N-乙酰半胱氨酸对肺微血管内皮细胞核因子κB活性影响的体外研究

Effective research of N-acetylcysteine on nuclear factor-κB binding activity and expression of Cyclooxygenase-2 in human pulmonary microvascular endothelial cells in vitro

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。

Objective To investigate the effects of N-acetylcysteine （NAC） on nuclear factor-κB （NF-KB） 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 hinding activity in HPMEC was observed by electrophoretic gel mobility shift assay （EMSA）. The protein expression of IKBαin HPMEC was examined by Western blot. NF-κB expression translocation was detected hy immunohistoehemistry. COX-2 expression of HPMEC was assessed by immunobloting and laser confocal microscopy. Results The proliferation of HPMEC cells eouhl be inhihited by NAC, al）sorhanee value in NAC＋TNF-αtreatment 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 IKB-α was down-regulated after treating with TNFα, expression of IKB-α in NAC trealment group was higher Ihan that in TN F-α treatment group, the difference was statislically significant （P 〈 0.01）. NF-KB expression was transloeated h＇om cytoplasm into nucleus after the cells were wated with TNF-α for 1 h; NF-KB expression of HPMEC was mainly in cytoplasm, rarely in nucleus. The expression of COX-2 after TNF-α lreat-ent was higher than that in NAC＋TNF-α 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 statisti cally 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.