骨化三醇对哮喘小鼠气道重塑及气道上皮细胞凋亡的影响

Effects of 1, 25-（OH）2D3 on airway remodeling and airway epithelial cell apoptosis in a murine model of asthma

目的 观察骨化三醇对支气管哮喘（简称哮喘）小鼠气道重塑及气道上皮细胞（AECs）凋亡的影响.方法 24只雌性清洁级Balb/c小鼠按随机数字表法均分为对照组、慢性哮喘组、骨化三醇干预组各8只：（1）慢性哮喘组：于第1、14天小鼠腹腔注射0.1ml致敏液[卵清白蛋白（OVA,20 μg）＋氢氧化铝凝胶（50μl）],从第21天开始雾化吸入1％ OVA溶液10 ml进行激发,30 min/次,3次/周,连续8周;（2）骨化三醇干预组：OVA致敏和激发同慢性哮喘组,每次激发前30 min予以100 ng骨化三醇腹腔注射;（3）对照组：使用生理盐水代替OVA进行致敏及激发.末次激发后24h内处死小鼠,取左肺多聚甲醛固定后石蜡包埋切片,分别行HE染色、阿利辛兰-过碘酸雪夫（AB-PAS）染色、Masson染色、α-平滑肌肌动蛋白（α-SMA）免疫组织化学染色,图像分析软件测定支气管基底膜周径、管壁总面积及AB-PAS、Masson、α-SMA染色阳性面积,结果均以基底膜周径将其标准化.石蜡切片经末端脱氧核糖核苷酸转移酶介导的dUTP缺口末端标记测定法（TUNEL）检测气道上皮细胞凋亡,B细胞淋巴瘤/白血病2 （Bcl-2）免疫组织化学染色法检测Bcl-2蛋白在气道上皮组织中的相对表达量.结果 慢性哮喘组、骨化三醇干预组小鼠出现慢性哮喘气道炎症及气道重塑典型改变.骨化三醇干预组管壁总面积及AB-PAS、Masson、α-SMA染色阳性面积与基底膜周径的比值分别为（14.12±2.13）、（3.72 ±0.57）、（4.31 ±0.65）、（3.27±0.46） μm2/μm,均显著低于慢性哮喘组的（19.24±1.70）、（5.23±0.90）、（7.63±1.55）、（5.40±0.69）μm2/μm而高于对照组的（7.79±1.01）、（0.05 ±0.03）、（1.37±0.25）、（1.40 ±0.24）.μm2/μm（均P＜0.01）.骨化三醇干预组气道上皮细胞凋亡指数显著低于慢性哮喘组而高于对照组[（14.89±1.75）％比（29.73±5.74）％、（0.45±0.38）％,均P＜0.01].骨化三醇干预组Bcl-2蛋白相对表达量显著高于慢性哮喘组而低于对照组（0.114±0.009比0.091±0.023、0.160±0.021,均P＜0.05）.结论 骨化三醇可减轻慢性哮喘小鼠的气道重塑及气道上皮细胞凋亡;其减少哮喘小鼠气道上皮细胞凋亡的机制涉及机体对线粒体凋亡途径重要分子Bcl-2的调控.

Objective To investigate the effects of calcitriol on airway remodeling and airway epithelial cell apoptosis in a murine model of bronchial asthma.Methods Twenty-four SPF female Balb/c mice were randomly allocated into three groups according to a random digits table with 8 mice in each group：the control group,the chronic asthma group and the calcitriol intervention group.（1） The chronic asthma group：the mice were sensitized by intraperitoneal injection of Ovalbumin （OVA,20 μg） and aluminium hydroxide （50.d） on days 1 and 14.From day 21,the mice were challenged by inhalation of 1% OVA solution （10 ml,30 min/time,three times a week for consecutively 8 weeks）.（2） The calcitriol intervention group：the mice were sensitized and challenged as above,and were given calcitriol 100 ng through intraperitoneal injection 30 min before every inhalation.（3） The control group：the mice were sensitized and challenged by saline instead of OVA.The mice were sacrificed 24 hours after the last challenge,and the left lung were removed,fixed with paraformaldehyde,embedded with paraffin and sectioned.HE staining,Alcian blue and Periodic acid Schiff （AB-PAS） staining,Masson staining,and α-smooth muscle actin （α-SMA） immunohistochemistry staining were conducted.Bronchial basement membrane perimeter （Pbm）,total bronchial wall area and positive areas of AB-PAS staining,Masson staining,and α-SMA staining were determined with image analysis software.The results were standardized with the basement membrane perimeter.Paraffin sections of mice lung tissue were detected with terminal transferase deoxyuridine triphosphate （dUTP） nick end labeling enzyme mediated method （TUNEL） for apoptosis of airway epithelial cells and with immunohistochemistry staining for expression of B cell lymphoma/lewkmia-2 （Bcl-2） in the airway epithelium.Results The mice in the chronic asthma group and calcitriol intervention group showed characteristic airway inflammation and airway remodeling of asthma.The ratios between the total bronchial wall area and positive areas of AB-PAS staining,Masson staining and α-SMA staining and the basement membrane perimeter in the calcitriol intervention group were （14.12 ± 2.13）,（3.72 ±0.57）,（4.31 ± 0.65） and （3.27 ± 0.46） μm2/μm,respectively,all of them were significantly lower than （19.24 ± 1.70）,（5.23 ±0.90）,（7.63 ± 1.55） and （5.40 ±0.69） μm2/μm in the chronic asthma group and higher than （7.79 ± 1.01）,（0.05 ± 0.03）,（1.37 ± 0.25） and （1.40 ± 0.24） μ m2/μm in the control group （all P 〈 0.01）.The airway epithelial cell apoptosis index in the calcitriol intervention group was significantly lower than that in the chronic asthma group and higher than the control group [（14.89 ± 1.75） % vs （29.73 ± 5.74） % and （0.45 ± 0.38） %,both P 〈 0.01].The relative expression of Bcl-2 in the calcitriol intervention group was significantly higher than that in the chronic asthma group and lower than the control group （0.114 ±0.009 vs 0.091 ±0.023 and 0.160 ±0.021,both P 〈0.05）.Conclusions Calcitriol attenuates airway remodeling and reduces the apoptosis of airway epithelial cells in a murine model of chronic asthma.The mechanism of calcitriol in reducing apoptosis of airway epithelial cells is by regulation of expression of the important molecule Bcl-2 protein in mitochondrial apoptotic pathway.