转化生长因子β1I型受体拮抗剂对支气管哮喘小鼠气道炎症及气道重塑的影响

Effects of TGF-β1 type I receptor antagonist on airway inflammation and remodeling in a murine model of chronic bronchial asthma

目的研究转化生长因子β1（TGF-β1）I型受体拮抗剂SB431542对慢性支气管哮喘（简称哮喘）小鼠模型气道炎症及气道重塑的影响，为哮喘的治疗提供新思路。方法将40只BALB／c小鼠随机分为正常组、哮喘组、布地奈德组及SB431542组，每组10只；卵清蛋白（OVA）致敏、激发建立慢性哮喘小鼠模型；布地奈德组和SB431542组分别给予布地奈德雾化吸入和SB431542滴鼻，每周3次。HE和Masson染色观察各组小鼠气道炎症及胶原沉积情况；AD-PAS染色观察杯状细胞增生情况；免疫组织化学半定量法测定气道壁a-平滑肌肌动蛋白（a—SMA）的表达；酶联免疫吸附试验（ELIsA）法检测BALF中IL-4、IL-5、TGF-81、MMP-9、TIMP-1水平及血清总IgE水平；免疫印迹法（Westernblot）测定各组小鼠肺组织中Smad3、p-Smad3及Smad7蛋白表达。结果哮喘组与正常组相比，嗜酸粒细胞浸润增多，气道管腔狭窄，平滑肌层增厚，胶原纤维增生；布地奈德组上述改变均较哮喘组轻；SB431542组嗜酸粒细胞浸润较哮喘组减轻，但仍高于正常组及布地奈德组，平滑肌层增厚、胶原纤维增生较哮喘组明显减轻（P〈0．05），与布地奈德组比较差异无统计学意义（P〉o．05）。与正常组比较，哮喘组小鼠支气管AB-PAS及a-SMA阳性染色面积／支气管基底膜周径显著增加（P〈0．05），布地奈德组及SB431542组小鼠支气管AB-PAS及a—SMA阳性染色面积／支气管基底膜周径低于哮喘组（P〈O．05）。哮喘组气道炎症指标（血清总IgE、IL-4、IL-5及TGF-β1）显著高于正常组（Pd0．05），布地奈德组上述指标低于哮喘组（P〈0．05），SB431542组与哮喘组比较差异无统计学意义（P〉0．05）。MMP-9、TIMP-1在哮喘组的表达明显高于正常组（P〈0．05），在布地奈德组及SB43542组的表达低于哮喘组（P〈O．05），两治疗组之间差异无统计学意义（P〉0．05）。Westernblot检测显示，各组小鼠肺组织Smad3的表达差异无统计学意义（P〉O．05）；哮喘组p-Smad3表达明显高于正常组（P〈0．05），布地奈德组及SB431542组p-Smad3表达低于哮喘组（P〈O．05）；与正常组比较，哮喘组Smad7表达降低（P〈0．05），布地奈德组及SB431542组Smad7表达高于哮喘组（P〈0．05），且这两组间差异无统计学意义（P〉0．05）。结论SB431542能显著减轻哮喘小鼠细胞外基质沉积及平滑肌增厚，延缓哮喘小鼠气道重塑进程，该作用可能部分与其调节MMP-9、TIMP-1的表达有关。SB431542对哮喘小鼠气道炎症浸润无明显改善，说明哮喘气道炎症可能不依赖于TGF-β1／Smads通路。

Objective To investigate the effects of TGF-β1 type I receptor antagonist on airway inflammation and remodeling in murine model of chronic bronchial asthma （asthma）, providing a potential approach for the therapy of asthma. Methods Forty BALB/c mice were randomly divided into 4 groups：a normal group, an asthma group, a budesonide group and a SB 431542 group, with 10 mice in each. The mice were sensitized and challenged with ovalbumin （OVA） to establish routine model of chronic asthma. The budesonide group and SB 431542 group were intervened with aerosolized budesonide and intranasal SB 431542 three times a week respectively. Alterations of the airway inflammation and collagen deposition were observed by the means of haematoxylin-eosin （HE） and Masson staining. The global cells were quantified by periodic acid schiff （PAS）. The expression of a-smooth muscle actin （a-SMA） in lungs was evaluated by immunohistochemistry. The levels of interleukin （IL）-4, IL-5, TGF- β1 ,MMP-9 and TIMP-1 in BALF, and the total level of IgE in serum were evaluated by enzyme linked immunosorbent assay （ELISA）. The protein expression of Smad3,phosphorylation of Smad3 （p-Smad3） and Smad7 were detected by Western blot. Results There were mass inflammatory cells infiltration, airway stenosis, bronchial smooth muscle hypertrophy and collagen fiber increasing in the asthmatic group, these alterations were lessen in budesonide group. The inflammatory cells infiltration in SB 431542 group were more relievable than asthma group, but still worse than budesonide group, while the bronchial smooth muscle hypertrophy and collagen fiber increasing were equal to budesonide group （ P〈 0.05）. A significant increase in the number of PAS-positive epithelial cells was found in the asthma group compared with the control group （ P〈0.05）. Treatment with budesonide or SB 431541 reduced the number of PAS-positive cells （ P 〈0.05）. The area of the a-SMA-stained smooth muscle layer in the asthmatic group were significantly larger than those in the control group （P 〈 0.05 ）, while administration of budesonide or SB 431542 reduced the a-SMA immunostained area （ P 〈0.05）. The levels of total serum IgE and BALF IL-4, IL-5, TGF-β1 were increased in asthma group compared to control group（ P 〈0.05）, but there were no significant differences between the asthma group and SB 431542 group （P 〈0.05）, while the levels of mentioned indexes above were decreased in budesonide group （ P 〈0.05）. MMP-9 and TIMP-1 levels were significantly raised in asthma group （ P 〈0.05）, both budesonide and SB 431542 intervene could reduced MMP-9 and TIMP-1 levels （ P 〈0.05）. The lung Smad3 protein were expressed similarly in all groups （ P 〈0.05）, but the p-Smad3 in budesonide group were higher then control group （ P 〈0.05）, while it was significantly decreased in the budesonide group and the SB 431542 group（ P 〈0.05）. The Smad7 protein was observed lower in asthma group compared to the control group（ P 〈0.05）, administration of budsonide or SB 431542 could restore the express of Smad7 protein （ P 〈0.05）, there were no significant differences between the budsonide group and SE 431542 group （ P 〈0.05）. Conclusions SB 431542 alleviated the extracellular matrix deposition and airway smooth muscle thickening, partially by regulating the levels of MMP-9 and TIMP-1. However, we did not observe significant improvement of inflammation infiltration in asthmatic mice airway, this may imply that airway inflammation in asthma is not dependent on TGF-β1/Smads signaling pathway.