内毒素休克大鼠核因子κB活化规律及其在生物蝶呤诱生中的作用

The pattern of nuclear factor-κB activation in rats with endotoxin shock and its role in biopterin-mediated nitric oxide induction

目的观察内毒素休克大鼠血浆及主要脏器核因子(NF)κB活化规律及其对生物蝶呤(BH4)和一氧化氮(NO)表达水平的影响,探讨内毒素休克时NF-κB信号通路对BH4诱生NO的分子调控机制及其与多器官功能损害的关系。方法将47只大鼠按表格随机法分为正常组(8只)、内毒素／脂多糖(LPS)组(24只,每观察时相点8只,均同时注射LPS制成休克模型)和拮抗组[15只,每观察时相点5只,均同时注射LPS并以吡咯烷二硫代氨基甲酸盐(PDTC)拮抗]。休克及拮抗组于注射LPS后2、6、12 h观察,并与正常组同法处死,无菌留取大鼠血标本及肝、肺、肾组织,测定组织中NF-κB活性和三磷酸鸟苷环水解酶Ⅰ(GTP-CHⅠ)和诱导型一氧化氮合酶(iNOS)mRNA表达水平、血浆和组织中的BH4含量及NO水平、肝脏和肾脏功能指标、肺组织髓过氧化物酶活性。结果与正常组(例如肺组织中NF-κB活性为26±6)比较,LPS组大鼠组织中NF-κB迅速活化(P<0．01),并于注射后2 h达峰值(肺组织中为291±44);LPS组各组织中GTP-CHⅠ和iNOS mRNA表达、BH4和NO水平也较正常组明显升高(P<0．05或0．01),至伤后12 h仍持续较高水平。此外,该组相应器官功能均受到不同程度的损害。应用PDTC的拮抗组大鼠各组织中NF-κB活性均较LPS组有所降低,GTP-CHⅠ、iNOS mRNA表达及BH4、NO水平显著受抑,肝、肺、肾功能明显改善。结论内毒素休克时机体内NF-κB通路高度活化,并对BH4／NO系统具有明显调节效应;可通过下调BH4介导的iNOS的过度活化抑制NF-κB信号途径,从而减轻组织炎性反应,对机体脏器功能起到保护作用。

Objective To investigate the pattern of nuclear factor-kappaB （NF-κB） activation in rats with lipopolysaccharide（LPS） shock, and to explore the mechanism of NF-κB signal pathway in the biop terin-mediated nitric oxide（NO） induction, as well as its role in the development of multiple organ dysfunction syndrome （MODS） secondary to endotoxin challenge. Methods Fourty-seven male Wistar rats were randomly divided into control group （ C, n = 8） , LPS group （ n = 24, with 8 rats at each time-points, and shock model was made by injection of same dosage of LPS） , and pyrrolidine dithiocarbamate （PDTC） treatment group （PDTC, n = 15, with 5 rats at each time-points, and the rats were injected with LPS and PDTC）. The rats were sacrificed at 2,6,12 post-injection hour（PIH）, and the blood and tissue samples from liver, lungs and kidneys were harvested for the determination of NF-κB activity, GTP cyclohydrolase Ⅰ （GTP-CH Ⅰ ）, and inducible nitric oxide synthase （iNOS） mRNA expression in the liver, lungs and kidneys, plasma and tissue content of biopterin and NO, as well as hepatic and renal function, and pulmonary myeloperoxydase activity. Results NF-κB DNA binding activity in LPS group was rapidly enhanced in liv er, lungs and kidneys after endotoxin challenge when compared with that in controls （ e. g. in pulmonary tissue it was 26 ±6） , and it reached the peak at 2 PIH, which was 291 ±44 in pulmonary tissue（ P 〈0. 01 ）. GTP-CH Ⅰ mRNA expression and biopterin levels in the liver, lung and kidney of each group were obviously higher than those in control group（ P 〈 0.05 or 0.01 ） , and it maintained at high levels at 12 PIH. Additionally, different degrees of dysfunction of the above mentioned organs was observed. Treatment with PDTC, an inhibitor of NF-κB signal transduction pathway, could reduce NF-κB DNA binding activity, inhibit GTP-CHⅠ and iNOS/NO mRNA expression, as well as BH4 and NO levels in various tissues. Meanwhile the multiple organ damage was significantly ameliorated by PDTC pretreatment. Conclusion Endotoxin challenge can rapidly lead to activation of NF-κB in various tissues, and NF-κB pathway might markedly up-regulate the production of biopterin/NO following endotoxic shock. Inhibition of NF-κB pathway attenuates inflammatory response and ameliorates multiple organ dysfunction, which might be associated with its downregulation of the excessive activation of iNOS mediated by biopterin.