苦参碱对支气管哮喘小鼠气道炎症和早期气道重塑的影响

Effects of matrine on airway inflammation and early airway remodeling in asthmatic mice

目的观察苦参碱对支气管哮喘（简称哮喘）小鼠早期气道重塑和炎症的影响。方法将50只BALB／C小鼠按随机数字表法分为空白对照组（A）、哮喘模型组（B）、地塞米松组（C）、苦参碱高剂量组（D，50mg／kg）和低剂量组（E，25mg／kg）5组。卵清白蛋白致敏建立哮喘小鼠模型，每次激发前，C、D、E组分别给予地塞米松和相应剂量的苦参碱进行灌胃；B组给予等剂量生理盐水；A组以等剂量的生理盐水致敏、激发及灌胃。肺组织切片染色，炎症细胞及黏液分泌评分、定量杯状细胞百分比，测定平滑肌面积、基底膜胶原面积。采用逆转录PCR和免疫组织化学检测转化生长因子-β1（TGF-β1）和结缔组织生长因子（CTGF）的mRNA和蛋白表达水平。采用SPSS13．0统计软件处理，符合正态分布和方差齐性的数据采用单因素方差分析（one—way ANOVA），多组间两两比较采用Dunnet-t检验；对等级数据和不符合正态分布或方差齐性的数据则进行秩和检验（Kruskal-Wallis法），多组间两两比较采用Mann-whiteney检验；相关性采用Spearman等级相关分析。结果A—E组炎症细胞评分（均数，四分位数）分别为1．5（1，2）、4（4，5）、2（1，3）、2（2，3）、3（2，3．3），黏液分泌评分分别为1．5（1，2）、5（4，6）、2（1，3）、2（2．5，4）、3（3，4），B组明显高于A组（Х^2值分别为21．3和22．6，均P〈0．01），C组明显低于B组（Х^2值分别为13．3和15．0，均P〈0．01），D、E组低于B组（Х^2值分别为9．1、10．9和9．8、9．7，均P〈0．05）；杯状细胞占上皮细胞百分比分别为（1．7±0．5）％、（54．7±15．5）％、（20．4±5．9）％、（31．7±7．6）％、（36．2±10．8）％，B组明显高于A组（t=12．0，P〈0．01），C、D组明显低于B组（t值分别为7．7和5．1，均P〈0．01），E组低于B组（t=4．2，P〈0．05）；5组平滑肌面积分别为（11．5±2．1）、（30．0±3．3）、（15．2±3．1）、（22．2±4.8）和（26．5±3．4）μm^2／μm，B组明显高于A组（t=11．4，P〈0．01），C、D、E组均明显低于B组（t值分别为9．1、4．7和2．2，均P〈0．01）；胶原沉积面积5组分别为（3．9±1．8）、（24．4±6．1）、（15．4±3．5）、（16．6±6．0）和（17．5±4．4）μm^2／μm，B组明显高于A组（t=9．3，P〈0．01），C、D组明显低于B组（t值分别为4．1、3．5，均P〈0．01），E组低于B组（t-3．2，P〈0．05）；5组TGF-β1 mRNA灰度值分别为160±25、247±37、174±23、195±25、207±42，CTGF mRNA灰度值5组分别为86±8、160±24、94±10、93±14、104±10，B组明显高于A组（t值分别为6．1、11．6，均P〈0．01），C、D、E组均明显低于B组（t值分别为3．7、2．7、5．1和10．6、8．6、10．3，均P〈0．01）。5组肺组织TGF-β1吸光度值分别为21±5、36±8、26±5、26±5和26±5，肺组织CTGF吸光度值分别为15±4、27±5、21±4、22±3和23±4，B组明显高于A组（t值分别为5．7和6．4，均P〈0．01），C、D组明显低于B组（t值分别为3．9、3．9和3．2、2．8，均P〈0．01），E组低于B组（t值分别为3．8和2．5，均P〈0．05）。各组小鼠胞质中TGF-β1与平滑肌面积、TGF—β1与胶原沉积面积、CTGF与平滑肌面积、CTGF与胶原沉积面积均呈正相关（r值分别为0．435、0．583、0．522和0．590，均P〈0．01）。结论苦参碱能够抑制哮喘的早期气道重塑和炎症，其抑制气道重塑的可能机制与TGF-β1到CTGF的信号转导通路有关。

