Department of Food and Nutrition, Brain Korea 21 PLUS Project, College of Human Ecology, Yonsei University, Seoul, 120-749, Korea.
Inflamm Res. 2014 May;63(5):347-56. doi: 10.1007/s00011-013-0706-0. Epub 2014 Jan 12.
To investigate whether glutamine deprivation induces expression of inflammatory cytokine interleukin-8 (IL-8) by determining NF-κB activity and levels of oxidative indices (ROS, reactive oxygen species; hydrogen peroxide; GSH, glutathione) in fibroblasts isolated from patients with ataxia telangiectasia (A-T).
We used A-T fibroblasts stably transfected with empty vector (Mock) or with human full-length ataxia telangiectasia mutated (ATM) cDNA (YZ5) and mouse embryonic fibroblasts (MEFs) transiently transfected with ATM small interfering RNA (siRNA) or with non-specific control siRNA.
The cells were cultured with or without glutamine or GSH.
ROS levels were determined using a fluorescence reader and confocal microscopy. IL-8 or murine IL-8 homolog, keratinocyte chemoattractant (KC), and hydrogen peroxide levels in the medium were determined by enzyme-linked immunosorbent assay and colorimetric assay. GSH level was assessed by enzymatic assay, while IL-8 (KC) mRNA level was measured by reverse transcription-polymerase chain reaction (RT-PCR) and/or quantitative real-time PCR. NF-κB DNA-binding activity was determined by electrophoretic mobility shift assay. Catalase activity and ATM protein levels were determined by O2 generation and Western blotting.
While glutamine deprivation induced IL-8 expression and increased NF-κB DNA-binding activity in Mock cells, both processes were decreased by treatment of cells with glutamine or GSH or both glutamine and GSH. Glutamine deprivation had no effect on IL-8 expression or NF-κB DNA-binding activity in YZ5 cells. Glutamine-deprived Mock cells had higher oxidative stress indices (increases in ROS and hydrogen peroxide, reduction in GSH) than glutamine-deprived YZ5 cells. In Mock cells, glutamine deprivation-induced oxidative stress indices were suppressed by treatment with glutamine or GSH or both glutamine and GSH. GSH levels and catalase activity were lower in Mock cells than YZ5 cells. MEFs transfected with ATM siRNA and cultured without glutamine showed higher levels of ROS and IL-8 than those transfected with negative control siRNA; increased levels of ROS and IL-8 were suppressed by the treatment of glutamine.
Glutamine deprivation induces ROS production, NF-κB activation, and IL-8 expression as well as a reduction in GSH in A-T fibroblasts, all of which are attenuated by glutamine supplementation.
通过测定 NF-κB 活性和氧化指标（ROS、活性氧；过氧化氢；GSH、谷胱甘肽）的水平，来研究谷氨酰胺剥夺是否会诱导来自共济失调毛细血管扩张症（A-T）患者的成纤维细胞表达炎性细胞因子白细胞介素-8（IL-8）。
我们使用了稳定转染空载（Mock）或全长人共济失调毛细血管扩张症突变（ATM）cDNA（YZ5）的 A-T 成纤维细胞，以及瞬时转染 ATM 小干扰 RNA（siRNA）或非特异性对照 siRNA 的小鼠胚胎成纤维细胞（MEFs）。
用或不用谷氨酰胺或 GSH 培养细胞。
使用荧光读取器和共聚焦显微镜测定 ROS 水平。通过酶联免疫吸附测定和比色测定法测定培养基中 IL-8 或鼠类白细胞介素-8 同系物，角质细胞趋化因子（KC）和过氧化氢的水平。通过酶促测定法评估 GSH 水平，通过逆转录聚合酶链反应（RT-PCR）和/或实时定量 PCR 测量 IL-8（KC）mRNA 水平。通过电泳迁移率变动测定法测定 NF-κB DNA 结合活性。通过 O2 生成和 Western 印迹测定法测定过氧化氢酶活性和 ATM 蛋白水平。
尽管谷氨酰胺剥夺会诱导 Mock 细胞中 IL-8 的表达并增加 NF-κB DNA 结合活性，但用谷氨酰胺或 GSH 或谷氨酰胺和 GSH 两者处理细胞会降低这两个过程。谷氨酰胺剥夺对 YZ5 细胞中的 IL-8 表达或 NF-κB DNA 结合活性没有影响。与谷氨酰胺剥夺的 YZ5 细胞相比，谷氨酰胺剥夺的 Mock 细胞具有更高的氧化应激指标（ROS 和过氧化氢增加，GSH 减少）。在 Mock 细胞中，谷氨酰胺剥夺诱导的氧化应激指标通过用谷氨酰胺或 GSH 或两者都处理而得到抑制。与 YZ5 细胞相比，Mock 细胞的 GSH 水平和过氧化氢酶活性较低。在没有谷氨酰胺的情况下转染 ATM siRNA 的 MEFs 比转染阴性对照 siRNA 的 MEFs 显示出更高水平的 ROS 和 IL-8；ROS 和 IL-8 的增加水平通过谷氨酰胺处理得到抑制。
谷氨酰胺剥夺会诱导 A-T 成纤维细胞中 ROS 的产生、NF-κB 的激活和 IL-8 的表达，以及 GSH 的减少，所有这些都可以通过补充谷氨酰胺得到缓解。
