Objective To investigate the effects of N-acetylcysteine (NAC) on nuclear factor-κB (NF-ΚB) DNA binding activity and expression of cyclooxygenase-2 (COX-2) in human pulmonary microvascular endothelial cell (HPMEC) in vitro. Methods HPMEC cell line was treated with NAC and tumor necrosis factor-α (TNF-α) in vitro, and the inhibitory action of NAC in cell proliferation was determined with MTT colorimetric assay by NAC (1 mmol/L × 1 h), TNF-α (100 ng/mL × 1 h) and NAC combined with TNF-α. NF-ΚB DNA binding activity in HPMEC was observed by electrophoretic gel mobility shift assay (EMSA). The protein expression of IΚBain HPMEC was examined by Western blot. NF-ΚB expression translocation was detected by immunohistochemistry. COX-2 expression of HPMEC was assessed by immunobloting and laser confocal microscopy. Results The proliferation of HPMEC cells could be inhibited by NAC, absorbance value in NAC+TNF-α treatment group was lower than that in TNF-α treatment group, the difference was statistically significant (P < 0.05). NF-ΚB DNA binding activity was up-regulated and IΚB-a was down-regulated after treating with TNFα, expression of IΚB-α in NAC treatment group was higher than that in TNF-α treatment group, the difference was statistically significant (P < 0.01). NF-ΚB expression was translocated from cytoplasm into nucleus after the cells were treated with TNF-α for 1 h; NF-ΚB expression of HPMEC was mainly in cytoplasm, rarely in nucleus. The expression of COX-2 after TNF-α treatment was higher than that in NAC+TNFα- treatment group and normal control group, the differences were all statistically significant (all P < 0.05); the difference between NAC+TNF-α treatment group and normal control group were not statistically significant (P > 0.05). Conclusion NAC can inhibit the proliferation of HPMEC cells with TNF-α treatment. NAC can reduce NF -ΚB DNA binding activity and expression of COX-2 in HPMEC.%目的 探讨N-乙酰半胱氨酸(NAC)对人肺微血管内皮细胞(HPMEC)核因子κB(NF-κB)结合活性和环氧合酶-2(COX-2)表达的影响机制.方法 体外培养HPMEC细胞株,采用四甲基偶氮唑盐(MTT)检测NAC对HPMEC增殖活化的抑制作用,分别采用NAC(1 mmol/L)处理1 h,肿瘤坏死因子α(TNF-α)(100 ng/mL)处理1 h,NAC+TNF-α联合处理.凝胶电泳移动抑制实验检测HPMEC NF-κB的结合活性;免疫蛋白质印迹检测相应的HPMEC胞质内NF-κB抑制蛋白(IκB-α)的表达;免疫细胞化学观察HPMEC NF-κB表达的核内转移;激光共聚焦检测NAC对HPMEC中COX-2表达的影响.结果 NAC对HPMEC的增殖活化有明显抑制作用,NAC+TNF-α联合处理组吸光度值明显低于TNF-α处理组,差异均有统计学意义(均P < 0.05).TNF-α刺激后具有诱导HPMEC NF-κB结合活性,且IκB-α表达明显减弱,NAC处理组IκB-α表达高于TNF-α处理组,差异有高度统计学意义(P < 0.01).TNF-α处理1 h后,HPMEC NF-κB的主要表达从细胞质转移至细胞核内;NAC预处理后联合TNF-α刺激,HPMEC NF-κB表达主要位于细胞质,出现核内转移极少.HPMEC经TNF-α处理后细胞内COX-2表达明显高于NAC+TNF-α联合处理组以及正常对照组,差异均有统计学意义(均P < 0.05);而NAC+TNF-α联合处理组与正常对照组比较,差异无统计学意义(P > 0.05).结论 NAC 可抑制HPMEC增殖活化;NAC可抑制HPMEC NF-κB结合活性、减少核内转移发生和COX-2的表达.
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