桔梗皂苷对慢性支气管炎小鼠气道重塑的干预作用研究

探讨桔梗皂苷(kikyosaponin,简称KS)对慢支小鼠气道重塑的干预作用机制.将50只健康小鼠分成正常对照组,模型组和桔梗皂苷低、中、高剂量组,除正常组外,其余4组动物均采用烟熏加浓氨水吸入法建立慢支模型,然后分别用药物进行治疗.实验结束后,取各组小鼠肺组织进行石蜡制片,苏木精-伊红(HE)染色分析气管管壁厚度变化,Masson染色分析肺组织胶原沉积,采用Image-Pro Plus 6.0图像分析软件测量总管壁厚度(WAt/Pbm)、内壁厚度(WAi/Pbm)、平滑肌厚度(WAm/Pbm)以代表气道重塑指标;生化法检测肺组织中羟脯氨酸(Hyp)含量;免疫组织化学染色法观察MMP-9在肺组织中的表达;蛋白印迹术分析MMP-9和TIMP-1在肺组织中的表达水平.HE染色显示,模型组肺组织中支气管明显狭窄,管腔及肺泡中含有大量炎性分泌物,且气道重塑的三项指标均异常增高;免疫组化和三色法显示,模型组小鼠支气管和肺泡细胞中有大量的MMP-9表达,且有胶原蛋白增加明显,生化检测发现模型组小鼠肺组织中Hyp含量亦异常升高,且MMP-9和TIMP-1表达水平升高显著,与正常对照组相比,差异十分显著(P0.01).在连续用药30 d后,与模型组相比,桔梗皂苷各剂量组能明显抑制慢支小鼠支气管胶原纤维增生,改善气道重塑各项指标;减少MMP-9和TIMP-1表达水平,使气管重塑得到显著的减轻(P0.05,P0.01,P0.01).结果显示,桔梗皂苷可明显减轻慢支小鼠气道重塑的病理改变,其干预机制可能是通过清除慢支小鼠肺组织中的炎性因子和自由基的含量,同时又能抑制MMP-9和TIMP-1的表达,从而达到减少胶原蛋白的沉积以减轻气管狭窄而达到逆转气道重塑之目的.

Study on intervention effects of kikyosaponin on the airway remodeling in chronic bronchitis(CB) mice

The authors have investigated the intervention effects of kikyosaponin(KS) on the airway remodeling of chronic bronchitis(CB) mice.Fifty healthy mice were divided into five groups: normal control group,chronic bronchitis model group and treatment groups with low dose of kikyosaponin, middle dose of kikyosaponin and high dose of kikyosaponin.Each group of mice inhaled dense smoke and stronger ammonia water to establish a model of chronic bronchitis except the normal control group,and then respectively was treated by kikyosaponin.After the experiment,the lung tissue of mice from each group was removed for paraffin section.Hematoxylin and eosin(HE) staining was performed to observe thickness of the airway wall,and Masson staining was used for detecting collagen deposition of lung tissues.Thicknesses of total airway,airway wall and airway smooth muscle were measured by IPP 6.0 for airway remodeling indicators.Hydroxy proline(Hyp)was detected by biochemical methods.Expression of MMP-9 of lung tissue was analyzed by immunohistochemical method.Expressions of MMP-9 and TIMP-1 of lung tissue were analyzed by western blot.HE staining showed that the bronchi of lung tissues in model group got narrow significantly.There were lots of inflammatory secretions in airway lumens and alveoli.The three airway remodeling indicators increased obviously.Immunohistochemical and staining methods showed the abundant expression of MMP-9,and the collagen deposition increased significantly in the model group.Biochemical methods revealed that there was an unusual increase in the concentration of Hyp,and expressions of MMP-9 and TIMP-1 rose significantly compared with the normal group.After treatment for 30 days,kikyosaponin could inhibit the collagen fibrous proliferation of mice bronchi and decrease airway remodeling indicators and expressions of MMP-9 and TIMP-1 in different doses of groups,which improved the airway remodeling(P<0.05,P<0.01,P<0.01).The results showed that kikyosaponin can obviously improve the pathological changes of airway remodeling in CB mice.We speculate that,through eliminating the inflammatory factors and free radicals in lung tissues of mice and inhibiting the expression of MMP-9 and TIMP-1,kikyosaponin can decrease the collagen deposition and alleviate the tracheal stenosis,and then finally reverse airway remodeling.