组蛋白脱乙酰酶2-核转录因子-κB/激活蛋白-1介导的重度哮喘对糖皮质激素不敏感的分子机制

Histone deacetylase 2-nuclear transcription factor-κB/activating protein-1-mediated molecular mechanism of glucocorticoid insensitivity in severe asthma

目的探讨重度哮喘患者对糖皮质激素不敏感的分子机制,尤其从糖皮质激素/糖皮质激素受体(glucocorticoid/glucocorticoid receptor,GC/GR)及组蛋白脱乙酰酶2(histone deacetylase 2,HDAC2)及其下游的炎性转录因子途径进行研究。方法临床招募健康对照者12例,轻中度哮喘患者12例,重度哮喘患者10例,收集其外周血,进行地塞米松(dexamethasone,Dex)抑制IL-8释放试验,检测重度哮喘患者外周血单个核细胞(peripheral blood molecule cells,PBMCs)是否存在激素敏感性下降。通过Western blot和细胞免疫荧光染色法检测PBMCs的GRα蛋白质水平及跨膜转运情况。HDAC2活性试剂盒检测PBMCs核蛋白的HDAC2活性,Western blot检测磷酸化NF-κB、激活蛋白1(activating protein-1,AP-1)的亚基磷酸化c-Jun及磷酸化c-Fos的水平。结果重度哮喘患者PBMCs在TNFα诱导IL-8释放实验中表现出对Dex敏感性下降;GR总蛋白质水平虽然代偿性升高,但GRα水平下降且向核内转移减弱;HDAC2活性降低,炎性转录因子NF-κB、AP-1亚基被激活。结论重度哮喘患者的PBMCs中存在激素不敏感,可能是通过HDAC2-NF-κB/Ap-1介导的炎症通路引起了GRα表达量下降和转运能力降低。

Objective To investigate the molecular mechanism of glucocorticoid insensitivity in patients with severe asthma,especially from the glucocorticoid/glucocorticoid receptor(GC/GR),histone deacetylase 2(HDAC2)and its downstream inflammatory transcription factor pathway.Methods Twelve healthy volunteers,twelve mild to moderate asthmatics and ten severe asthmatics were recruited.The peripheral blood was collected for dexamethasone inhibition of IL-8 release test.GRαexpression and transmembrane transport ability in peripheral blood molecule cells(PBMCs)were detected by Western blot and immunofluorescence staining respectively.HDAC2 activity of PBMCs nuclear protein was analyzed by HDAC2 activity kit.Western blot was applied to measure the expression of phosphorylated NF-κB,phosphorylated c-Jun and phosphorylated c-Fos subunit of activating protein-1(AP-1).Results PBMCs of severe asthmatics showed decreased sensitivity to dexamethasone in TNF-αinduced IL-8 release experiment.They also showed decreased GRαexpression and its nuclear transfer,decreased HDAC2 activity,and activated NF-κB and activating protein-1(AP-1)subunit,which contributed to GC insensitivity.Conclusion GC insensitivity in PBMCs of patients with severe asthma may be mediated by HDAC2-NF-κB/AP-1 signaling pathway which reduced the amount and transport capacity of GRα.