巯基化合物对内毒素诱导大鼠枯否细胞NF-κB活性和肿瘤坏死因子释放的影响

Effects of thiol on NF-κB activity in and release of TNF-α from endotoxin-activated cultured rat kupffer cells

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 determine whether thiol can affect the activity of nuclear factor kappa B （NF-kB ） in and release of TNF-afrom lipopolysaccharide （LPS） -activated kupffer cells （KCs） and ascertain whether these effects are mediated through glutathione （GSH）. Methods KCs were isolated from livers of healthy male adult SD rats weighing 250-300g and cultured for 12h. The cultured KCs were exposed to LPS 10 ng·ml^-1 . The effects of different concentrations of glutathione monoethylester （GSHEE） and N-acetylcysteine （NAC） on NF-kB activity in and release of TNF-afrom KCs were determined. The effect of pretreatment with GSH synthesis inhibitor - BSO on the inhibitory effect of NAC on inflammation was investigated. NFkB activity was determined by electrophoretic mobility shift assay （EMSA） and intracellular GSH content and TNF-alevel in supernatant by HPLC. Results Intracellular GSH content remained unchanged after exposure of KCs to LPS. Both GSHEE and NAC increased intracellular GSH level and inhibited NF-kB activity and release of TNF-a. BSO blocked the increase in GSH induced by NAC but did not affect the inhibitory effect of NAC on TNF-urelease. Conclusions LPS does not affect GSH level in KCs. NAC can inhibit NF-kB activity in and TNF-urelease from KCs and increase intracellular GSH level but the inhibitory effect is not mediated through GSH.