Role of interleukin-1 receptor antagonist in stress response of rats

我们研究了白细胞介素-1受体拮抗剂（IL-1 ra）mRNA的反义寡核苷酸进入大鼠脑侧脑室是否可以减少脑中细胞因子的浓度，以及是否可以通过施用反义寡核苷酸来抑制应激诱导的细胞因子水平的增强。使用ANTISENSER（Biognostik，Gottingen，德国）确定反义寡核苷酸和错配寡核苷酸的排列。以大鼠肺泡巨噬细胞NR 8383为靶点，观察反义寡核苷酸的抑制作用。将2纳摩尔的寡核苷酸注射到大鼠（Sprague-Dawley，雄性，12-14周龄）的左侧脑室中。注射后12 h，在乙醚和戊巴比妥麻醉下将大鼠断头，从颅骨取出脑，并在冰上用10倍体积的培养基匀浆。离心（15，000 rpm，10 ...更多信息 min）并在-80 ℃下储存直至测量IL-1 ra水平。在一系列研究应激暴露影响的实验中，从制动60分钟的大鼠中提取上清液。实验符合庆应义塾大学医学部实验动物护理和使用指南中提出的标准。向培养基中添加反义寡核苷酸显著抑制了NR 8383产生的IL-1 ra浓度。与不匹配组相比，脑室注射反义寡核苷酸并没有降低脑内IL-1 ra的浓度。应激暴露并不引起大鼠脑内IL-1 ra水平的升高，应用反义寡核苷酸对应激暴露大鼠脑内IL-1 ra水平也无影响，提示改变脑内注射时间对反义寡核苷酸的抑制作用是必要的。少

We investigated whether administration of antisense oligonucleotide for interleukin-1 receptor antagonist (IL-1ra) mRNA into the lateral ventricle of rat brain could diminish concentrations of the cytokine in the brain, and whether stress-induced enhancement of the cytokine levels could be inhibited by administrating the antisense oligonucleotide. Arrangement of both antisense oligonucleotide and miss-matched one was determined by using ANTISENSER (Biognostik, Gottingen, Germany). Inhibitory effect of antisense oligonucleotide was estimated by NR8383 derived from rat alveolar macrophage. Two nano mole of oligonucleotide was injected into the left lateral ventricle of rats (Sprague-Dawley, male, 12-14 week old). The rats were decapitated under anesthesia with ether and pentobarbital at 12 h after the injection, and the brains were removed from the skull and homogenized with 10-times volume of culture medium on ice. Supernatant of the medium was collected after centrifuge (15,000 rpm, 10 … More min) and stored at -80 * until measurement of IL-1ra levels. In the series of experiments to investigate effect of stress exposure, supernatant was extracted from rats that had been immobilized for 60 min. The experiments conform to the standards put forth in the Guideline for the Care and Use of Laboratory Animals of Keio University School of Medicine.Adding antisense oligonucleotide into the culture medium significantly inhibited concentration of IL-1ra produced by NR8383. Administration of antisense oligonucleotide into the rat ventricle did not decrease in the concentration of IL-1ra in the brain, compared with that of miss-matched one. Stress exposure did not enhance the levels of IL-1ra in the rat brain, and administration of antisense oligonucleotide had no effect on the concentration of IL-1ra in the brain of the stress-exposed rats.These results suggest that an alteration of timing for injection into the brain of animals was necessary to lead inhibitory effect of antisense oligonucleotide sufficiently. Less