细胞外信号调节激酶信号转导途径在哮喘大鼠气道重塑中作用及糖皮质激素的调控

Role of external signal regulated kinase signal transduction pathway in airway remodeling of rats with asthma and regulation by glucocorticoids

目的研究细胞外信号调节激酶(ERK)信号转导途径在支气管哮喘气道重塑过程中作用,探讨糖皮质激素对 ERK 信号转导途径及哮喘气道重塑的调控。方法 SD 大鼠随机分为对照组(30只)、哮喘组(30只)、治疗组[包括地塞米松干预组(DM 组)及布地奈德干预组(BUD 组),各10只]三大组,通过卵白蛋白(OVA)致敏和激发复制哮喘模型;分别在雾化激发4周、8周及12周三个时间观察点取哮喘组、对照组鼠各10只(DM 组在雾化8周、BUD 组在雾化12周取标本)各组均在末次激发后取血及肺组织用于指标测定。图像分析软件测定支气管壁厚度(Wat)和平滑肌厚度(Wam),ELISA 法测定血清中 AB 型血小板源性生长因子(PDGF-AB)的含量,免疫组化测定肺组织中磷酸化的 ERK(Phospho-ERK,P-ERK)与原癌基因 c-fas 产物即 c-Fos 蛋白表达水平,Western Blot 法测定 ERK 的磷酸化水平。结果各时间点哮喘组 Wat 和 Wam 均高于相应对照组(P<0.01),治疗组低于哮喘组(P<0.01);各时间点哮喘组血清 PDGF-AB 含量均较相应对照组高(P<0.01或 P<0.05),DM 组较低于8周哮喘组(P<0.01),BUD 组与12周哮喘组比较差异无统计学意义(P>0.05);各时间点哮喘组 P-ERK 和 c-Fos 的平均吸光度(4、8、12周哮喘组分别为27.2±4.5、31.1±2.2、32.5±2.1和32.5±4.8、30.0±2.0、30.9±1.6)均较相应对照组高(P<0.01),治疗组低于哮喘组(P<0.01);各时间点哮喘组 P-ERK 的吸光度值(4、8、12周哮喘组分别为3.5±0.4、3.8±0.4、3.5±0.5)较相应对照组高(P<0.01),治疗组低于哮喘组(P<0.01)。结论哮喘大鼠 PDGF-AB 含量、ERK 磷酸化水平及 c-Fos 表达高;糖皮质激素抑制了哮喘大鼠 ERK 磷酸化及 c-Fos 表达,同时War、Wam 也明显抑制,提示糖皮质激素在哮喘气道重塑过程中具有抑制作用。

Objective Airway remodeling in asthma makes treatment of asthma very difficult, and study of its pathogenesis becomes very important. The present study aimed to explore the role of external signal regulated kinase （ERK） signal transduction pathway in airway remodeling in rats asthma model and regulatory effects of glucocorticoids on ERK signal transduction pathway and airway remodeling. Methods Totally 80 male Sprague-Dawlay rats （6- 8 weeks old,weighing about 120 g） were randomly divided into control groups （ 30 rats ）, asthma groups （ 30 rats ） and treated groups [ including a group intervened with dexamethasone （DM group） and budesonide （BUD group）, each had 10 rats]. The rats were sensitized for inducing asthma by intraperitoneal injection of ovalbumin and AI （OH） 3 and were repeatedly exposed to aerosolized ovalbumin for 4, 8, or 12 weeks [respectively called 4, 8 or 12 wk asthma group （A4, A8 or A12 group）, each had 10 rats] ; and correspondingly control rats were intraperitoneally injected with 0. 9% NaCI, then were repeatedly exposed to 0. 9% NaCI for 4, 8, or 12 weeks [ respectively called 4, 8 or 12 wk control group （C4, C8 or C12 group）, each had 10 rats]; DM group rats were repeatedly exposed to aerosolized ovalbumin for 8 wk, and BUD group rats for 12 wk. Total bronchial wall thickness （War） and smooth muscle thickness （Warn） were measured by an image analysis system. Concentrations of PDGF-AB in serum were measured by sandwich ELISA. Phospho-ERK （P-ERK） and c-Fos were detected by immunohistochemical technique; lung tissue extracts were analyzed for phosphorylation of ERK by Western blotting. Results Wat and Wain in all asthma groups were significantly higher than those in corresponding control groups （P 〈 0. 01, respectively）, those of the treated groups were significantly lower than asthma groups （P 〈 0. 01 ）. The concentrations of PDGF-AB in serum of asthma groups [ （228± 18） pg/ml, （293 ±77） pg/ml, （225 ±66） pg/ml for A4, A8, A12 groups, respectively] were all significantly higher than those of the control groups [ （ 160 ± 14） pg/ml, （ 165 ±29） pg/ml and （ 164±27） pg/ml for C4, C8, C12 group, respectively] （P 〈0. 01 or P 〈0. 05） ; the value of DM group [ （ 157 ±46） pg/ml] was significantly lower than that of the group A8 （ P 〈 0. 01 ）, no significant difference was found when the values of BUD group [ （208±40） pg/ml] was compared with that of A12 group（P 〉0. 05）. Mean absorbance values （ by immunohistochemistry） of P-ERK and c-Fos in asthma groups were significantly higher than those in corresponding control groups （P 〈 0. 01, respectively）, DM group had a significantly lower value than group A8 （P 〈 0. 01 ）, BUD group had a significantly lower value than group A12 （ P 〈 0. 01 ） ; absorbance （ by Western blot） of P-ERK in A4, A8, A12 group was significantly higher than that in C,4 and C8 group, the value of DM group was significantly lower than that of group A8 （P 〈0. 01 ）, and that of BUD group （ 1.8 ± 0.2） was significantly lower than that of group A12 （P〈0.01）. Conclusion Asthmatic rats have higher concentrations of PDGF-AB in serum and phosphorylation of ERK and c-Fos; glucocorticoids inhibit phosphorylation of ERK and c-Fos in asthmatic rats, and to some extent also inhibit Wat and Warn.