Liu Cui-qing, Cao Lei, Zheng Hua-cheng, Jia Xi-qun, Kang Li-min, Li Lan-feng, Liu Su-zhe
Department of Neonatology, Childrens' Hospital of Hebei Provence, Shijiazhuang 050031, China.
Zhonghua Er Ke Za Zhi. 2006 Aug;44(8):602-6.
Inflammatory reaction and injury in immature lungs are associated with activation of nuclear factor-kappa B (NF-kappaB) to trigger proinflammatory cytokine release, but the mechanism thereof is not fully understood. The present study was conducted to understand possible relationship between expression of NF-kappaB and its inhibitor and severity and outcome of neonates with hyaline membrane disease (HMD).
Serial samples of bronchoalveolar lavage fluid (BALF) were obtained during mechanical ventilation from 31 preterm infants with HMD. These infants were divided into two groups: survivors group [n = 22, birth weight (1500 +/- 320) g and gestational age (31.2 +/- 1.8) weeks] and nonsurvivors group [birth weight (1340 +/- 280) g, gestational age (30.8 +/- 2.1) weeks]. Nineteen preterm infants [birth weight (1470 +/- 280) g, gestational age (30.6 +/- 1.9) weeks] without respiratory disorders were enrolled as control subjects. Alveolar macrophages (AM) were isolated by differential adherence. AM was cultured and treated with lipopolysaccharide (LPS) for 1 hr. Then, nuclear extracts of AM were analyzed by electrophoretic mobility shift assay (EMSA) for NF-kappaB expression. NF-kappaB inhibitor (IkappaB-alpha protein) in cytoplasmic extracts was detected by using Western blotting and IL-1beta and IL-8 in BALF by enzyme-linked immunosorbent assay (ELISA).
NF-kappaB complexes were observed by EMSA, they were characterized by competition with cold oligonucleotide and p65-specific antibodies. The addition of an excess of cold oligonucleotide, corresponding to the NF-kappaB binding site, turned off the signal of the band, showing that the band was specific. An excess of an irrelevant oligonucleotide (corresponding to the SP-1) did not show any effect. The addition of an anti-p65 antibody caused the supershift of the two upper bands. After EMSA, the NF-kappaB complexes were quantified by using a ImageQuant software. NF-kappaB expression in AM at 24 hrs was higher in all the patients with HMD as compared with control subjects (survives/control, 34.1 vs 11.4 RDU, P < 0.01; nonsurvivors/control, 55.2 vs 11.4 RDU, P < 0.01). The NF-kappaB expression in AM at 72 hrs was higher than that in control subjects but not for nonsurvivors (survivors/control, 47.8 vs 25.6 RDU, P < 0.01; nonsurvivors/control, 21.8 vs 25.6, P > 0.05). The NF-kappaB expression in AM from nonsurvivors was depressed at 72 hrs as compared to 24 hrs (21.8 vs 55.2, P < 0.01), whereas the NF-kappaB expression in AM from survivors was still higher at 72 hrs than that at 24 hrs (47.8 vs 34.1, t = 4.43, P < 0.01).
Altered NF-kappaB activation in AM of BALF of neonates with HMD was observed, and it may be mediated by decreased IkappaB synthesis, increased IkappaB degradation, or both. In HMD nonsurvivors NF-kappaB translocation was hampered upon LPS activation.
未成熟肺中的炎症反应和损伤与核因子-κB（NF-κB）激活有关，可触发促炎细胞因子释放，但其机制尚未完全阐明。本研究旨在了解NF-κB及其抑制剂的表达与透明膜病（HMD）新生儿严重程度及预后之间的可能关系。
对31例机械通气的HMD早产儿在机械通气期间获取系列支气管肺泡灌洗（BALF）样本。这些婴儿分为两组：存活组[n = 22，出生体重（1500±320）g，胎龄（31.2±1.8）周]和非存活组[出生体重（1340±280）g，胎龄（30.8±2.1）周]。19例无呼吸系统疾病的早产儿[出生体重（1470±280）g，胎龄（30.6±1.9）周]作为对照。通过差异贴壁法分离肺泡巨噬细胞（AM）。将AM培养并用脂多糖（LPS）处理1小时。然后，通过电泳迁移率变动分析（EMSA）分析AM的核提取物中NF-κB的表达。通过蛋白质印迹法检测细胞质提取物中的NF-κB抑制剂（IκB-α蛋白），并通过酶联免疫吸附测定（ELISA）检测BALF中的IL-1β和IL-8。
通过EMSA观察到NF-κB复合物，其特征在于与冷寡核苷酸和p65特异性抗体竞争。加入过量对应于NF-κB结合位点的冷寡核苷酸可使条带信号消失，表明该条带具有特异性。加入过量不相关的寡核苷酸（对应于SP-1）未显示任何影响。加入抗p65抗体导致两条上部条带发生超迁移。EMSA后，使用ImageQuant软件对NF-κB复合物进行定量。与对照组相比，所有HMD患者24小时时AM中NF-κB表达均较高（存活者/对照组，34.1对11.4相对密度单位，P <0.01；非存活者/对照组，55.2对11.4相对密度单位，P <0.01）。72小时时AM中NF-κB表达高于对照组，但非存活者除外（存活者/对照组，47.8对25.6相对密度单位，P <0.01；非存活者/对照组，21.8对25.6，P> 0.05）。与24小时相比，非存活者72小时时AM中NF-κB表达降低（21.8对55.2，P <0.01），而存活者72小时时AM中NF-κB表达仍高于24小时时（47.8对34.1，t = 4.43，P <0.01）。
观察到HMD新生儿BALF的AM中NF-κB激活改变，可能由IκB合成减少、IκB降解增加或两者共同介导。在HMD非存活者中，LPS激活后NF-κB易位受阻。
