Departments of Biomedical Sciences, Ohio University Heritage, College of Osteopathic Medicine, and Edison Biotechnology Institute, Ohio University, Athens, OH, USA.
Crit Care Med. 2012 Mar;40(3):886-94. doi: 10.1097/CCM.0b013e318236ef8b.
One form of sepsis, or endotoxic shock, is a hyperactivated systemic response caused by excessive expression of proinflammatory mediators, which results from Gram-negative bacterial lipopolysaccharide-stimulated Toll-like receptor-4 signaling. This lipopolysaccharide signaling is known to consist of a MyD88-dependent nuclear factor-κB-mediated pathway that results in production of proinflammatory mediators (tumor necrosis factor-α, interleukin-6, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, inducible nitric oxide synthase, cyclooxygenase-2) and a MyD88-independent interferon regulatory factor-mediated pathway that regulates production of Type 1 interferon-inducible proteins (interferon γ-induced protein-10, monocyte chemotactic protein-1). In prior studies, phenylmethimazole markedly decreased virally induced Toll-like receptor-3 expression and signaling and significantly suppressed murine colitis in an experimental model wherein lipopolysaccharide is known to play an important role.
In this study, we probed the hypothesis that phenylmethimazole inhibits lipopolysaccharide-mediated Toll-like receptor-4 signaling and is efficacious in attenuating inflammatory changes and improving survival in an in vivo murine model of endotoxic shock.
Experimental animal model.
University laboratory.
Male C57BL/6J mice weighing 18-22 g.
Phenylmethimazole (1 mg/kg) was administered intraperitoneally to mice before a lethal lipopolysaccharide challenge (25 mg/kg). RAW264.7 mouse macrophage cells were pretreated with phenylmethimazole followed by lipopolysaccharide stimulation.
: Macroscopic observations revealed that phenylmethimazole was significantly protective in controlling clinical manifestations of endotoxic shock and death under conditions wherein flunixin of meglumine and prednisolone were marginally effective. A combination of enzyme-linked immunosorbent assay, Northern blot, reverse transcriptase-polymerase chain reaction, immunohistochemistry, and Western blot analyses showed that phenylmethimazole attenuated lipopolysaccharide-induced increases in production of proinflammatory cytokines (tumor necrosis factor-α, interleukin-6, interferon-γ), endothelial cell adhesion molecules (intercellular adhesion molecule-1, vascular cell adhesion molecule-1), inducible nitric oxide synthase and cyclooxygenase-2, interferon regulatory factor-1, interferon-inducible proteins (interferon γ-induced protein-10, monocyte chemotactic protein-1), and signal transducer and activator of transcription-1 phosphorylation in multiple tissues in mice. Consistent with these observations, electrophoretic mobility shift assay demonstrated that phenylmethimazole inhibited in vitro lipopolysaccharide-induced nuclear factor-κB and interferon regulatory factor-1 activation in RAW 264.7 mouse macrophages.
Collectively, these results provide direct evidence that phenylmethimazole diminishes lipopolysaccharide-induced MyD88-dependent as well as MyD88-independent signaling pathways and is protective in an experimental model of endotoxic shock.
败血症（包括内毒素休克）是一种全身过度激活的反应，是由革兰氏阴性菌脂多糖刺激 Toll 样受体 4 信号通路导致的促炎介质过度表达引起的。这种脂多糖信号通路由 MyD88 依赖性核因子-κB 介导的途径组成，导致促炎介质（肿瘤坏死因子-α、白细胞介素-6、细胞间黏附分子-1、血管细胞黏附分子-1、诱导型一氧化氮合酶、环氧化酶-2）的产生，以及由 MyD88 非依赖性干扰素调节因子介导的途径调节 I 型干扰素诱导蛋白（干扰素 γ 诱导蛋白-10、单核细胞趋化蛋白-1）的产生。在先前的研究中，苯并咪唑显著降低了病毒诱导的 Toll 样受体 3 表达和信号，并在实验模型中显著抑制了脂多糖起重要作用的小鼠结肠炎。
本研究旨在探讨苯并咪唑抑制脂多糖介导的 Toll 样受体 4 信号通路的假说，并在脂多糖诱导的内毒素休克的体内小鼠模型中证明其减轻炎症变化和提高存活率的功效。
实验动物模型。
大学实验室。
体重 18-22 克的雄性 C57BL/6J 小鼠。
苯并咪唑（1mg/kg）在给予致死性脂多糖（25mg/kg）前经腹腔注射给予小鼠。用苯并咪唑预处理 RAW264.7 小鼠巨噬细胞，然后用脂多糖刺激。
宏观观察表明，苯并咪唑在控制内毒素休克的临床表现和死亡方面具有显著的保护作用，而氟尼辛甲胺和泼尼松龙的效果则略有改善。酶联免疫吸附试验、Northern blot、逆转录聚合酶链反应、免疫组织化学和 Western blot 分析表明，苯并咪唑减弱了脂多糖诱导的促炎细胞因子（肿瘤坏死因子-α、白细胞介素-6、干扰素-γ）、内皮细胞黏附分子（细胞间黏附分子-1、血管细胞黏附分子-1）、诱导型一氧化氮合酶和环氧化酶-2、干扰素调节因子-1、干扰素诱导蛋白（干扰素 γ 诱导蛋白-10、单核细胞趋化蛋白-1）和信号转导和转录激活因子 1 磷酸化的产生在小鼠的多种组织中。与这些观察结果一致，电泳迁移率变动分析表明，苯并咪唑抑制了体外脂多糖诱导的 RAW264.7 小鼠巨噬细胞中核因子-κB 和干扰素调节因子-1 的激活。
综上所述，这些结果为苯并咪唑可减弱脂多糖诱导的 MyD88 依赖性和非依赖性信号通路提供了直接证据，并在实验性内毒素休克模型中具有保护作用。
