慢性阻塞性肺疾病中转录因子ATF3/ATF4与Nrf2的表达及相互作用

Expression and interactions of transcription factors ATF3/ATF4 and Nrf2 in chronic obstructive pulmonary disease

目的:研究活化转录因子3(ATF3)、活化转录因子4(ATF4)和红系衍生核因子相关因子2(Nrf2)在慢性阻塞性肺疾病(COPD)患者体内的表达变化,探讨ATF3、ATF4与Nrf2蛋白之间的相互作用。方法:肺组织标本取自40例肺肿瘤行肺叶切除者,于远离病灶处取材,诊断符合COPD者入选COPD组(20例),不符合者入选对照组(20例)。取两组患者的肺组织,原位杂交和免疫组化检测ATF3、ATF4和Nrf2 mRNA及蛋白表达水平。复制COPD大鼠模型,应用免疫共沉淀法(Co-IP)研究ATF3、ATF4与Nrf2蛋白之间的相互关系。结果:(1)COPD组患者第1秒用力呼气容积/用力肺活量(FEV1/FVC)和第1秒用力呼气容积占预计值百分比(FEV1%预计值)明显低于对照组(均P<0.01)。(2)COPD大鼠肺功能(FEV0.3、FEV0.3/FVC和PEF)较对照组显著恶化(均P<0.01)。(3)免疫组化显示COPD组ATF3、ATF4和Nrf2蛋白水平较对照组升高(均P<0.01)。(4)原位杂交结果显示ATF3和ATF4 mRNA表达水平在COPD组显著高于对照组(均P<0.01),而Nrf2 mRNA在两组表达无明显差异(P>0.05)。(5)Co-IP结果显示在Nrf2抗体捕获的免疫沉淀中,ATF3和ATF4抗体均可杂交出明显的蛋白条带(P<0.05)。结论:氧化应激状况下ATF3和ATF4表达明显上调,通过与Nrf2之间的相互作用,可能转录调控Nrf2靶基因的表达,在COPD的氧化/抗氧化失衡中发挥生物学作用。

AIM：To investigate the expression of activating transcription factor 3（ATF3）,activating transcription factor 4（ATF4） and NF-E2-related factor 2（Nrf2）,and the protein interactions of ATF3/ATF4 with Nrf2 in lung tissues of the patients with chronic obstructive pulmonary disease（COPD）.METHODS： Lung specimens were obtained from 40 patients who underwent lobectomy for lung tumor,and the samples were taken from the areas remote to the lesion,of whom 20 had concurrent COPD and 20 without COPD.In situ hybridization and immunohistochemistry were used to detect the mRNA and protein expression of ATF3,ATF4 and Nrf2 in the pulmonary tissues.The rat model of COPD was established by the method of intra-tracheal dripping with both fumigation and lipopolysaccharide（LPS）.Meanwhile,the associations of ATF3,ATF4 and Nrf2 proteins were investigated by the method of co-immunoprecipitation（Co-IP） in the rats with COPD.RESULTS： Forced expiratory volume in one second/forced vital capacity（FEV1/FVC） and FEV1 to predictive value（FEV1 % Pred） of the patients in COPD group were significantly lower than those in control group（all P〈0.01）.The pulmonary functions（FEV0.3,FEV0.3/FVC and PEF）were markedly aggravated in COPD rats（all P〈0.01）.Immunohistochemical results showed that the protein levels of ATF3,ATF4 and Nrf2 were higher in COPD group than those in control group（all P〈0.01）.In situ hybridization results showed that the mRNA expression of ATF3 and ATF4 in COPD group was conspicuously higher than that in control group（all P〈0.01）,while the mRNA expression of Nrf2 in COPD group had no significant difference compared with that in control group（P〉0.05）.Co-IP results showed that ATF3 and ATF4 were obviously positive in Nrf2-captured immunoprecipitates（P〈0.05）.CONCLUSION： ATF3 and ATF4 are significantly up-regulated in oxidative stress.Via interactions with Nrf2,ATF3 and ATF4 may play roles in the transcriptional control of the target gene expression in the oxidative imbalance in COPD.