固本防哮饮对哮喘缓解期小鼠气道上皮紧密连接蛋白及CCSP的影响

  目的  研究固本防哮饮对哮喘缓解期小鼠气道上皮紧密连接蛋白及Clara细胞分泌蛋白(CCSP)的影响, 探讨其防治缓解期哮喘的可能机制。  方法  联合使用卵清蛋白(OVA)及呼吸道合胞病毒(Respiratory syncytial virus, RSV)建立哮喘缓解期小鼠模型, 将36只Balb/c小鼠随机分为正常组、模型组、固本防哮饮低剂量组、固本防哮饮中剂量组、固本防哮饮高剂量组和孟鲁司特钠组, 每组6只。模型建立完成后, 固本防哮饮各剂量组分别予不同剂量(12、24、36 g·kg-1)固本防哮饮, 孟鲁司特钠组予2.6 mg·kg-1孟鲁司特钠，各组均灌胃28 d。末次给药后24 h留取样本。使用苏木精-伊红(HE)染色法观察各组小鼠肺组织病理; Western blot、qPCR及免疫组化检测各组小鼠肺组织中闭锁小带蛋白-1(ZO-1)等气道上皮紧密连接蛋白表达; ELISA法检测肺组织及肺泡灌洗液(BALF)中CCSP的表达。  结果  HE染色结果显示, 与正常组相比, 模型组小鼠肺组织中可见明显的炎性细胞浸润(P < 0.01), 固本防哮饮中、高剂量组小鼠肺组织中炎性细胞浸润减少(P < 0.05，P < 0.01);与正常组相比，模型组中ZO-1、闭合蛋白(Occludin)和桥粒芯胶蛋白(Desmocollin)的蛋白表达水平显著降低(P < 0.05，P < 0.01)，免疫组化结果显示上述指标蛋白在模型组中表达减少(P < 0.01, P < 0.001)且排列松散, 固本防哮饮各剂量组对上述蛋白表达有不同程度地回调; 同时，模型组中ZO-1、ZO-2、纽带蛋白(Vinculin)、连环蛋白(Catenin) mRNA的表达水平显著降低(P < 0.05，P < 0.01)，固本防哮饮高剂量组ZO-1、ZO-2、Vinculin、Catenin mRNA表达水平显著升高(P < 0.05);与正常组相比, 模型组小鼠肺组织及BALF中CCSP的表达显著下降(P < 0.01), 低、高剂量固本防哮饮显著增加肺组织及BALF中CCSP的表达(P < 0.05, P < 0.01)。  结论  固本防哮饮能够上调气道上皮紧密连接蛋白以及CCSP的表达水平, 修复上皮损伤, 恢复上皮屏障功能, 从而防治哮喘。 

Effects of Guben Fangxiao Decoction on Airway Epithelial Tight Junction Protein and CCSP in Mice of Asthma in Remission Stage

  OBJECTIVE  To observe the effects of Guben Fangxiao Decoction (GBFXD) on airway tight junction protein and CCSP, and to explore the possible mechanism of GBFXD on airway inflammation in remission stage of asthma.  METHODS  Mouse model of asthma in remission stage was established by sensitization and stimulation of OVA combined with respiratory syncytial virus (RSV) infection. Mice were randomLy divided into six groups (n=6): control group, model group, GBFXD low-dose group (GBFXD-L), GBFXD medium-dose group (GBFXD-M), and GBFXD high-dose group (GBFXD-H) and montelukast group.The mice were developed asthma model and given different concentrations of GBFXD (12, 24, 36 g·kg-1)and montelukast sodium (2.6 mg·kg-1) by gavage according to groups for 28 days. The mice were sacrificed and samples were obtained after the intervention. Hematoxylin-eosin (HE) staining was used to observe the pathological features of lung tissues in each group. The expression of CCSP in lung tissues and alveolar lavage fluid (BALF) was determined by ELISA. Western blot, qPCR and immunohistochemical assay were used to detect the expression of ZO-1 and other airway epithelial tight junction proteins in lung tissues of mice in each group.  RESULTS  Compared with the control group, the model mice in remission stage are mainly infiltrated by inflammatory cells in the lung tissue (P < 0.01), and the infiltration of inflammatory cells in the lung tissues of mice was alleviated after the treatment (P < 0.01). The protein expression levels of ZO-1, Occludin and Desmocollin in the model group were significantly lower than those in the normal group (P < 0.05, P < 0.01), immunohistochemical results showed that the expression of the above indexes was reduced and the proteins were loosely arranged in the model group. Meanwhile, the mRNA expression levels of ZO-1, ZO-2, Vinculin and Catenin in the model group significantly decreased (P < 0.05, P < 0.01). Compared with the normal group, the expression of CCSP in the model group significantly decreased in lung tissue and BALF (P < 0.01), while the expression of CCSP in BALF and lung tissue was significantly increased by the intervention of low and high doses of GBFXD (P < 0.05, P < 0.01).  CONCLUSION  GBFXD can repair the epithelial injury and restore the epithelial barrier function by up-regulating the expression of epithelial tight junction protein and CCSP, an indicator of epithelial cell permeability change, so as to reduce the impact of various external pathogenic factors on the airway and preventing and treating asthma.