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目的观察巯基化合物对内毒素诱导大鼠枯否细胞(KC)NF-κB活性和肿瘤坏死因子-α (TNF-α)释放的影响。方法采用胶原酶灌注和percoll密度梯度离心法分离得到高纯度大鼠KC,在过夜培养后冲洗两遍(不含血清),加入内毒素(LPS)10ng／ml刺激原代培养的KC,观察不同浓度谷胱甘肽乙基酯(GSHEE)及N-乙酰半胱氨酸(NAC)对大鼠KC TNF-α释放和KC内谷胱甘肽(GSH)及NF- κB活性的影响。并观察使用谷胱甘肽合成抑制剂BSO后,对NAC抑制炎症因子的影响,采用凝胶滞留电泳法测NF-κB活性,采用高效液相色谱分析法测GSH水平。结果 LPS诱导KC中GSH水平无变化;GSHEE和NAC可提高KC中GSH水平(P<0．05),降低NF-κB活性和TNF-α释放(P<0．05)。 NAC与BSO合用时KC中GSH水平无变化,TNF-α释放降低(P<0．05)。结论NAC通过抑制KC中 NF-κB的激活和TMF-α的释放调节KC功能,但这种抑制作用并非通过增加GSH介导。
Objective To investigate the effects of sulfhydryl compounds on the activity of NF-κB and the release of tumor necrosis factor-α (TNF-α) in Kupffer cells induced by endotoxin in rats. Methods High purity rat KC was isolated by collagenase perfusion and percoll density gradient centrifugation, washed twice (without serum) after overnight culture, primary cultured KC was stimulated by LPS (10ng / ml) (GSHEE) and N-acetylcysteine (NAC) on the release of KC and the content of glutathione (GSH) and NF-κB in KC were determined. The effect of NAC on the inflammatory cytokines was observed after using glutathione syntheses inhibitor BSO. The activity of NF-κB was measured by gel-retention electrophoresis and the level of GSH was measured by HPLC. Results GSH levels in KC induced by LPS were unchanged. GSHEE and NAC increased GSH level in KC (P <0.05), and decreased NF-κB activity and TNF-α release (P <0.05). There was no change in GSH level and TNF-α release in NAC and BSO group (P <0.05). Conclusion NAC regulates KC function by inhibiting the activation of NF-κB and the release of TMF-α in KC, but this inhibition is not mediated by increased GSH.