表皮生长因子受体对支气管哮喘大鼠气道炎症的影响

Effects of epidermal growth factor receptor on airway inflammation in a rat asthmatic model

目的探讨表皮生长因子受体（EGFR）在支气管哮喘（简称哮喘）大鼠急慢性气道炎症的作用以及阻断EGFR的激活对气道炎症的影响。方法将45只SD大鼠按随机数字表法分为对照组（A1、A2、A4组）、哮喘组（B1、B2、B4组）和治疗组（C1、C2、C4组），每组5只，其中1、2和4分别表示激发1、2和4周。治疗组在每次卵清白蛋白激发前1h给予酪氨酸激酶抑制剂（TKI）金雀异黄素（genistein）20mg／kg腹腔注射。末次激发后收集BALF行细胞总数及分类计数。肺组织HE染色并行气道黏膜下炎症评分。免疫组织化学及免疫荧光染色观察气道上皮EGFR表达及其酪氨酸磷酸化（活化的EGFR）程度。两组数据间的比较采用单因素方差分析，多组间比较采用叮检验。结果（1）B组各时段BALF中细胞总数及各类细胞绝对计数均高于A组相应时段组（均P〈0．05），嗜酸粒细胞在2周末达高峰；C组各时段BALF中细胞总数[分别为（48±6）、（51±9）和（57±12）×10^5]及嗜酸粒细胞计数[分别为（2．5±0．5）、（2．7±0．6）和（2．6±0．5）×10^5]均低于相应B时段组[细胞总数分别为（70±10）、（88±8）和（72±10）×10^5，嗜酸粒细胞数分别为（5．6±0．8）×10^5、（6．6±0．6）×10^5和（4．3±0．4）×10^5]，差异有统计学意义（均P〈0．05）；C组各时段BALF中中性粒细胞及淋巴细胞计数与相应时段B组比较差异无统计学意义；C1组BALF中上皮细胞数[（2．5±0．5）×10^5]低于B1组[（4．9±0．7）×10^5]（q=4．671，P〈0．05），C4组BALF中上皮细胞数[（5．7±1．2）×10^5]高于B。组[（4．3±0．4）×10^5]（q=4．012，P〈0．05）。（2）C4组气道黏膜下炎症评分（3．6±0．6）低于B4组（5．1±0．6）（q=4．923，P〈0．05）。（3）B组各时段各级气道EGFR蛋白表达均高于相应时段A组（均P〈0．01）；气道上皮EGFR活化程度A组分别为（1．84±0．26）、（1．43±0．29）和（1．67±0．32），B组分别为（3．69±0．43）、（3．57±0．29）和（4．46±0．47），C组分别为（3．12±0．24）、（3．00±0．28）和（2．69±0．54），B组各时段气道上皮EGFR活化程度均高于相应时段A组（均P〈0．05）；C组各时段气道上皮EGFR活化程度均弱于相应时段B组（均P〈0．05）。（4）相关分析显示气道上皮EGFR表达及其活化程度均与BALF中细胞总数、嗜酸粒细胞、中性粒细胞及淋巴细胞绝对计数，以及气道炎症评分呈明显正相关。结论哮喘大鼠气道上皮EGFR参与气道炎症，TKI金雀异黄素有一定抑制哮喘大鼠急慢性气道炎症的作用。

Objective To explore the possible roles of epidermal growth factor receptor （EGFR） in the process of acute and chronic airway inflammation in a rat asthmatic model. Methods Forty-five Sprague- Dawley （SD） rats were randomly divided into control groups （subgroups A1, A2, A4 ）, asthmatic groups （subgroups B1, B2, B4） and treatment groups （subgroups C1, C2, C4 ）, with 5 mice in each subgroup. Mice in the asthmatic and treatment groups were exposed to OVA challenge for 1 week, 2 weeks and 4 weeks. Rats in the treatment groups received intraperitoneal injection of a tyrosine kinase inhibitor Genistein （Rongli China ） with the dose of 20 mg/kg 1 hour before OVA exposure. Total cell counts and cell differentials in bronchoalveolar lavage fluid （BALF） were performed. A semi-quantified method of airway inflammation score was used to evaluate airway inflammation by hematoxylin-eosin （HE） staining. Expression of EGFR and tyrosine phosphorylation （EGFR activation） in airway epithelium at different times of OVA exposure were evaluated by immunohistochemical and immunofluorescence. All data were expressed as mean ＋ SD. One-way ANOVA was used for comparison between 2 groups and post-hoc multiple comparisons of means were performed by using Least Significant Difference. Results （ 1 ） The total cell counts and cell differentials in the BALF of subgroups B1 ,B2 and B4 were higher than those of subgroups A1 , A2 and A4. The total cell counts and eosinophils （EOS） in the BALF of subgroups Cl, C2 and C4 [Total cells （48±6） ×10^5, （51 ±9） ×10^5, （57±12） ×10^5; EOS （2.5±0.5）×10^5, （2.7±0.6） ×10^5, （2. 6 ±0. 5）×10^5, respectively] decreased significantly as compared to those of subgroups Bl, B2 and B4 [Total cells （70±10） ×10^5, （88±8）×10^5, （72±10）×10^5; EOS （5.6±0.8） ×10^5, （6.6±0.6） ×10^5, （4. 3 ±0. 4） ×10^5] , all P 〈0. 05. There was no significant difference in the counts of neutrophils and lymphocytes in BALF between the treatment groups and the asthmatic groups. The count of epithelial cells in group C1 [ （2. 5± 0. 5 ） ×10^5 ] was lower than that in group B1 [ （4. 9 ± 0. 7） ×10^5 ], q = 4. 671, P 〈 0. 05. But that in group C4 [ （5.7 ± 1.2） ×10^5 ] was higher than that in group B4 [ （4. 3±0.4） ×10^5 ], q = 4. 012, P 〈0.05. （2） The airway inflammation score in group C4 （3.6 ±0. 6） was less than that in group B4（5.1 ±0.6）, q =4. 923, P〈0.05. The scores of group Cj and C2 were less than those of group B2 and B2, but the differences did not reach statistical significance. （3） The expression of EGFR and tyrosine phosphorylation in airway epithelium of the OVA sensitized subgroups were increased statistically as compared to the control subgroups （ all P 〈 0.05）. Genistein decreased tyrosine phosphorylation of EGFR in subgroups C1, C2 and C4[ （3.12±9. 24）, （3.00 ±0. 28） , （2. 69±0. 54） ] as compared to subgroups B1, B2 and B, [ （3.69 ± 0. 43 ）, （ 3.57 ± 0.29 ）, （4. 46± 0. 47 ）, respectively 3 （ all P 〈 0.05 ）. （4） There were positive correlations between expression and activation of EGFR in airway epithelium and total cell counts, EOS counts, neutrophil and lymphocyte counts in BALF, and airway inflammation scores （ all P 〈 0. 05）. Conclusions EGFR is involved in airway inflammation of asthmatic rats. Tyrosine kinase inhibitor Genistein inhibits acute and chronic airway inflammation in the asthmatic model.