Taylor B S, Shao L, Gambotto A, Ganster R W, Geller D A
Department of Surgery, University of Pittsburgh, PA 15261, USA.
Surgery. 1999 Aug;126(2):142-7.
Nitric oxide is overexpressed in nearly every organ during sepsis and it has profound biologic effects. Previously, we showed that maximal inducible nitric oxide synthase (iNOS) expression is up-regulated by a combination of cytokines and that this effect is mediated by the transcription factor NF-kappa B. Therefore the purpose of this study was to establish whether gene transfer of the inhibitory molecule I kappa B would result in the abrogation of cytokine-induced iNOS expression.
Cultured hepatocytes were infected with an adenoviral vector containing the I kappa B alpha gene (Ad5I kappa B) and after an 18-hour recovery period were stimulated with the cytokine mixture of tumor necrosis factor-alpha (500 U/mL) plus interleukin 1 beta (200 U/mL) plus interferon gamma (100 U/mL).
As expected, cytokine mixture induced significant hepatocyte nitrite (NO2-) and iNOS messenger RNA production. Cells infected with the I kappa B alpha gene showed a dose-dependent decrease in NO2- and iNOS messenger RNA levels. Western blot analysis showed a marked decrease in iNOS protein levels in the presence of Ad5I kappa B alpha. Gel shift assays of nuclear extracts demonstrated that Ad5I kappa B alpha decreased the cytokine-induced DNA binding activity for NF kappa B.
NF kappa B is an important regulator of cytokine-induced NO expression. These results identify a novel therapeutic approach where gene transfer of the inhibitory molecule I kappa B alpha can be used to down-regulate cytokine-induced iNOS expression as well as other NF kappa B-dependent genes that are up-regulated during the inflammatory response.
在脓毒症期间，一氧化氮在几乎每个器官中均过度表达，且具有深远的生物学效应。此前，我们发现细胞因子组合可上调诱导型一氧化氮合酶（iNOS）的最大表达，且此效应由转录因子核因子κB（NF-κB）介导。因此，本研究的目的是确定抑制分子IκB的基因转移是否会导致细胞因子诱导的iNOS表达被消除。
用含IκBα基因的腺病毒载体（Ad5IκB）感染培养的肝细胞，在18小时恢复期后，用肿瘤坏死因子-α（500 U/mL）加白细胞介素1β（200 U/mL）加干扰素-γ（100 U/mL）的细胞因子混合物进行刺激。
正如预期的那样，细胞因子混合物诱导肝细胞亚硝酸盐（NO2-）和iNOS信使核糖核酸产生显著增加。用IκBα基因感染的细胞显示NO2-和iNOS信使核糖核酸水平呈剂量依赖性降低。蛋白质免疫印迹分析显示，在存在Ad5IκBα的情况下，iNOS蛋白水平显著降低。对核提取物的凝胶迁移试验表明，Ad5IκBα降低了细胞因子诱导的NF-κB的DNA结合活性。
NF-κB是细胞因子诱导的NO表达的重要调节因子。这些结果确定了一种新的治疗方法，即抑制分子IκBα的基因转移可用于下调细胞因子诱导的iNOS表达以及在炎症反应期间上调的其他NF-κB依赖性基因。
