慢性阻塞性肺病患者外周血单个核细胞自噬与疾病状态的相关性研究

Correlation between autophagy of peripheral blood mononuclear cells and clinical parameters in chronic obstructive pulmonary disease patients

目的自噬在慢性炎性疾病中起到重要作用,比较慢性阻塞性肺病(COPD)和健康人外周血单个核细胞(PBMCs)自噬相关分子表达,并探索PBMCs自噬与COPD相关临床参数之间的相关性。方法前瞻性纳入了2015年9月至2018年3月COPD患者和80例健康志愿者,分别采集外周血后采用Ficoll密度梯度离心法分离外周血PBMCs,采用Western-Blot方法分别检测PBMCs中P62、LC3Ⅰ/Ⅱ和Beclin-1蛋白的表达,采用酶联免疫吸附实验(ELISA)方法检测外周血中白细胞介素(IL)-6、IL-8和肿瘤坏死因子(TNF)-α含量。结果与健康对照组相比,COPD患者PBMCs中P62表达显著下调(P62/GAPDH=0.212±0.089比0.378±0.176,P=0.0011),而LC3Ⅰ/Ⅱ(4.94±1.85比3.85±1.06,P=0.0249)和Beclin-1(Beclin-1/GAPDH=0.578±0.126比0.149±0.035,P<0.01)的表达显著上调。COPD患者血浆中IL-6[(11.96±3.46)ng/L比(2.70±1.72)ng/L,P<0.01]、IL-8[(20.38±18.44)ng/L比(6.00±4.08)ng/L,P=0.0016]和TNF-α[(29.50±28.18)ng/L比(11.08±12.07)ng/L,P=0.0106]浓度显著高于健康对照组。COPD患者PBMCs自噬与外周血促炎性因子显著相关,与第一秒用力呼气量占预计值百分比呈负相关。结论PBMCs自噬水平是COPD严重程度的可靠生物标志物,是潜在的治疗靶点。

Objective Autophagy serves a role in the pathogenesis of chronic inflammatory diseases.The aim of the present study was to compare the autophagy levels of the peripheral blood mononuclear cells(PBMCs)in patients with chronic obstructive pulmonary disease(COPD)and healthy individuals and to assess the association between autophagy and the clinical parameters of COPD.Methods Samples of peripheral blood from 20 patients with stable COPD and 20 healthy controls were collected.PBMCs were harvested using Ficoll density gradient centrifugation.Levels of the autophagy-associated proteins ubiquitin-binding protein P62(P62),microtubule-associated proteins 1A/1B light chain 3A(LC3Ⅰ/Ⅱ)and beclin-1 in PBMCs were detected by western blotting.Enzyme-linked immunosorbent assay kits were used to detect the serum concentrations of interleukin(IL)-6,IL-8 and tumor necrosis factor(TNF)-α.Results Western blotting demonstrated that the protein expression of P62 decreased(P62/GAPDH=0.212±0.089 vs.0.378±0.176,P=0.001 1),but LC3Ⅰ/Ⅱ(4.94±1.85 vs.3.85±1.06,P=0.024 9)and beclin-1(Beclin-1/GAPDH=0.578±0.126 vs.0.149±0.035,P<0.01)levels increased in patients with COPD compared with healthy controls.Serum levels of IL-6[(11.96±3.46)ng/L vs.(2.70±1.72)ng/L,P<0.01],IL-8[(20.38±18.44)ng/L vs.(6.00±4.08)ng/L,P=0.001 6]and TNF-α(29.50±28.18 vs.11.08±12.07,P=0.010 6)increased in patients with COPD.The extent of PBMC autophagy was negatively correlated with FEV1%predicted,but positively correlated with levels of proinflammatory cytokines.The levels of autophagy in PBMCs in patients with COPD increased and were negatively correlated with FEV1%predicted and positively correlated with circulating levels of pro-inflammatory cytokines.Conclusions Autophagy may serve a role as a biomarker of the severity of COPD or as a therapeutic target for treatment of COPD.