摘要
【研究目的】 进一步观察哮喘时肺表面活性物质（Pulmonary Surfactant, PS）系统的变化，探讨PS与哮喘之间的关系，并应用药物盐酸氨溴索（ambroxol,商品名：沐舒痰）进行干预，观察其对哮喘大鼠气道炎症、肺表面活性物质（Pulmonary Surfactant, PS）系统的影响，探讨其在哮喘防治中的作用。
【研究方法】 本实验以改良方法用卵白蛋白致敏、激发建立大鼠哮喘模型，并分成三组，即正常对照组、哮喘组、哮喘( 盐酸氨溴索治疗组（简称治疗组），进行下列实验： 1.予卵白蛋白或生理盐水雾化吸入激发哮喘，测定气道内压的变化，验证哮喘模型，并观察盐酸氨溴索对哮喘大鼠呼吸力学的影响； 2.激发哮喘后，行支气管肺泡灌洗(bronchial alveolar lavage，BAL)，测定支气管肺泡灌洗液(bronchial alveolar lavage fluid，BALF)内细胞计数、分类计数，H-E染色及AB-PAS染色观察肺组织病理改变及杯状细胞增生状况； 3. 氯仿-甲醇萃取BALF中的PS并测定其总磷脂（TPL）、饱和磷脂（DSPC）和总蛋白质（TP）含量，膜天平测量BALF中PS表面活性； 4. Western Blot法测定BALF中肺表面活性物质结合蛋白－A（surfactant-associated proteins A ，SP-A）含量，免疫组化方法观察各组肺组织及气道中SP-A的变化。 5. 对BALF中表面活性指标与SP-A、DSPC及DSPC/TP、TP的进行多元相关分析。
【研究结果】1.哮喘激发前各组气道内压无显著差异（P＞0.05），抗原激发后哮喘组气道内压显著增加，气道内压增加的百分数显著高于对照组（119.2％±30.1％ vs 18.4％±4.7％，P＜0.01）,治疗组气道内压增加的百分数明显低于哮喘组( 58.8％±19.0％ vs 119.2％±30.1％，P＜0.01）； 2.哮喘组BALF细胞总数较对照组显著增加（20.29±4.55×106 vs 6.35±1.17×106，P＜0.001）,BALF中巨噬细胞、中性粒细胞、淋巴细胞、嗜酸性粒细胞均较对照组明显增多（P＜0.001），治疗组BALF细胞总数明显低于哮喘组（12.70±2.03×106 vs 20.29±4.55×106，P＜0.001），哮喘组大鼠肺组织病理可见明显支气管收缩征象及其周围炎症细胞浸润，治疗组气道炎症明显减轻，AB/PAS染色显示哮喘组大鼠各级气道杯状细胞均较正常组明显增多（P＜0.01），可见气道粘液潴留，治疗组大鼠各级气道杯状细胞较哮喘组均明显减少（P＜0.01），未见粘液潴留； 3.哮喘组BALF中
TPL及DSPC含量与正常对照组无明显差异（P＞0.05），TP含量显著增加（172.67±74.53μg/ml vs 85.41±21.18μg/ml，P＜0.01），PS表面活性较对照组明显降低，表现为最小表面张力增高、稳定系数降低（P＜0.001），治疗组BALF中TPL含量较正常组显著增加（134.56±8.76μg/ml vs 116.05±10.20， P＜0.01）,TPL及DSPC含量均较哮喘组显著增加（TPL：134.56±8.76μg/ml vs 117.17±10.22μg/ml，P＜0.01；DSPC：68.66±33.16μg/ml vs 28.12±10.81μg/ml，P＜0.01），TP含量较哮喘组显著降低（106.11±22.41μg/ml vs 85.41±21.18，P＜0.01），PS表面活性较哮喘组及对照组显著增加（P＜0.01）； 4.哮喘组BALF中SP-A含量较正常组明显减少（SP-A光密度值17.01±4.53 vs 30.26±3.89，P＜0.001），治疗组BALF中SP-A含量较哮喘组明显增多（SP-A光密度值23.36 ±2.22 vs 17.01±4.53，P＜0.01），SP-A免疫组化检测结果与Western Blot结果一致； 5.多元线性相关分析提示BALF表面活性与DSPC及SP-A含量呈显著正相关，与TP含量呈显著负相关。
【结论】 1.大鼠哮喘发作时BALF中总磷脂含量无明显变化，SP-A含量减少，蛋白渗出增多，表面活性降低，存在PS相对不足及功能不良，BALF表面活性与支气管肺泡灌洗液中DSPC、SP-A、TP含量相关； 2. 盐酸氨溴索可刺激哮喘大鼠PS磷脂的合成及分泌，改善PS功能，对SP-A无明显刺激作用； 3. 应用盐酸氨溴索可减轻大鼠哮喘发作程度，改善哮喘气道炎症，对哮喘的防治有一定作用。
【Objective】 To investigate the relationship between pulmonary surfactant(PS) system and asthma, the present study was designed to observe the change of PS system in asthma rat. To evaluate the protective effects of Ambroxol in asthma, we observed the effects of Ambroxol treatment on PS system and airway inflammation of chronic asthmatic rat.
