H2型松弛素对慢性支气管哮喘小鼠气道重塑与肺组织细胞周期蛋白D1表达的影响

Effects of H2 relaxin on airway remodeling and expression of cyclin D1 in a murine model of chronic asthma

目的研究H2型松弛素（H2 Relaxin）对慢性支气管哮喘（简称哮喘）模型小鼠气道重塑及肺组织细胞周期蛋白D1（cyclinD。）表达的影响，探索松弛素在哮喘治疗中的可能作用。方法将40只BALB／c小鼠按随机数字表法分为健康对照组、哮喘组、阴性对照组（vehicle）、松弛素组4组，每组10只；卵清白蛋白（OVA）致敏、激发建立慢性哮喘小鼠模型；vehicle组和松弛素组每日分别给予生理盐水和松弛素（0．25mg·kg-1·d-1）皮下注射。HE和马森（Masson）染色观察各组小鼠气道炎症及胶原沉积情况；采用免疫组织化学半定量法测定气道壁α-平滑肌肌动蛋白（α-SMA）的表达；酶联免疫吸附测定（ELISA）法检测肺组织羟脯氨酸含量；用逆转录一PCR（RT—PCR）及免疫印迹法（Westernblot）分别测定各组小鼠肺组织中cyclinD、mRNA和蛋白表达水平。结果哮喘组、vehicle组与健康对照组相比，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，而松弛素组上述改变较此2组轻。与健康对照组相比，哮喘组、vehicle组小鼠支气管仪一SMA阳性染色面积／支气管基底膜周径显著增加（均P〈0．05），松弛素组小鼠支气管α-SMA阳性染色面积／支气管基底膜周径低于哮喘组和vehicle组（均P〈0．05）；哮喘组和vehicle组小鼠羟脯氨酸含量[每克肺组织中分别为（0．68±0．10）mg、（0．67±0．10）mg]高于健康对照组的（0．26±0．05）mg（q值分别为16．61和16．01，均P〈0．01），松弛素组小鼠羟脯氨酸含量为（0．40±0．06）mg，低于哮喘组和vehicle组（q值分别为10．88和10．26，均P〈0．05）；Westernblot检测结果显示，cyclinD．在哮喘组、vehicle组和松弛素组的表达（分别为1．38±0．18、1．50±0．10和0．724-0．13）高于健康对照组的0．38±0．10（q值分别为13．00、14．65和4．49，均P〈0．05），而松弛素组表达量低于哮喘组与vehicle组（q值分别为8．51和10．16，均P〈0．05）。哮喘组与vehicle组各项检测指标间差异无统计学意义（P〉0．05）。结论松弛素可以显著减轻慢性哮喘小鼠气道炎症反应和平滑肌层的增厚，延缓哮喘小鼠气道重塑进程，该作用可能部分与其下调cyclinD1表达有关。

Objective To investigate the effects of H2 Relaxin （Relaxin）on airway remodeling and the expression of cyclin D1 in a murine model of chronic asthma. Methods Forty BALB/c mice were randomly divided into 4 groups ： a normal control group, an asthma group, a vehicle control group and a relaxin treatment group, with 10 mice in each. The mice were sensitized and challenged with ovalbumin （OVA） to establish the chronic asthmatic model. The vehicle control group and the relaxin treatment group were subcutaneously injected with saline and relaxin （0. 25 mg·kg-1 ·d-1 ） respectively. Alteration of the airway inflammation and collagen deposition were observed by haematoxylin-eosin（HE） and Masson staining. Hydroxyproline in the lung was measured by enzyme linked immunosorbent assay （ELISA）. The expression of α-smooth muscle actin （ct-SMA） in lungs was evaluated by immunohistochemistry. The protein expression and the mRNA of cyclin D1 were detected by Western blot and RT-PCR respectively. Results There were inflammatory cell infiltration, airway stenosis, bronchial smooth muscle hypertrophy and increased collagen deposition in the asthmatic group and the vehicle control group; but these changes were significantly ameliorated in the relaxin treatment group. The area of the α-SMA-stained smooth muscle layer in the asthmatic group and the vehicle control group was significantly greater than that in the control group （ all P 〈 0. 05）, while administration of relaxin decreased the α-SMA immunostained area （ all P 〈 0. 05 ）. The lung hydroxyproline content in the asthmatic and the vehicle groups [ （0. 68 ± 0. 10） mg/g lung tissue, （0. 67 _± 0. 10） mg/g lung tissue ] was significantly greater than that in the control group I （ 0. 26 ± 0. 05 ） mg/g lung tissue] （q = 16. 61,16. 01 respectively, all P 〈 0. 01 ） . In contrast, treatment with relaxin significantly reduced the lung hydroxyproline content [ （0.40 ± 0. 06） mg/g lung tissue ] compared with aforementioned 2 groups （ q = 10. 88,10. 26 respectively, all P 〈 0. 05 ）. The results of the Western blot analysis showed that the expression level of cyclin D1 in the asthmatic and the vehicle groups [ （ 1.38 ± 0. 18 ）, （ 1.50 ± 0. 10） ] was higher than that in the control group（0. 38 ±0. 10） （q = 13.00,14. 65 respectively,all P 〈0.05） ,while it was significantly decreased in the relaxin group（0. 72 ±0. 13） （q = 8. 51,10. 16 respectively,all P 〈0. 05）. There were no differences in all of the parameters between the asthmatic group and the vehicle group （P 〉 0. 05 ）. Conclusion Relaxin alleviated airway inflammation, airway smooth muscle thickening and airway remodelling in a murine model of chronic asthma, partially by down-regulating the expression level of cyclin D1.