摘要
目的建立烟雾暴露的哮喘大鼠模型及尼古丁诱导人肺腺癌A549细胞糖皮质激素抵抗的实验模型,观察p38丝裂原活化蛋白激酶(p38MAPK)抑制剂SB203580对烟雾暴露的哮喘大鼠的影响,并分别从体内实验、体外实验两个方面探讨SB203580影响糖皮质激素敏感性的机制。
方法Wistar大鼠随机分为4组,即正常对照组、哮喘组、烟雾暴露的哮喘组及SB203580干预组。动物肺功能仪测定大鼠呼气阻力、吸气阻力及肺顺应性,观察肺组织病理学改变,通过ELISA检测大鼠肺组织匀浆中IL-4、IL-5和IL-8的水平。经RT-PCR检测大鼠肺组织糖皮质激素受体(GR)、HSP90和p38MAPK mRNA的表达,经Western blot检测大鼠肺组织GR、HSP90和p38MAPK蛋白的表达。培养A549细胞,将其随机分为四组：A组：空白对照组,B组：1umol/L的地塞米松,C组：1umol/L的地塞米松+1umol/L的尼古丁,D组：1umol/L的地塞米松+1umol/L的尼古丁+1umol/L的SB203580,采用免疫荧光染色技术分析GR亚细胞定位。
结果与烟雾暴露的哮喘组相比,SB203580干预组大鼠的气道阻力显著下降,肺顺应性显著升高,差异有统计学意义(P<0.05)；气道炎症明显减轻；肺组织中IL-4、IL-5和IL-8的含量显著下降,差异有统计学意义(P<0.05)；肺组织GRmRNA的表达无明显变化,HSP90和p38MAPK mRNA的表达显著下降,差异有统计学意义(P<0.05)；肺组织GR、HSP90和p38MAPK蛋白表达呈现与mRNA表达相一致的结果。C组A549细胞GR的核浆比为0.08±0.05,D组A549细胞GR的核浆比为0.59±0.25,两组之间的差异有统计学意义(P<0.05)。
结论p38MAPK抑制剂SB203580可以改善烟雾暴露的哮喘大鼠的气道炎症,减轻其支气管收缩反应。其通过促使GR核转位提高糖皮质激素敏感性。
Objective To establish model of cigarette smoke exposure to asthmatic rats and glucocorticoid resistance induced by nicotine in alveolar epithelioid cells A549cells. Investigate the effect of a p38MAPK inhibitor SB203580on the cigarette smoke exposure to asthmatic rats and study the mechanism to the change of glucocorticoid sensitivity induced by p38MAPK inhibitor SB203580.
Method Wistar rats were randomly divided into four groups:normal group, asthmatic group, cigarette smoke exposure to asthmatic group and SB203580group. Rat lung function was measured by animal pulmonary function meter. ELISA was used to detect the level of cytokines in homogenate of rats'lungs. GR、HSP90and p38MAPK mRNA expression was detected by RT-PCR, and GR、HSP90and p38MAPK protein expression was detected by western blot. A549cells were randomly divided into four groups:group A:normal group, group B:1umol/L dexamethasone (DEX), group C lumol/L DEX+1umol/L nicotine, group D:lumol/L DEX+1umol/L nicotine+lumol/L SB203580.Immunofluorescence staining was used to study the colocalization of glucocorticoid receptor(GR) in A549cells.
Results Compared to the cigarette smoke exposure to asthmatic group, the rats of SB203580group had a lower airway resistance and a higher pulmonary compliance, the difference was statistically significant (P<0.05). SB203580treatment attenuated airway inflammation. The results of ELISA showed the level of IL-4, IL-5and IL-8was lower in SB203580group than cigarette smoke exposure to asthmatic group. In RT-PCR experiment, the mRNA expression of HSP90and p38MAPK was significantly lower in SB203580group than cigarette smoke exposure to asthmatic group (P<0.05). However, the difference of GR mRNA expression between SB203580group and cigarette smoke exposure to asthmatic group wasn't statistically significant. Western blot detecting protein expression found the GR、HSP90and p38MAPK protein expression was the same as mRNA expression. The ratio of the GR amount within A549nucleus versus that in cytoplasm was0.08±0.05in group C and0.59±0.25in group D. The difference was statistically significant (P<0.05).
Conclusion It is suggested that p38MAPK inhibitor SB203580may be effective on reducing airway inflammation and bronchial contractile response in cigarette smoke exposure to asthmatic rats. The mechanism of SB203580improving the glucocorticoid sensitivity may be improving nuclear translocation of GR to elevate glucocorticoid sensitivity.
引文
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