【Methods】 The rat asthma model were established with ovalbumin sensitize and challenge method. 3 group were studied: control group, asthma group and Ambroxol treated group(treated group):
1. The rats were challenged with aerosol of ovalbumin or saline and the change of intratracheal pressure of sensitized rats. 2. Bronchial alveolar lavage (BLA) were performed after challenge. The total and differential white blood cell counts of bronchial alveolar lavage fluid (BALF) were carried out. Lung tissue section were stained with hematoxylin and eosin for general morphology and Alician Blue-Periodic Acid Schiff (AB-PAS) for identification of goblet cell. The pathologic changes were observed inender optical microscope. 3.PS was extracted from bronchial alveolar lavage fluid (BALF) using chloroform/methanol and the amount of total phospholipid (TPL). disaturated phosphatidylcholine (DSPC), total protein (TP) were determined. Surface activity of PS in BALF were measured with Wilhelmy film balance; 4.The concentration of pulmonary surfactant protein A(SP-A) in the BALF and the distribution of SP-A in the lung were assayed by Western Blot and immumohistochemistry(IHC) methods, respectively, 5. The bivariate co-relationships between the surface activity of PS in BALF and the amounts of DSPC、TP、SP-A in BALF were analysed.
【Result】1. The intratracheal pressure of sensitized rats in 3 groups before antigen challenge were similar. The intratracheal pressure of rats in asthma
group significantly increased after antigen challenge and the percentages of intratracheal pressure increasing were higher than those in the control group（119.2％±30.1％ vs 18.4％±4.7％，P<0.01）.The percentages of intratracheal pressure increasing in treated group were significantly decreased than those in the asthma group（58.8％±19.0％ vs 119.2％±30.1％，P<0.01）. 2. The total cell numberS were significantly increased in the asthma group than those in the control group(P<0.001）The number of eosinophils and netrophils were significantly increased in the asthma group than those in the control group and the number of macrophages and lymphocytes likewise. The total cell numbers were significantly decreased in the treated group than those in the asthma group(P<0.001）.The bronchoconstriction and inflammatory cells infiltration surrounding bronchi in the asthma group were observed. Goblet cell hyperplasia（GCH）and mucus retarded in airway lumen in the asthma group were also observed. The airway inflammation and GCH were significantly alleviated in treated group. 3. TPL and DSPC in asthma group were similar to those in the control group(P>0.05）. The concentration of TP in BALF increased obviously in asthma group than those in other two groups(p<0.01).We also found that the of PS in BALF had a significantly decrease in asthma group. It indicated an increase in minimum surface tension(STmin) and a decrease in stability index(SI). The concentration of TPL in BALF were much higher in treated group than those in the of other two groups(p<0.01). The concentration of DSPC in BALF were much higher in treated group than those in the asthma groups(p<0.01). The PS in BALF of treated group revealed a better surface activity. 4. When compared with normal rats, there were marked reductions of SP-A levels in BALF of asthmatic rats (p<0.01).The SP-A levels in BALF of treated rats were higher than those of asthmatic rats. 5. The surface activity correlated positively with the DSPC and SP-A levels in BALF and correlated negatively with the TP levels in BALF.
【Conclusions】1.These results indicated the involvement of pulmonary surfactant system in the allerigic bronchial inflammation of asthmat
引文
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