Probiodrug AG, Weinbergweg 22, Halle/Saale, 06120, Germany.
J Neuroinflammation. 2011 Jul 29;8:86. doi: 10.1186/1742-2094-8-86.
Elevated brain levels of the pleiotropic cytokine interleukin-6, which is mainly secreted from activated local astrocytes, contribute to pathological events including neuroinflammation and neurodegeneration. Thus, inhibition of pathological IL-6 expression provides a rationale strategy for targeting the onset or further progression of neurological disorders including Alzheimer's disease, multiple sclerosis, Parkinson's disease and traumatic brain injury. The purpose of this study was to identify and to characterize new potent inhibitors of astrocytic IL-6 expression for further therapeutic development of novel anti-inflammatory and neuroprotective drugs.
Oncostatin M (OSM)-treated human glioma U343 cells were used as model for induction of astrocytic IL-6 expression. This model was characterized by immunoblotting, siRNA technique, ELISA and qRT-PCR and used to screen low molecular weight compound libraries for IL-6-lowering effects. To validate bioactive compounds identified from library screens, bacterial lipopolysaccharide was used to induce IL-6 expression in cultivated primary astrocytes and in mice in vivo. To dissect underlying molecular mechanisms, protein extracts from OSM-treated U343 cells were analyzed by phospho-specific immunoblotting and immunocytochemistry as well as by co-immunoprecipitation.
OSM-treatment (100 ng/ml; 24 h) led to 30-fold increase of IL-6 secretion from U343 cells. The temporal profile of IL-6 mRNA induction displayed a biphasic induction pattern with peak synthesis at 1 h (6.5-fold) and 16 h (5.5-fold) post stimulation. IL-6 protein release did not show that biphasic pattern and was detected as early as 3 h post stimulation reaching a maximum at 24 h. The screen of compound libraries identified a set of heteroarylketones (HAKs) as potent inhibitors of IL-6 secretion. HAK compounds affected the second peak in IL-6 mRNA synthesis, whereas the first peak was insensitive to HAK treatment. HAK compounds also suppressed lipopolysaccharide-induced IL-6 expression in primary murine astrocytes as well as in brain and plasma samples from lipopolysaccharide-treated mice. Finally, HAK compounds were demonstrated to specifically suppress the OSM-induced phosphorylation of STAT3 at serine 727 and the physical interaction of pSTAT3S727 with p65.
Heteroarylketone compounds are potent inhibitors of IL-6 expression in vitro and in vivo and may represent a new class of potent anti-inflammatory and neuroprotective drugs.
多效细胞因子白细胞介素-6（IL-6）的脑内水平升高，主要由活化的局部星形胶质细胞分泌，导致包括神经炎症和神经退行性变在内的病理事件。因此，抑制病理性 IL-6 表达为靶向包括阿尔茨海默病、多发性硬化症、帕金森病和创伤性脑损伤在内的神经疾病的发病或进一步进展提供了合理的策略。本研究的目的是鉴定和表征新的有效的星形胶质细胞 IL-6 表达抑制剂，以进一步开发新型抗炎和神经保护药物。
用奥曲肽（OSM）处理的人神经胶质瘤 U343 细胞作为诱导星形胶质细胞 IL-6 表达的模型。该模型通过免疫印迹、siRNA 技术、ELISA 和 qRT-PCR 进行了表征，并用于筛选降低 IL-6 表达的低分子量化合物库。为了验证从文库筛选中鉴定出的生物活性化合物，使用细菌脂多糖在培养的原代星形胶质细胞和体内的小鼠中诱导 IL-6 表达。为了剖析潜在的分子机制，用磷酸特异性免疫印迹和免疫细胞化学以及共免疫沉淀分析 OSM 处理的 U343 细胞的蛋白提取物。
OSM 处理（100ng/ml；24 小时）导致 U343 细胞中 IL-6 分泌增加 30 倍。IL-6 mRNA 诱导的时间曲线呈双峰诱导模式，刺激后 1 小时（6.5 倍）和 16 小时（5.5 倍）达到峰值。IL-6 蛋白释放未显示出双峰模式，在刺激后 3 小时即可检测到，在 24 小时达到最大值。化合物文库的筛选鉴定了一组杂芳基酮（HAKs）作为 IL-6 分泌的有效抑制剂。HAK 化合物影响 IL-6mRNA 合成的第二个峰值，而第一个峰值对 HAK 处理不敏感。HAK 化合物还抑制脂多糖诱导的原代小鼠星形胶质细胞以及脂多糖处理的小鼠脑和血浆样本中的 IL-6 表达。最后，证明 HAK 化合物特异性抑制 OSM 诱导的 STAT3 丝氨酸 727 磷酸化和 pSTAT3S727 与 p65 的物理相互作用。
杂芳基酮化合物在体外和体内均能有效抑制 IL-6 的表达，可能代表一类新的有效的抗炎和神经保护药物。
