| BackgroundBronchial asthma is a chronic airway inflammatory disease mediated by a variety of cells and cell components, which is one of the most common chronic respiratory diseases in the world and also one of the highest lifetime prevalence of chronic diseases. Asthma severely affects personal health and brings in huge social burden. Global research scholars focus on its pathogenesis and prevention methods. Airway remodeling is one of the characteristics of asthma, which is considered to be an important cause of irreversible respiratory airflow limitation and continued airway hyperresponsiveness, resulting in pathological basis of refractory asthma. The mechanism of current commonly used drug for the treatment of asthma in improving the efficacy of airway remodeling is not exact, and the research on the pathogenesis and treatment method of airway remodeling in asthma research is a hotspot. Vitamin D has traditionally been considered essential nutrient for the human body, but recent studies have found that it ban be used as a steroid hormone and its role is much more than this. In asthma, the current study has found that VitD may be related to occurrence and control of asthma, and glucocorticoid sensitivity of asthma patientsthrough gene, anti infection, affecting lung development and enhancing glucocorticoid response mechanism, and studies have shown that VitD could reduce airway remodeling. Airway epithelial injury in the pathogenesis of asthma is getting more and more attention, and a series of studies have shown an association between asthma and clinical manifestations with airway epithelial physical barrier and biological barrier function. Epithelial cell apoptosis is one of the manifestations of epithelial injury, and apoptosis of AECs under physiological conditions is required for maintaining the homeostasis of body, but excessive apoptosis of AECs under pathological conditions is harmful, which is relatedto airway inflammation, airway remodeling, and airway hyperresponsiveness. The current research on asthma airway epithelial cell apoptosis degree is reported and inconsistent with each other. Many studies show that the effect of VitD on apoptosis is bidirectional, and effects of VitD on apoptosis of airway epithelial cells has not been reported. In this study, we investigated whether VitD can inhibit airway remodeling in asthma, and airway epithelial cell apoptosis, and effect of VitD on apoptosis of asthmatic airway epithelial cells through the establishment of asthma airway remodeling mice model.ObjectivesTo investigate the effects of 1,25-(OH)2D3on airway remodeling and airway epithelial cell apoptosis of asthma in a murine model.Methods1. Sixties clean grade female Balb/c mice weightng of 17g-19g were randomly divided into 3 groups:normal control group (group A), asthma group (B group) and vitD intervention group (C group) with 20 mice in each group. Ovalbumin (OVA) sensitization and excitation method was employed to construct murine model of chronic asthma mice.24 h after the last challenge, half of the mice were intraperitoneally injected for anesthesia, and trachea was cut open for intubation. Pulmonary function at basal condition and different concentrations (0.025,0.05; 0.1,0.2mg/kg) of methacholine (Ach) were measuredincluding inspiratory resistance (Ri), expiratory resistance (Re), and compliance (Cdyn); The other half of the mice were sacrificed to detect relevant indicators of airway remodeling. HE staining of lung tissues was conducted and observed under light microscope. The complete bronchial cross-section was found and analyzed by using image analysis software for testing of bronchial basement membrane perimeter (Pbm), and total bronchial wall area (Wat). The bronchial pipe wall thickness was expressed as Wat/Pbm. Lung tissues were performed AB-PAS staining, Masson trichrome staining, and a smooth muscle actin (a-SMA) for the immunohistochemical staining to observe airway mucus secretion, collagen fibers hyperplasia and airway smooth muscle thickness. The results were expressed as AB-PAS positive staining area/Pbm (AB-PAS (+) area/Pbm, Massonpositive staining area/Pbm (Wcol/Pbm), a-SMA staining positive area/Pbm (alpha-SMA (+) area/Pbm).2. Mouse lung tissue paraffin sections was used by terminal transferase dUTP nick end labeling enzyme mediated method (TUNEL method) to detect apoptosis of airway epithelial cells, and the apoptosis was expressed as index (AI); immunohistochemistry staining was used to detect expression of Bcl-2 in the airway epithelium, and the average optical density (OD) value represented the expression。