H2型松弛素对哮喘小鼠肺组织Epac表达的影响及其意义

Effects of H2 relaxin on the expression of Epac in a murine model of chronic asthma

目的探讨H2型松弛素对支气管哮喘（简称哮喘）模型小鼠肺组织环磷酸腺苷直接活化的交换蛋白（Epac）表达的影响及其在气道重塑中的意义。方法将32只BALB／c小鼠按随机数字表法随机分为对照组、哮喘组、安慰剂组和松弛素组4组，每组8只。卵清蛋白致敏、激发建立慢性哮喘小鼠模型；安慰剂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染色观察各组小鼠气道炎症情况；免疫组织化学观察气道平滑肌层增殖细胞核抗原（PCNA）和d平滑肌肌动蛋白（α-SMA）表达情况；Western印迹法测定各组小鼠肺组织Epac和细胞外信号调节激酶（ERK）1／2磷酸化表达水平。结果与对照组相比，哮喘组和安慰剂组小鼠气道管腔狭窄，嗜酸粒细胞浸润增多，平滑肌层增厚，而松弛素组上述改变较此2组为轻。哮喘组及安慰剂组支气管平滑肌细胞PCNA阳性细胞比例（34．8％±6．1％、33．5％±6．6％）和仪．SMA阳性染色面积／支气管基底膜周径[（1，70±0．25）、（1．54±0．24）μm2／μm]显著大于对照组[9．9％±2．6％，（0．51±0．16）μm2／μm]（均P〈0．05），而松弛素组[22．9％4-5．2％，（1．06±0．25）μm2／μm]均小于哮喘组和安慰剂组（均尸〈0．05）。哮喘组和安慰剂组Epac表达（0．62±0．12、0．68±0．11）均低于对照组（1．50±0．17）（均P〈0．05），而松弛素组（1．08±0．15）高于哮喘组和安慰剂组（均P〈0．05）。哮喘组和安慰剂组ERK1／2磷酸化水平（1．45±0．13、1．36±0．09）均高于对照组（0．384-0．17）（均P〈0．05），而松弛素组（0．72±0．06）低于哮喘组和安慰剂组（均P〈0．05）。哮喘组和安慰剂组各项指标差异均无统计学意义（均P〉0．05）。结论松弛素可能通过活化Epac、抑制ERK1／2磷酸化，减轻慢性哮喘小鼠气道炎症和气道平滑肌细胞增生。

Objective To explore the effects of H2 relaxin on the expression of exchange protein directly activated by cyclic adenosine monophosphate （Epac） in a routine model of chronic asthma and the roles in the management of airway remodelling. Methods Thirty-two BALB/c mice were randomly divided into 4 groups of normal control, asthma, vehicle control and relaxin treatment （ n = 8 each ）. They were sensitized and challenged with ovalbumin to establish a chronic asthmatic model. The vehicle control and relaxin treatment groups were subcutaneously injected with saline and relaxin （ 0. 25 mg ~ kg- 1 . d - 1 ） respectively. Alteration of airway inflammation was observed by hematoxylin-eosin （HE） staining. The airway expressions of proliferating cell nuclear antigen （PCNA） and α-smooth muscle actin （cL-SMA） were evaluated by immunohistochemistry. The protein expression of Epac and phosphorylated extracellular signal regulated kinasesl/2 （p-ERK1/2） were detected by Western blot. Results Compared to those in the normal control group, massive infiltration of inflammatory cells, airway stenosis, bronchial smooth muscle hypertrophy were present in the asthmatic and vehicle control groups. The above-mentioned changes were significantly ameliorated in the relaxin treatment group. The percentage of PCNA positive cells （34. 8% ± 6. 1%, 33.5% ±6. 6% ） and the expression of α-SMA （ （1.70 ±0. 25）, （1.54 ±0. 24） i^m2/p,m） in the asthmatic and vehicle control groups were significantly higher than those in the normal control group （9. 9% ±2. 6%, （0. 51 ±0. 16） μm2/μm） （all P 〈 0. 05） while administration of relaxin decreased the airway expression levels of PCNA and a-SMA （22. 9% ± 5.2%, （1.06 ± 0. 25） μm2/μm） （all P 〈 0. 05 ）. The results of Western blot showed that the expression levels of Epac in the asthmatic and vehicle groups （0. 62 ± 0. 12, 0.68 -±0. 11） were lower than those in the control group （1.50 ±0. 17） （all P 〈0.05） while it significantly increased in the relaxin group （ 1.08 ± 0. 15 ） （ all P 〈 0. 05）. The levels of phosphorylation of ERK1/2 in the asthmatic and vehicle groups （1.45 ± 0. 13, 1.36 ± 0. 09） were higher than those in the control group （0. 38 ± 0. 17） （all P 〈 0.05 ） while it decreased in the relaxin treatment group （0. 72 ± 0. 06） （ all P 〈 0. 05）. No differences existed in all parameters between the asthmatic and vehicle groups （P 〉0. 05）. Conclusion Relaxin alleviates the airway inflammation and airway smooth muscle cell proliferation in a murine model of chronic asthma probably through activating Epac and inhibiting the phosphorylation of ERK1/2.