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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 elucidate the mechanism of the effect of NAC on nuclear factor κB (NF-κB) binding activity and cyclooxygenase 2 (COX-2) expression in hepatic stellate cells (HSCs). Methods Rat HSC-T6 cells were cultured in vitro. The inhibitory effect of NAC on HSC proliferation was detected by MTT assay. The cells were treated with NAC (1 mmol / L) for 1 h respectively. NAC and tumor necrosis factor (TNF) α were intervened (NAC for 1 h followed by TNFα for 1 h); TNFα 100 ng / ml for 1 h. Gel electrophoretic mobility shift assay was used to detect the binding activity of NF κB. The corresponding cytoplasmic NF-κB inhibitory protein (IκBα) expression was detected by immunoblotting. Immunohistochemistry was used to observe the nuclear translocation of HSC-T6NF-κB. Effect of NAC on the Expression of COX-2 in HSC-T6 Cells by Laser Scanning Confocal Scanning. Results NAC had obvious inhibitory effect on HSC. TNFα can induce NF-κB binding activity, while NAC can significantly inhibit TNFα-induced NF-κB binding activity. The expression of IκBα was decreased after TNFα treatment, and the expression of IκBα was enhanced after NAC treatment. After stimulated by TNFα for 1 h, NF κB expression was transferred from the cytoplasm to the nucleus. After pretreatment with NAC, the cells were stimulated with TNFα. The expression of NF-κB mainly located in the cytoplasm with few nuclear translocation. The expression of COX 2 in HSC T6 treated with TNFα was significantly higher than that in NAC and TNFα combined treatment group and normal control group (P <0.05). There was no significant difference between NAC and TNFα combined treatment group and normal control group (P> 0.05). 05). Conclusion NAC can inhibit HSC proliferation, inhibit HSC-NF κB binding activity and COX-2 expression.