内毒素肝损伤过程中NF--kB和AP--1活性变化及其对IL--6表达的调控

Kinetics of the activation of NF-kB and AP-1 and their regulation of IL-6 expression during LPS-induced liver injury

目的:探讨内毒素肝损伤过程中,Kupffer细胞(KC)内主要转录因子核因子-kB(nuclear factor-kappa B,NF-kB)、活化蛋白-1(activator protein-1,AP-1)活性的变化规律及其在炎细胞因子IL-6表达中的调控作用。方法:健康昆明种小鼠随机分组如下:LPS尾静脉注射3h的量效关系:正常对照组和低(1mg/kg)、中(5mg/kg)、高(10mg/kg)三个内毒素剂量组;注射5mg/kg LPS的时效关系:正常对照、0.5h、1h、3h、5h、8h组;吡咯啉烷二硫代氨基甲酸盐(pyrrolidine dithiocarbamate,PDTC)干预组(3h):正常对照、5mg/kg LPS、200mg/kg PDTC、200mg/kg PDTC +5mg/kg LPS.EMSA法检测KC中NF-kB、AP-1活性,ELISA法检测肝组织中IL-6的表达水平。结果:内毒素肝损伤过程中KC中NF-kB活性与LPS处理3h的量效关系为:1mg/kg LPS组即可观察到NF-kB活性,5mg/kg LPS组达峰值,10mg/kgLPS组仍可维持高活化状态;用5mg/kg LPS处理后的时效关系为:0.5h即可检测到NF-kB活性,3h活性明显增强,并可持续到至少8h;PDTC对NF-kB活性有明显抑制作用。内毒素肝损伤过程中KC中AP-1活性与LPS处理3h的量效关系为:1mg/kg LPS组即可检测到AP-1活性,5mg/kg LPS组活性与1mg/kg LPS组相比稍有减弱,10mg/kg LPS组又明显增强,三剂量组AP-1活性与对照组相比均显著增强;用5mg/kg LPS处理后的时效关系为:0.5h即可检测到AP-1活性,1h达峰值,3h减弱后,5h增强,8h又有所减弱,表现出明显的波动现象,但各时相点AP-1活性均显著强于对照;PDTC对AP-1活性有明显促进作用。内毒素肝损伤过程中,内毒素的不同剂量和作用的不同时间肝组织内IL-6水平均表现为先升高后降低的变化趋势,且各处理组均明显高于正常对照;PDTC能明显抑制IL-6的释放。相关分析显示IL-6水平的变化与NF-kB活性变化呈显著正相关,与AP-1活性变化则无明显相关性。结论:内毒素肝损伤过程中,KC中NF-kB、AP-1均不同程度活化,但NF-kB对IL-5的表达可能具有一定的调控作用,而AP-1可能并不参与IL-6表达的调控。

AIM: To explore the kinetics of the activation of NF-kB and AP-1 and their regulations of IL-6 expression during LPS induced liver injury. METHODS: Kunming mice were divided randomly as follows: dose-effect of LPS infusion 3h: normal control group, low(1mg/kg), intermediate(5mg/kg), high(10mg/kg)LPS induced groups; time-effect of 5mg/kg LPS infusion: normal control group, 0.5h, 1h, 3h, 5h, 8hgroups; PDTC intervened groups(3h): normal control group, 5mg/kg LPS, 200 mg/kg PDTC and 200 mg/kg PDTC+5 mg/kg LPS groups. NF-kB and AP-1 activities of KC were determined with EMSA and expression levels of IL-6 were measured with ELISA. RESULTS: NF-kB activation could be detected in 1mg/kg LPS group, reached the highest level in 5mg/kg LPS group, and persisted in 10mg/kg LPS group. The DNA-binding activity was observable at 0.5h after LPS infusion, increased significantly at 3h, and persisted for at least 8h. In addition, antioxidant PDTC could inhibit the activation of NF-kB significantly. AP-1 activation could be detected in 1mg/kg LPS group, slightly reduced in 5mg/kg LPS group compared with that of the 1mg/kg LPS group, and significantly increased in 10mg/kg LPS group. Its DNA-binding activity was detectable at 0.5h after LPS infusion, peaked at 1h, reduced after 3h, increased after 5h, then reduced after 8h again, all exhibiting higher activity than the control but with some fluctuations. PDTC could significantly promote the activation of AP-1. The kinetics of IL-6 level in liver tissues during LPS-induced liver injury were that IL-6 level after 3h of infusion increased first and then reduced; the same trend was observed in the time-course on IL-6 level after LPS infusion; PDTC could significantly inhibit the release of IL-6. Correlation analyses revealed that IL-6 level was significantly and positively correlated with the activation of NF-kB, while not significantly correlated with the activation of AP-1. CONCLUSIONS: NF-kB and AP-1 in KC are activitied during LPS-induced liver injury to some extent. But NF-kB may have some regulation on the expression of IL-6, while AP-1 may not.