细胞外信号调节激酶在内毒素休克大鼠生物喋呤诱生中的作用机制探讨

The mechanisms of extracellular-signal regulated protein kinase pathway in biopterin induction in rats with endotoxic shock

目的观察细胞外信号调节激酶 (ERK) 通路抑制剂对生物喋呤 (BH4)和一氧化氮(NO)表达及核因子-κB (NF-κB) 活化的影响,探讨内毒素休克时ERK信号通路与NF-κB的交汇作用及其对BH4诱生NO的调控机制.方法采用内毒素休克模型,60只大鼠随机分为正常对照组(n=8)、内毒素休克组(n=32)和ERK抑制剂PD98059拮抗组(n=20).留取动物肝、肺、肾组织进行NF-κB活性分析以及三磷酸鸟苷环水解酶Ⅰ(GTP-CHⅠ)、诱生型一氧化氮合酶 (iNOS) 基因表达的检测,并测定组织及血浆中BH4、NO水平.结果内毒素攻击可导致动物肝、肺、肾组织GTP-CHI基因表达和BH4水平明显升高,至伤后24 h仍持续于较高水平;与之相应,组织iNOS基因表达和NO水平亦明显升高;各组织NF-κB 迅速活化,并于2 h达峰值.采用PD98059处理后,内毒素休克动物肾组织GTP-CHⅠ mRNA表达明显受抑,肝、肺组织GTP-CHⅠ mRNA表达仅呈现降低趋势;血浆及肝、肾组织中BH4水平12 h显著降低;同样,各组织iNOS mRNA表达及NO水平早期亦显著降低.此外,PD98059处理组动物肝组织2～6 h、肺组织2 h、24 h和肾组织24 h时相点 NF-κB活性显著降低.结论内毒素休克时抑制ERK通路,能部分下调BH4和NO表达与NF-κB的活化,表明ERK与NF-κB通路间可能存在交汇作用,共同参与了BH4诱生NO的调控作用.

Objective To observe the influence of treatment with the inhibitor of extracellular-sigual regulated protein kinase （ ERK ） signal transduction pathway on the expression of biopterin/nitric oxide （NO） as well as the activation of nuclear factor-kappaB （NF-kappaB） , and to clarify the potential cross-talk regulation mechanisms between ERK and NF-kappaB pathway in biopterin-mediated NO induction in rats with endotoxic shock. Methods Using an endotoxic shock model, 60 male Wistar rats were randomly divided into normal controls （n = 8） , endotoxic shock group （n = 32 ） and PD98059 treatment group （n = 20）. At serial time points animals in each group were sacrificed, and tissue samples from liver, lungs as well as kidneys were harvested to detect NF-kappaB activity, guanosine triphosphate-cyclohydrolase （GTP-CHⅠ） and inducible nitric oxide synthase （iNOS） mRNA expression. Biopterin and NO levels in plasma and tissues were also assayed. Results It was found that after lipopolysaccharide （LPS） challenge, GTP- CH Ⅰ mRNA expression and biopterin levels significantly elevated in liver, lungs and kidneys, keeping at high values up to 24 h, so did the values of iNOS mRNA expression and NO levels. NF-kappaB DNA binding activity was enhanced rapidly in various tissues, peaking at 2 h after LPS challenge. Treatment with PD98059, an inhibitor of ERK signal transduction pathway, could significantly inhibit GTP-CH Ⅰ mRNA expression in kidneys, and GTP-CH Ⅰ mRNA expression in liver and lungs showed certain down-regulationtendency. At the same time, biopterin level was significantly decreased in plasma, liver and kidneys at 12 h. Similarly, iNOS/NO induction at early stage markedly decreased in various tissues. In addition, treatment with PD98059 reduced NF-kappaB DNA binding activity in liver, lungs, as well as kidneys at 245 h, 2 h, 24 h and 24 h after LPS challenge, respectively. Conclusions Inhibition of ERK pathway could partially inhibit the production of biopterin/NO as well as the activation of NF-kappaB pathway, which indicated that cross-talk regulation seems to be existed between ERK and NF-kappaB pathway, and they might be involved in the regulatory process of biopterin-mediated nitric oxide induction in rats with endotoxic shock.