, which are AQP1, AQP3, AQP4, AQP5, AQP8 and AQP9. AQPs provide a major pathway for osmotically-driven water movement across epithelial and endothelial barriers in the airways, alveoli and pleura. Cell-specific and regulated expression of AQPs in the lung and airways has provided indirect evidence supporting their physiological role. The possibility remains that new functions of AQPs in the lung and airways will emerge, perhaps related to the new roles of AQPs in dendritic cells migration and apoptosis. We postulate that if we can affect the water movement across DCs to interfere its migration and apoptosis through regulating expression of AQPs in DCs so that we should control the allergic airway inflammation.Studies on effect of AQPs in lung under different pathological condition have been done, while little about the role of AQPs in asthma has been shown. We want to affluence the physiological significance of AQPs in lung tissue by studying the effect of AQPs on bronchial asthma. We approached the changes of AQPs and MUC5AC expression in lung tissue during allergic airway inflammation in a murine model of asthma and the regulation of dexamethasone on them. We emphasize the effect on allergic airway inflammation and synthesis and secretion of MUC5AC by the change of AQP5 expression. According to the founction of AQP5 on the dendritic cells, we indicate the possible mechanism by which AQP5 knockout ameliorate allergic airway inflammation. Then a new target was issued for the more effective prevention and cure strategy.Methods1. Setting up the murine model of bronchial asthma.72 male C57BL/6 mice were divided into 3 groups: control group, asthma group and Dex group. Mice were sensitized on days 1 and 15 by intraperitoneal injection of 20μg ovalbumin emulsified in 2.6 mg aluminum hydroxide with a total volume of 200μl. On days 25-29, after the initial sensitization, the mice were challenged for 30 min with an aerosol of 1% (w/v) OVA in PBS (or with PBS as a control) using an ultrasonic nebulizer. The Dex group was administrated with 1mg/kg dexamethasone by intraperitoneal injection 30 min before every challenge. We valued the pathological section of lung, AB-PAS score and wet/dry ratio, ELGV and lung density. The concentration of IL-13 and IFN-γin serum and BALF were quantified by ELISA according to the manufacturer's protocol. 2. Changes of AQPs and MUC5AC expression in lung tissue from the murine model of bronchial asthma and the regulation by dexamethasone. mRNA expression of AQP1, AQP3, AQP4, AQP5 and MUC5AC was relatively quantitified by real-time PCR. Distribution of AQPs in lung was stained by immunohistochemistry. The concentration of MUC5AC protein in BALF was valued by ELISA.3. If AQP5 knockout affect the allergic airway inflammation and synthesis and secretion of MUC5AC in murine asthma model.The AQP5+/+ mice and the AQP5?/? mice were obtained enough by mating of those heterozygous mice after gene identification by PCR. They were devided into 4 groups as AQP5+/+control group, AQP5+/+ OVA group, AQP5?/? control group and AQP5?/? OVA group, n=24/each group. We valued the pathological section of lung, AB-PAS score and wet/dry ratio, ELGV and lung density. The concentration of IL-13, Eotaxin and IFN-γin serum and BALF were quantified by ELISA. mRNA expression of MUC5AC was relatively quantitified by real-time PCR. Distribution of MUC5AC in lung was stained by immunohistochemistry. The concentration of MUC5AC protein in BALF and lung tissue was valued by ELISA.4. Effect of AQP5 on the murine marrow DCs.The murine marrow DCs were isolated from AQP5+/+ and AQP5?/? mice. All DCs were collected and enriched by density centrifugation over immunomagnetic beads. FACS analysis revealed that almost all cells coexpressed the DCs marker CD11c. mRNA expression of AQP5 was valued by RT-PCR and protein expression of AQP5 was revealed by FACS in DCs. The DCs from AQP5+/+ and AQP5?/? mice were divided into two groups. FACS analyzed expression of the cell surface molecular CD80, CD86, CD40. Antigen special T cells were purifed from splenocytes of DO10.11 mice. Mixed lymphocyte reaction was measured by [3H]TdR. For measurement of antigen uptake activity of immature DCs from different group by FACS, OVA-FITC was used as antigen.Results1. Setting up murine model of bronchial asthma successfully which was regulated by dexamethasone efficiently.Total cell counts, eosinophil and lymphocyte in BALF and lung tissue of asthma group were increased significantly while dexamethasone decrease them. The levels of IFN-γand IL-13 were regulated in dexamethasone-treated group [(89.25±9.71) and (52.37±8.69) pg/ml] and had significant difference with asthma group[(66.71±7.51) and (85.49±9.23) pg/ml]( P < 0.05). The valuation of the pathological section of lung, AB-PAS score and wet/dry ratio, ELGV and lung density showed that the murine model of bronchial asthma was successful and dexamethasone regulated it efficiently.2. Changes of AQPs and MUC5AC expression in lung tissue from the murine model of bronchial asthma and the regulation by dexamethasone. Expression of AQP1, AQP4, AQP5 in lung was decreased (both mRNA level and protein level) while AQP3 and MUC5AC was increased (mRNA level and protein level) more obviously 24h after the last OVA-exposure in AQP5+/+ mice than that of control mice and the difference was significant (P < 0.05). There was significant negative regulation by dexamethasone on the expression of AQP1, AQP3, AQP5 and MUC5AC in lung of asthmatic mice while no effect on expression of AQP4 was showed.3. AQP5 knockout affect the allergic airway inflammation and synthesis and secretion of MUC5AC in murine asthma model.There is obvious increase in concerntration of protein and MUC5AC in BALF from AQP5-/- control mice compared with AQP5+/+ control mice (P < 0.05), while no difference in other valuement between them. While specifically in asthma model groups, AQP5 knockout not only significantly inhibited OVA-induced pulmonary inflammation by 35.9%, synthesis and secretion of MUC5AC by 66.8% and 53.1% in lung tissue and BALF separately although no difference in mRNA expression of MUC5AC, but also reduced the wet-to-dry ratio of lung by about 60.2% compared with AQP5+/+ allergic model mice (P < 0.05). Interestingly we also found that the wet/dry ratio and ELGV were regulated obviously by AQP5 knockout in asthma model group (P < 0.05).4. Effect of AQP5 on the murine marrow DCs.With the simultaneous induction by GM-CSF and IL-4, isolated DCs from different group were cultured in vitro successfully and showed clinical morphology of mature DCs. After collection by density centrifugation over immunomagnetic beads, CD11C+ cells were increase from 54.8%±5.12% to 95.13%±2.43%. We also found AQP5 was expressed on the surface of DCs. Furthermore, we evaluated expression of the cell surface molecular CD80, CD86, CD40 and mixed lymphocyte reaction which was decreased and the antigen uptake activity of immature dendritic cells (DCs) which was downregulated obviously from AQP5?/? control mice by 18%, 25%, 41%, 42% after different incubation time (P < 0.05).Conclusion1. We set up murine model of bronchial asthma successfully by OVA sensitization and challenge. ELGV may be as a measurement to value the lung compliance in asthma model indirectly.2. mRNA and protein of AQP1, AQP4 and AQP5 were decreased obviously in lung tissue from murine model of bronchial asthma, while AQP3 was increased.3. mRNA and protein of MUC5AC were increased obviously in lung tissue from murine model of bronchial asthma.4. Dexamethasone treatment regulated allergic airway inflammation, mucus secretion and the expression of AQP1, AQP3, AQP5 and MUC5AC, except AQP4.5. The AQP5+/+ mice and the AQP5?/? mice were obtained enough by mating of those heterozygous mice after gene identification by PCR.6. AQP5 knockout attenuated allergic airway inflammation, synthesis and secretion of MUC5AC and lung edema in murine asthma model.7. There was AQP5 expressed on DCs. AQP5 knockout reduce the expressions of costimulated molecule, stimulation on antigen specific T cells and the endocytic activity of DCs which maybe attributed to the effect on allergic airway inflammation, synthesis and secretion of MUC5AC and lung edema in murine asthma model.Innovation1. We indicate ELGV may be as a measurement to value the lung compliance in asthma model indirectly.2. Value the changement of expression of AQPs in lung tissue from murine asthma model systematically and the regulation on AQPs by dexamethasone.3. We found AQP5 knockout attenuated allergic airway inflammation, synthesis and secretion of MUC5AC and lung edema in murine asthma model.4. We indicate AQP5 knockout attributed to the effect on allergic airway inflammation, synthesis and secretion of MUC5AC and lung edema in murine asthma model which maybe by reducing the expressions of costimulated molecule, stimulation on antigen specific T cells and the endocytic activity of DCs.On the whole, our studies affluence the physiological significance of AQPs in lung tissue and pathological mechanism of asthma. We indicate a new target for treatment of asthma. Furthermore study on the exact role of AQPs in asthma and their regulation on allergic airway inflammation and mucin secretion should be of great significance for academic research and clinical treatment.
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