慢性阻塞性肺疾病患者肺组织中ATF3与ATF4对γ-GCS表达的影响

Effects of ATF3/ATF4 on Expression of γ-GCS in Lung Tissues of Patients with Chronic Obstructive Pulmonary Disease

目的探讨慢性阻塞性肺疾病(简称慢阻肺)患者肺组织中活化转录因子3(ATF3)、活化转录因子4(ATF4)的表达变化,以及ATF3和ATF4对γ-谷氨酰半胱氨酸合成酶(γ-GCS)表达的影响,探讨其在慢阻肺发病中的作用。方法收集伴或不伴慢阻肺并行肺叶切除的肺癌患者临床肺组织标本40例(慢阻肺组和对照组各20例),取材标本均为远离原发肺癌病灶5 cm以上、肉眼观察无肺癌浸润的外周肺组织。所有患者术前均详细询问病史,进行体格检查、胸部X线片或肺部CT以及肺功能检测。应用原位杂交及免疫组化检测患者肺组织中ATF3、ATF4、γ-GCS重链亚基(γ-GCS-HS)m RNA及蛋白的表达,应用免疫共沉淀法(CO-IP)检测ATF3、ATF4与γ-GCS-HS蛋白之间的相互作用,并对ATF3、ATF4 m RNA及蛋白与γ-GCS-HS m RNA及蛋白进行相关分析。结果慢阻肺患者肺组织中ATF3、ATF4、γ-GCS-HS m RNA及蛋白呈强阳性表达,较对照组显著升高(P<0.01)。在γ-GCS-HS抗体捕获的免疫沉淀中,ATF3、ATF4抗体均可杂交出明显的蛋白条带,且慢阻肺组较对照组明显增强(P<0.01)。相关分析显示肺组织中ATF3、ATF4蛋白表达与γ-GCS-HS m RNA及蛋白表达均呈明显正相关(P<0.01);ATF3、ATF4、γ-GCS-HS蛋白表达与FEV1%pred、FEV1/FVC均呈明显正相关(P<0.01)。结论在慢阻肺患者发病过程中,氧化应激诱导转录因子ATF3和ATF4过表达,ATF3和ATF4可能通过影响γ-GCS m RNA和蛋白的表达而发挥抗氧化保护作用。

Objective To investigate the expressions of activating transcription factor 3 （ATF3） and ATF4, and their effects on γ-glutamylcysteine synthetase （γ-GCS） in lung tissues of patients with chronic obstructive pulmonary disease （COPD）. Methods Non-cancerous lung tissue specimens （ 5 cm or above away from tumor） were collected from 40 lung cancer patients who underwent pulmonary lobectomy between December 2008 and December 2009. The patients were divided into a COPD group and a control group according to whether they were complicated with COPD. The clinical data were collected including the history in detail, physical examination, chest X-ray or lufig CT, and pulmonary function test. The mRNA and protein expressions of ATF3, ATF4, and heavy subunit of γ-GCS （γ-GCS-HS） in lung tissues were detected by in situ hybridization and immunohistochemistry. At the same time, the interaction between ATF3, ATF4 and γ-GCS-HS protein was investigated by co-immunoprecipitation method. The correlation of ATF3 and ATF4 with γ-GCS-HS was analyzed. Results The mRNA and protein expressions of ATF3, ATF4 and γ-GCS-HS in lung tissues of the COPD group were strongly positive, and significantly higher than those in the control group （P 〈 0.01）. Theco-immunoprecipitation showed that ATF3 and ATF4 antibodies could cross the clear protein bands in the immune precipitation captured by γ-GCS-HS, and the intensity in the COPD group was significantly enhanced than that in the control group （P 〈 0.01）. Correlation analysis showed that the protein expressions of ATF3 and ATF4 were significantly positively correlated with the mRNA and protein expression ofγ-GCS-HS （all P 〈 0.01）. The protein expressions of ATF3, ATF4 and γ-GCS-HS in lung tissues were positively correlated with FEV1%pred and FEV/FVC （P 〈 0.01）. Conclusions Oxidative stress induces overexpression of ATF3 and ATF4 which may paly role in the pathogenesis ofCOPD. ATF3 and ATF4 may play antioxidative effect by affecting the mRNA and protein expression of γ-GCS.