Rresults1. Lung function:the values of Ri in A, B and C groups were (1.16±0.06), (1.49 ±0.08), and (1.29±0.05) cmH2O/(ml/s); The values of Re were (1.02±0.15), (1.48±0.16)) and (1.29±0.07) cmH20/(ml/s). The values of Cdyn were (0.03±0.03), (0.02± 0.04), and (0.02±0.02) mL/cmH2O-The values of Ri and Re in B and C groups were higher than those of group A, and the values of compliance (Cdyn) were lower than those of A group, and the differences were statistically significant (P values were <0.05); Ri and Re in group C was lower than that in group B (P<0.05). When stimulated by Ach, B and C group showed airway hyperresponsiveness. Under the concentration gradient of Ach excitation, the percentage change of Ri, Re under basal level in B, C group had no significant difference; when the concentration of Ach reached 0.2mg/kg, the percentage changeof Cdyn ofgroup C was less than that of group B (P<0.05).2.Lung tissue pathology(HE staining):under the light microscope, the bronchial epithelium mucosa in A group, and muscularis mucosa and lung tissues had structural integrity, and the bronchial lumen was neat. There was no obvious inflammatory cell infiltration. For the mice in group B, there was stenosis, and airway wall was significantly thickened, and there was thickening of bronchial mucosa. There was visible mucus plug and bronchial epithelial shedding, and the airway smooth muscle was thickened. The peribronchial and submucosal lays had lots of inflammatory cell infiltrations. Tracheal wall thickening and perivascular inflammation in C group was decreased compared with the mice in group B. TheWat/Pbm of the mice in A, B and C groups were(7.79±1.01), (19.24±1.70), (14.12±2.13)um2/um, respectively. The values of B and C groups were significantly higher than those of group A (P<0.05), and the value of C group was lower than that in group B (P<0.05).3. Mucus secretion(AB-PAS staining):AB-PAS positive signal was blue (acidic mucus), red (neutral mucus) or mixed with purple red. Under the microscope in group A, there was no or only a point AB-PAS positive signal. AB-PAS positive signal area of B groupincreased significantly. AB-PAS positive signal area ofC group was more than A group, but compared with the B group, it was less.The values of AB-PAS(+)area/Pbm of mice in A, B and C group were (0.04±0.02), (5.22±0.90) and (3.71±0.56)um2/um, respectively. The values in B and C groups were significantly higher than those of group A (P<0.01), and the value of C group was lower than that in group B (P<0.05).4. Collagen fibers deposition(Masson staining):Airway subepithelial collagen fiber was dyed green. collagen fibers of A group was a filmy layer around the trachea. airway subepithelial collagen deposition of B group was significantly increased, and there was formation of a thick and dense layer. When compared with A group, collagen depositionof C group was significantly increased, but compared with the B group, it was decreased. The values of Wcol/Pbm of mice in A, B and C groups were (1.37±0.25)、 (7.63±1.55)、(4.31±0.65) um2/um, respectively. The values of B and C groups were significantly higher than those of group A (P<0.05), and the value of C group was lower than that in group B (P<0.05).5.Airway smooth muscle thickness(α-SMA immunohistochemical staining):Airway smooth muscle layer was stained brown yellow. In group A, the positive region was visible as a thin layer around the trachea. In B and C group,the positive region was thickened, and that in C group was less than that in B group.The valurs of a-SMA(+)area /Pbm of mice in A, B and C group were (1.40±0.24), (5.40±0.69) and (3.27±0.46) um2/ um, respectively. The values of B and C groups were significantly higher than those of group A (P<0.05), and the value of C group was lower than that in group B (P<0.05).6. AECs apoptosis(TUNEL staining):Brown nuclear staining was positive. In group A, TUNEL (+) cells was not or rarely seen. In group B and group C, TUNEL (+)cells were significantly increased, and TUNEL (+) cells ratio in group Cwas less than that in group B. The AI values of the mice airway epithelial cells in the A, B and C groups were (0.0045±0.0038), (0.2973±0.0574), (0.1489±0.0175), respectively. The AI values of B and C groups showed significant differences (P<0.01).7. Bcl-2 immunohistochemical staining:Bcl-2 immunohistochemical staining: positive cell cytoplasm and cell nucleus was brown yellow. The high expression of Bcl-2 protein was seen in AECs of group A. In group B, Bcl-2 in AECs was little expression. In group C, Bcl-2 inAECs was expressed, but the content was lower than that of A group.The immunohistochemical OD values of the mice airway epithelial Bcl-2 in A, B and C groups were (0.1603±0.0209), (0.0912±0.0230), (0.1139±0.0086), respectively. The OD values of B and C groups were significantly lower than those of group A (P <0.05), and the OD value of C group was higher than that in group B (P<0.05).Conclusions1.1,25-(OH)2D3 attenuatesairway remodeling of chronic asthma in a murine model;2. The apoptosis rate ofepithelial cell of chronic asthma in a murine model is increased;3.1,25-(OH)2D3 can reduce the apoptosis of airway epithelial cells in chronic asthmain a murine model;4. The mechanism of 1,25-(OH)2D3 in reducing apoptosis of airway epithelial cells in chronic asthma is by up-regulation of expression ofBcl-2 proteinin a murine model. |
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