摘要
目的 阐明N乙酰半胱氨酸(NAC)对肝星状细胞(HSC)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 体外培养大鼠 HSC-T6 细胞株,MTT法检测 NAC对HSC增殖的抑制作用。分别予NAC(1 mmol/L)处理 1 h;NAC和肿瘤坏死因子(TNF)α联合干预(先予 NAC处理1 h,再予TNFα干预1 h);TNFα100 ng/ml处理1 h。凝胶电泳移动抑制实验检测NF κB的结合活性。免疫蛋白质印迹检测相应的胞质内NF-κB抑制蛋白(IκBα)表达。免疫组化观察 HSC-T6NF-κB表达的核转移。激光共聚焦检测NAC对 HSC-T6 中 COX-2 表达的影响。结果 NAC对 HSC具有明显的抑制作用。TNFα可诱导 NF-κB结合活性,而 NAC可显著抑制 TNFα诱导的 NF-κB结合活性。TNFα处理后 IκBα表达减弱,NAC处理后 IκBα表达增强。TNFα刺激 1 h后,NF κB表达从细胞质转移至细胞核内。NAC预处理后再予TNFα刺激,NF κB表达主要位于细胞质,很少发生核转移。HSC T6经TNFα处理后细胞内COX 2表达明显高于NAC和TNFα联合处理组以及正常对照组(P<0.05);NAC和TNFα联合处理组与正常对照组差异无统计学意义(P> 0. 05)。结论 NAC可抑制HSC增殖,抑制HSC-NF κB结合活性和COX-2表达。
Objective To determine the effect of N-acetylcysteine (NAC, an antioxidant) on (nuclear) factor-κB (NF-κB) DNA binding activity and expression of cyclooxygenase-2 (COX-2) in hepaticstellate cell (HSC) in vitro. Methods HSC-T6 cell line was treated with NAC or tumor necrosis factor (TNF)α, and the cell proliferation was determined with MTT colorimetric assay. HSC-T6 cells were pre-incubated with NAC for 1 h prior to exposure to TNFα and then with TNFα for 1 h. DNA binding (activity) of NF-κB was analyzed by electrophoretic gel mobility shift assay (EMSA). The protein expression of IκBα was examined by Western blot. NF-κB expression translocation was detected by immunohistochemistry. Moreover, the cells were treated with NAC (10^(-3)mol/L) and TNFα (100 ng/ml) for 24 h. COX-2 expression was assessed by immunobloting and laser confocal microscopy. Results The proliferation of HSC-T6 cells can be inhibited by NAC. EMSA revealed that there was an up-regulation in NF-κB DNA binding activity after HSC were treated with TNFα. NAC treatment can markedly reduce DNA binding activity of NF-κB induced by TNFα. Western blot showed that protein expression of IκBα (decreased) in TNFα treatment group. NAC treatment significantly increased the protein expression of IκBα. Immunohistochemical staining showed that NF-κB expression was translocated from cytoplasm into (nucleus) after the cells were treated with TNFα for 1 h. The effect can be inhibited by NAC. Laser (con-)(focal) microscopy showed that COX-2 expression can be intensified by TNFα and reduced by NAC. Conclusions The proliferation of HSC-T6 cells can be inhibited by NAC. NAC can reduce NF-κB DNA binding (activity) and expression of COX-2 in HSC.
出处
《中华消化杂志》
CAS
CSCD
北大核心
2005年第2期83-86,共4页
Chinese Journal of Digestion
基金
国家自然科学基金资助项目(30270610)
