糖皮质激素诱导的亮氨酸拉链蛋白(GILZ)在人气道上皮细胞中的表达及对MAPK信号通路的影响

目的:探讨糖皮质激素诱导的亮氨酸拉链蛋白(GILZ)在人气道上皮细胞9HTE0中的表达以及对MAPK信号通路因子Raf-1、Mek1/2、Erk1/2的影响。方法:采用RT-PCR及Western blot法检测地塞米松(Dex)作用后GILZ mRNA及蛋白的表达,同时细胞免疫荧光法检测GILZ蛋白的定位;合成三条GILZ-SiRNA分别转染人气道上皮细胞9HTE0,用Q-PCR及Western blot法筛选出沉默效果最佳的一条;Western blot法检测MAPK信号通路因子Raf-1、Mek1/2、Erk1/2磷酸化蛋白及其总蛋白的表达。结果:Dex能够明显刺激人气道上皮细胞9HTE0GILZ mRNA及蛋白的表达,GILZ蛋白主要定位在细胞浆中,Dex抑制了Raf-1、Mek1/2、Erk1/2磷酸化蛋白的表达,而GILZ-SiRNA转染后,Raf-1.Mek1/2、Erk1/2磷酸化水平有所回升。结论:糖皮质激素作为支气管哮喘一线用药,虽然能够诱导GILZ表达并发挥一定的抗炎作用,但同时GILZ却抑制了在气道上皮修复中起重要作用的MAPK信号通路的激活,这为临床上研究哮喘防治新措施提供了理论依据。

The expression of glucocorticoid-induced leucine zipper(GILZ) and its effect to MAPK signaling pathway in human airway epithelial cells

Objective:To explore the expression of glucocorticoid-induced leucine zipper and the effect of GILZ to Raf-1,Mek1/2,Erk1/2 of MAPK signaling pathway in human airway epithelial 9HTE0 cells.Methods:RT-PCR was used to detect GILZ mRNA.GILZ protein was tested by Western blot and located by immunofluorescence.Three synthetic GILZ-SiRNAs were respectively transfected into 9HTE0 cells to screen out the best GILZ-SiRNA.Western blot assayed the phosphorylated and total proteins of Raf-1,Mek1/2,Erk1/2.Results:GILZ mRNA and protein increased obviously with Dexamethasone stimulation and GILZ protein mainly expressed in cell cytoplasm of 9HTE0 cells.The phosphorylation of Raf-1,Mek1/2,Erk1/2 protein of which Dexamethasone inhibited the expression rebounded after GILZ-SiRNA was transfected.Conclusion:Glucocorticoid as the fist line treatment of asthma induced the expression of GILZ that played a certain anti-inflammatory effect,but GILZ inhibited the activation of MAPK signaling pathway which promoted the repair process of airway epithelial cells.This provided a theoretical basis for new therapeuticmeasures of clinical research in asthma.