Arima H, Sakamoto T, Aramaki Y, Ishidate K, Tsuchiya S
School of Pharmacy, Tokyo University of Pharmacy and Life Science, Hachioji, Japan.
J Pharm Sci. 1997 Oct;86(10):1079-84. doi: 10.1021/js970099g.
The effects of antisense oligonucleotides (ODNs) on nitric oxide (NO) production induced by lipopolysaccharide (LPS) were investigated using thioglycollate-induced mouse peritoneal macrophages. Antisense phosphorothioate ODNs (S-oligo) corresponding to a sequence in the neighborhood of the AUG initiation codon of a mouse inducible nitric oxide synthase (iNOS) mRNA, which has a G-quartet motif in its antisense sequence, inhibited NO induction in a dose-dependent manner. Antisense phosphodiester ODNs (D-oligo), 5'- and 3'-terminal phosphorothioate-modified antisense ODNs and control scramble and missense S-oligos had no such effect. In addition, control nonsense and two mismatched S-oligos, which include G-quartet motif in their sequences, inhibited NO induction to approximately 50% of those in the control. Antisense S-oligo showed the inhibitory effect on NO production by exposure of macrophages to various concentrations of LPS. Western blot analysis using anti-mouse inducible nitric oxide synthase (iNOS) antibody revealed that antisense S-oligo specifically removed an immunoreactive band at 130 kDa. In addition, the results of reverse transcription-polymerase chain reaction (RT-PCR) revealed that the antisense effect originated from a specific reduction of the targeted iNOS mRNA by hybridization with the antisense S-oligo. Furthermore, no ODNs affected beta-actin mRNA and tumor necrosis factor alpha (TNF-alpha) expression in macrophages stimulated by LPS. These findings demonstrated that antisense S-oligo inhibited NO production derived from iNOS expression in macrophages by an antisense mechanism, including the aptameric effect partially mediated by the G-quartet motif.
使用巯基乙酸盐诱导的小鼠腹腔巨噬细胞，研究了反义寡核苷酸（ODNs）对脂多糖（LPS）诱导的一氧化氮（NO）产生的影响。与小鼠诱导型一氧化氮合酶（iNOS）mRNA的AUG起始密码子附近序列相对应的反义硫代磷酸酯ODNs（S-oligo），其反义序列中具有G-四联体基序，以剂量依赖的方式抑制NO的诱导。反义磷酸二酯ODNs（D-oligo）、5'-和3'-末端硫代磷酸酯修饰的反义ODNs以及对照乱序和错义S-oligos均无此作用。此外，对照无义S-oligos和两个错配的S-oligos，其序列中包含G-四联体基序，将NO的诱导抑制至对照的约50%。反义S-oligo通过使巨噬细胞暴露于各种浓度的LPS，对NO的产生显示出抑制作用。使用抗小鼠诱导型一氧化氮合酶（iNOS）抗体的蛋白质印迹分析表明，反义S-oligo特异性去除了130 kDa处的免疫反应条带。此外，逆转录-聚合酶链反应（RT-PCR）的结果表明，反义作用源于与反义S-oligo杂交导致的靶向iNOS mRNA的特异性减少。此外，没有ODNs影响LPS刺激的巨噬细胞中β-肌动蛋白mRNA和肿瘤坏死因子α（TNF-α）的表达。这些发现表明，反义S-oligo通过反义机制抑制巨噬细胞中源自iNOS表达的NO产生，包括部分由G-四联体基序介导的适体效应。
