腺病毒E1A蛋白对核因子-κB活化的影响及N-乙酰半胱氨酸的干预作用

N-acetyl-L-cysteine inhibits adenoviral E1A-involved transactivation of nuclear factor-κB in rat alveolar epithelial cells

目的探讨腺病毒E1A蛋白（E1A蛋白）对转录因子核因子-κB（NF-κB）、活化蛋白-1（AP-1）转录活性的影响以及抗氧化剂N-乙酰半胱氨酸（NAC）干预作用。方法构建稳定表达E1A蛋白的大鼠肺泡上皮细胞（E1A组）及对照质粒转染细胞（对照组），每组5×10’个细胞，试验重复3次。采用脂多糖和肿瘤坏死因子-α（TNF-α）进行刺激，NAC进行干预，荧光素酶报告基因检测系统分析核因子-κB和AP-1转录活性的变化，Western blot法检测核因子-κB和AP-1蛋白的表达。两个样本均数的比较采用t检验，多个样本均数的比较采用单因素方差分析，组间两两比较采用LSD—t检验，结果荧光素酶活性（相对光单位）：刺激前E1A组为9698±98，在脂多糖和TNF-α刺激后分别为101195±234和170385±443，均明显高于对照组（2077±107、67846±332和95743±211）；刺激前转染AP-1的E1A组为9034±78，在脂多糖和TNF—α刺激后分别为26343±398和31731±332，均明显高于对照组（2845±93、10772±432和11005±556）。细胞内核因子-κB蛋白表达（积分吸光度值）：刺激前E1A组分别为79．3±4．6和80．3±3．8，在脂多糖和TNF-α刺激后分别为81．8±3．9～89．9±1．6和94．1±1．9～99．8±1．6，均明显高于对照组（刺激前分别为68．3±3．8和69．4±4．3，刺激后分别为70．1±2．8～80．8±3．6和73．4±4．9～83．2±6．7）；在脂多糖和TNF-α作用后E1A组细胞核内AP-1蛋白表达的积分吸光度值（72±4—73±4和83±4～86±8）与对照组（71±4～744-7和84±6～86±6）无明显差别。给予NAC预处理后再用脂多糖和TNF—α刺激，E1A组核因子-κB蛋白表达的积分吸光度值（1．98±0．20和1．90±0．20）明显低于脂多糖或TNF-α单独作用组（3．20±0．10和3．30±0．10）。结论腺病毒E1A蛋白持续表达可引起细胞内核因子-κB的过度活化，抗氧化物质NAC能明显拮抗E1A蛋白上调核因子-κB活化的作用，推测其机制可能与氧化应激有关。

Objective The relationship between latent adenvorius infection and airway inflammation had not been well documented. The aim of this study was to illustrate the roles of adenovirus E1A protein on the transactivation of NF-κB, AP-1 in response to inflammatory stimuli and the effect of N-Acetylcysteine （NAC） upon the transactivation of NF-κB and AP-1 in cells stably expressing E1A protein. Methods Rat alveolar epithelial cells stably expressing adenoviral E1A or control plasmid were developed. For isolation of nuclear extracts, 5 ×10^5 ceils were plated and grown overnight in 60 mm dishes. Experiments were repeated 3 times. The cell model of stably expressing adenoviral E1A was stimulated by LPS or TNF-α and treated with NAC, a precursor for cysteine. The NF-κB and AP-1 transcriptional activity were measured by LUC report system. The expression of NF-κB and AP-1 were measured by Western blot. Differences between groups were assessed for significance by Student＇ t test, and multiple comparisons were made by one-way ANOVA. Results The luciferase activity drived by NF-κB element was （9 698 ± 98） RLU in untreated E1A-positive clones and （101 195 ±234）, and （170 385 ±443） RLU in LPS and TNF-α-stimulated cells, which were significantly higher than that of the control group 2 077 ±107, 67 846 ±332, 95 743 ±211 respectively. The luciferase activity drived by AP-1 element was 9 034 ±78 RLU in untreated E1A-positive clones and 26 343 ±398 and 31 731 ±332 RLU in LPS and TNF-α-stimulated cells, which were significantly higher than that of the control group 2 845 ±93, 10 772 ±432, 11 005 ±556 respectively. The densitometry of the NF-κB expression in E1A-positive clones were 79. 3 ±4. 6 and 80. 3 ±3. 8 respectively without treatment and were 81.8 ±3.9 -89. 9 ±1.6 and 94. 1 ±1.9 to 99. 8 ±1.6 respectively under LPS or TNF-α stimulation, which were significantly higher than that of the control group （ 68. 3 ±3. 8, 69.4 ±4. 3 respectively） without stimulation and 70. 1 ±2. 8 to 80. 8 ±3.6, 73.4 ±4. 9 to 83.2 ±6. 7 respectively under stimulation. The level of AP-1 expression did not show difference upon treatment with LPS or TNF-α in either cell clones. The densitometry of the NF-κB expression in E1A-positive clones were 3. 2 ±0. 1 and 3.3 ±0. 1 respectively under EPS and TNF-α-stimulation and 1.98 ±0. 2 and 1.9 ±0. 2 respectively upon treatment for LPS and TNF-α with NAC pre-incubation. Conclusions These results indicate that E1A protein upregulated NF-κB transcription activity induced by LPS and TNF-α in rat alveolar epithelial cells and this effect could be repressed by NAC. The mechanisms underlying transactivation of NF-κB involved by E1A may be related to oxidative stress.