| Objective:To study the expression changes of aquaporin-4(AQP4) and mucin5AC (MUC5AC) in bronchial and lung tissue of asthmatic mice and the effect of dexamethasone(DEX) on them, and to explain the role of AQP4and MUC5AC in the pathogenesis of asthma. Methods:24five-week-old C57BL/6mice were randomly divided into three groups: control group, asthma group and dexamethasone treated group(DEX group),8mice in each group. The mice in asthma group were immunized on days0and14by intraperitoneal injection of0.2mL freshly prepared ovalbumin (OVA) and aluminum hydroxide suspended solution (1g/L OVA+lOg/L aluminum hydroxide), and were challenged by intranasal administration of50μL lg/L OVA solution on days21,22and23. Mice in DEX group, sensitized and challenged by method described above, were administered an extra intraperitoneal injection of1.0mg/kg dexame-thasone at30min before OVA instillation at every time. The mice in control group were given intraperitoneal injection and intranasal administration of an equal volume of sterile normal saline in a same manner.24h after the final OVA or saline challenge, mice were sacrificed and the samples were collected. The lung morphological changes were observed with hematoxylin and eosin (HE) staining and airway goblet cell and mucus secretion were evaluated with alcian blue-periodic acid-schiff (AB-PAS) staining. The quantitative analysis of pathological changes was performed with scoring method described by Underwood. The expression of AQP4and MUC5AC protein in bronchial and lung tissue was measured by immunohistostaining method. Quantitative real-time PCR was used to evaluate AQP4and MUC5AC mRNA expression. The content of AQP4, MUC5AC and housekeeping genes beta-actin mRNA were measured with SYBR-green I as fluorescence indicator and relative expression quantity of AQP4, MUC5AC mRNA was calculated using the double delta delta cycle threshold (Ct) method. The statistical analyses were performed using SPSS13.0statistical data processing software. All datum were expressed with mean±SD. A one-way analysis of variance followed by Student-Newman-Keuls (SNK) was performed to determine the statistical significance of groups. Correlation between variables was determined by linear correlation analyses methed.P<0.05was considered significant difference. Results:(1) HE staining:Asthma group showed the inflammatory cells infiltration around the bronchioles, bronchial mucosa and perivascular tissues, stricture with mucus plug of Lumen, shedding of airway epithelial cells, hypertrophy of airway smooth muscle, whereas control group exhibited no these features.The above changes of DEX group were significantly improved when compared with asthma group. The pathological score of asthma group (4.2±0.676) was significantly higher than that of the control group (0.6±0.507), the difference was statistically significant(P<0.05), DEX group score(2.4±0.737)was significantly lower than asthma group, but higher than control group (P<0.05).(2) AB-PAS staining:There was marked goblet cell hyperplasia and mucus hypersecretion within the bronchi from asthma group, but not within the bronchi from the control group. Treatment with dexame-thasone significantly attenuated the OVA-induced mucus secretion and goblet cell hyperplasia. The pathological score of asthma group (3.4±0.672) was significantly higher than that of the control group (0.7±0.723)(P<0.05). DEX group (2.1±0.545) was obviously reduced when compared with the asthma group (P<0.05).(3) Immunohisto-chemistry:AQP4protein was expressed on the basolateral side of bronchial epithelia, submucosal microvessels of airways and type I alve-olar epithelial cell, and MUC5AC protein was mainly expressed in submucosal layer of airways. There were obviously decreased expression of AQP4and significantly increased expression of MUC5AC in the asthma group relative to the control group.Treatment with dexame-thasone increased the expression of AQP4and attenuated the expression of MUC5AC when compared with the asthma group.(4) Quantitative real time PCR:AQP4mRNA expression in asthma group (1.1923±0.1933) was significantly lower than that in control group (4.4220±2.1953)(P<0.05). There was significantly increased expression of AQP4mRNA in DEX group (2.4132±1.0997) compared with the asthma group (P<0.05). MUC5AC mRNA expression in asthma group (9.1876±3.0894) was significantly higher than that in control group (1.0945±0.1920) and there was significantly reduced expression of MUC5AC mRNA in DEX group (2.4132±1.0997) compared with the asthma group (P<0.05), but was still higher than that in control group (P<0.05).(5) Linear correlation analyses showed a negative correlation between AQP4and MUC5AC mRNA expression in asthma group and DEX group (r-0.758,-0.591, allP<0.05). Conclusions:(1) C57BL/6mouse model of asthma was successfully established;(2) Ovalbumin-induced mice models of asthma were characterized by airway inflammation and mucus hypersecretion;(3) The expression of AQP4was reduced and the expression of MUC5AC was increased in bronchial and lung tissues of asthmatic mice, which might be involved in airway inflammation and mucus hypersecretion of acute allergic asthma;(4) Dexamethasone could up-regulate AQP4expression and down-regulate of MUC5AC expression in asthmatic mice, which might be one of the mechanisms for inhibiting airway inflammation and mucus hypersecretion. |
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