Yu Zaifang, Predina Jarrod D, Cheng Guanjun
Zaifang Yu, Jarrod D Predina, Guanjun Cheng, Thoracic Oncology Research Laboratory, 1016B ARC, University of Pennsylvania, Philadelphia, PA 19104, United States.
World J Biol Chem. 2013 May 26;4(2):18-29. doi: 10.4331/wjbc.v4.i2.18.
To explore the possibility that nucleotide oligomerization domain 1 (NOD1) pathway involved in refractoriness of interferon-β signaling in mouse respiratory epithelial cells induced by the anticancer xanthone compound, 5,6-dimethylxanthenone-4-acetic acid (DMXAA).
C10 mouse bronchial epithelial cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mmol/L glutamine, 100 units/mL penicillin, 100 g/mL streptomycin. Pathogen-free female BALB/c mice were used to explore the mechanisms of refractoriness of interferon-signaling. Mouse thioglycollate-elicited peritoneal macrophages, bone marrow derived macrophages and bone marrow derived dendritic cells were collected and cultured. The amount of interferon (IFN)-inducible protein-10 (IP10/CXCL10), macrophage chemotactic protein (MCP1/CCL2) and interleukin (IL)-6 secreted by cells activated by DMXAA was quantified using enzyme-linked immunosorbent assay kits according to the instructions of the manufacturers. Total RNA was isolated from cells or nasal epithelium with RNeasy Plus Mini Kit, and cDNA was synthesized. Gene expression was checked using Applied Biosystems StepOne Real-Time Polymerase Chain Reaction System. Transfection of small interfering RNA (siRNA) control, NOD1 duplexed RNA oligonucleotides, and high-mobility group box 1/2/3 (HMGB1/2/3) siRNA was performed using siRNA transfection reagent.
DMXAA activates IFN-β pathway with high level of IFN-β dependent antiviral genes including 2', 5'-oligoadenylate synthetase 1 and myxovirus resistance 1 in mouse thioglycollate-elicited peritoneal macrophages, bone marrow derived macrophages and bone marrow derived dendritic cells. Activation of IFN-β by DMXAA involved in NOD1, but not HMGB1/2/3 signal pathway demonstrated by siRNA. NOD1 pathway plays an important role in refractoriness of IFN-β signaling induced by DMXAA in mouse C10 respiratory epithelial cells and BALB/c mice nasal epithelia. These data indicate that DMXAA is not well adapted to the intrinsic properties of IFN-β signaling. Approaches to restore sensitivity of IFN-β signaling by find other xanthone compounds may function similarly, could enhance the efficacy of protection from influenza pneumonia and potentially in other respiratory viral infections.
NOD1 pathway may play an important role in refractoriness of IFN-β signaling in mouse respiratory epithelial cells induced by DMXAA.
探讨核苷酸寡聚化结构域1（NOD1）通路在抗癌呫吨酮化合物5,6 - 二甲基呫吨酮 - 4 - 乙酸（DMXAA）诱导的小鼠呼吸道上皮细胞中干扰素 - β信号难治性中的作用。
C10小鼠支气管上皮细胞在补充有10%胎牛血清、2 mmol/L谷氨酰胺、100单位/mL青霉素、100 μg/mL链霉素的杜尔贝科改良伊格尔培养基中培养。使用无特定病原体的雌性BALB/c小鼠来探究干扰素信号难治性的机制。收集并培养小鼠巯基乙酸盐诱导的腹腔巨噬细胞、骨髓来源的巨噬细胞和骨髓来源的树突状细胞。根据制造商的说明，使用酶联免疫吸附测定试剂盒对DMXAA激活的细胞分泌的干扰素（IFN）诱导蛋白10（IP10/CXCL10）、巨噬细胞趋化蛋白（MCP1/CCL2）和白细胞介素（IL）-6的量进行定量。使用RNeasy Plus Mini试剂盒从细胞或鼻上皮中分离总RNA，并合成cDNA。使用Applied Biosystems StepOne实时聚合酶链反应系统检查基因表达。使用siRNA转染试剂进行小干扰RNA（siRNA）对照、NOD1双链RNA寡核苷酸和高迁移率族蛋白盒1/2/3（HMGB1/2/3）siRNA的转染。
DMXAA在小鼠巯基乙酸盐诱导的腹腔巨噬细胞、骨髓来源的巨噬细胞和骨髓来源的树突状细胞中激活IFN - β通路，同时高水平表达包括2',5'-寡腺苷酸合成酶1和抗黏液病毒蛋白1在内的IFN - β依赖性抗病毒基因。siRNA结果表明，DMXAA激活IFN - β涉及NOD1通路，而非HMGB1/2/3信号通路。NOD1通路在DMXAA诱导的小鼠C10呼吸道上皮细胞和BALB/c小鼠鼻上皮中IFN - β信号难治性中起重要作用。这些数据表明，DMXAA与IFN - β信号的内在特性不相适应。寻找其他呫吨酮化合物以恢复IFN - β信号敏感性的方法可能具有相似作用，可增强预防流感肺炎以及潜在预防其他呼吸道病毒感染的保护效果。
NOD1通路可能在DMXAA诱导的小鼠呼吸道上皮细胞中IFN - β信号难治性中起重要作用。