Objective To observe the influence of matrine on airway inflammation and early airway remodeling in asthmatic mice. Methods Fifty BALB/c mice were randomly divided into 5 groups ： a normal control group （A） , an asthmatic group （B） , a dexamethasone （DXM） group （ C, 2 mg/kg） , a high-dose matrine group （D, 50 mg/kg） and a low-dose matrine group （E, 25 mg/kg）. The mice model of asthma in the B, C, D, and E groups was established by ovalbumin （OVA） intraperitoneal injections and aerosolizations. Intra-gastric administrations of different medications in C, D, E groups and 0. 9% sodium chloride in B group were carried out 1 hour before provocation. 0. 9% sodium chloride was used for intraperitoneal injection, aerosolization and intra-gastric administration in group A. The lung tissue slices were stained, and then the grade of inflammation around the wall of bronchi, mucous secretion, and the percentage of goblet-cells were counted. The areas of bronchial smooth muscle and of collagen deposition in airway wall were analyzed. The transcriptions and protein expressions of transforming growth factor-β1 （TGF- β1 ） and connective tissue growth factor （CTGF） were measured respectively by reverse transcriptase-polymerase chain reaction （RT-PCR） and immunohistochemistry. Results In the A, B, C, D, E groups, the grades of inflammation were 1.5 （ 1,2 ）, 4 （ 4,5 ） , 2 （ 1,3 ） , 2 （ 2,3 ）, 3 （ 2,3. 3 ） , respectively; the degrees of mucous secretions were 1.5（ 1,2）, 5（4,6）, 2（ 1,3）, 2（2. 5,4）, 3（3,4） ,respectively. These airway inflammatory parameters in group B were significantly higher than in group A （ Х^2 = 21.3, 22. 6, P all 〈 0. 01 ）, while they were remarkably decreased in group C compared to group B （ Х^2 = 13.3, 15.0, Pall 〈 0. 01 ）. These parameters in group D and group E were also lower than those in group B （ Х^2 = 9. 1, 10. 9 ; 9. 8, 9. 7 ; P all 〈0. 05 ）. The percentage of goblet cells in airway epithelium was （ 1.7 ± 0. 5 ） %, （54.7±15.5）%, （20.4 ±5.9）%, （31.7 ±7.6）% and （36.2 ± 10.8）%, respectively; it was significantly higher in group B than in group A （ t = 12. 0, P 〈 0. 01 ）, and remarkably lower in groups C and D than in group B （t =7.7, 5.1, P all 〈0. 01）, and lower in group E than in B group （t =4. 2, P 〈 0. 05 ）. In these 5 groups, the area of bronchial smooth muscle was （ 11.5 ± 2. 1 ） μm^2/μm, （30. 0 ± 3. 3 ） μm^2/μm, （ 15.2 ± 3. 1 ） μm^2/μm, （22. 2 ± 4. 8） μm^2/μm and （ 26. 5 ± 3.4 ） μm^2/μm, respectively ; it was significantly higher in group B than in group A （ t = 11.4, P 〈 0.01 ）, and remarkably lower in groups C, D and E than in group B （ t = 9. 1, 4. 7, 2. 2, P all 〈 0. 01 ）. The area of collagen deposition was （3. 9 ± 1.8）μm^2/μm, （24.4 ± 6. 1 ） μm^2/μm, （ 15.4 ± 3.5 ） μm^2/μm, （ 16. 6 ± 6. 0） μm62/μm and （ 17. 5 ±4. 4） μm^2/μm, respectively; it was also significantly higher in group B than in group A （t =9. 3, P 〈 0.01 ）, and remarkably lower in groups C and D than in group B （ t = 4. 1, 3.5, P all 〈 0.01 ）, and lower in group E than in B group （ t = 3.2, P 〈 0. 05 ）. The mRNA levels of TGF-β1 were 160 ± 25,247 ± 37, 174 ± 23, 195 ± 25 and 207 ± 42, respectively, and those of CTGF were 86 ± 8, 160 ± 24, 94 ± 10, 93 ± 14 and 104 ± 10, respectively in the 5 groups. The levels were remarkably increased in group B, as compared to group A （t =6. 1, 11.6, P all 〈0. 01）, and the levels in groups C, D and E were remarkably decreased, as compared to group B, the difference being significant （ t = 3.7, 2.7, 5.1 ; 10. 6, 8. 6, 10. 3 ; P all 〈 0. 01 ）. The protein level of TGF-β1 in lung tissues was 21 ± 5, 36 ± 8, 26 ± 5, 26 ± 5 and 26 ± 5, respectively, and that of CTGF was 15 ±4, 27 ±5, 21 ±4, 22 ±3 and 23 ±4, respectively in the 5 groups. The levels in B group were significantly increased, as compared to group A （ t = 5. 7, 6. 4, P all 〈 0. 01 ）, and those in groups C and D were significantly decreased （t = 3.9, 3.9 ; 3.2, 2. 8, P all 〈 0. 01 ） , and that in group E was also lower （ t = 3.8, 2. 5, P all 〈 0. 05 ） , as compared to group B. In all the groups, the protein levels of TGF-β1 and CTGF were positively correlated with the area of bronchial smooth muscle and with the area of collagen deposition （ r = 0. 435, 0. 583, 0. 522, 0. 590, P all 〈 0. 01 ） . Conclusions Matrine inhibited airway inflammation and early airway remodeling in asthmatic mice. The signal transduction of TGF-β1 and CTGF maybe involved.