D'Agostino P, Arcoleo F, Barbera C, Di Bella G, La Rosa M, Misiano G, Milano S, Brai M, Cammarata G, Feo S, Cillari E
Institute of General Pathology, University of Palermo, Italy.
Eur J Pharmacol. 1998 Apr 10;346(2-3):283-90. doi: 10.1016/s0014-2999(98)00046-6.
Here we investigate the effects of tetracycline base and of a semi-synthetic tetracycline derivative, doxycycline, on the induction of inducible nitric oxide synthase and, hence, on the production of nitric oxide (NO) by lipopolysaccharide in J774 macrophage cultured in vitro. The treatment of J774 line with tetracycline base (6.25-250 microM) or doxycycline (5-50 microM) dose-dependently decreased the lipopolysaccharide-stimulated (1 microg/ml) inducible NO synthase activity and, consequently, nitrite formation. For instance, the inhibition was 70% for tetracycline base at 250 microM and 68% for doxycycline at 50 microM. The inhibitory effect of tetracyclines was due neither to a reduction in the viability of the cells, studied as colorimetric 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (MTT) reduction assay, nor to an indiscriminate inhibition of total protein synthesis, but to a specific decrease in inducible NO synthase protein content in the cells, as attested by the significant reduction of the expression of inducible NO synthase, assayed by sodium-dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. However, no effect of tetracyclines on inducible NO synthase mRNA accumulation could be demonstrated in lipopolysaccharide-stimulated macrophage line, suggesting that the inhibitory effect of tetracyclines on NO synthesis involves post-transcriptional events. The reduction in lipopolysaccharide-stimulated nitrite accumulation produced by tetracyclines was significantly less when they were applied 6 h after lipopolysaccharide and absent 12 h after lipopolysaccharide, indicating that tetracyclines modify an early event in inducible NO synthase activation operating after mRNA transcription. The findings presented in this study indicate that the modulation of NO synthesis is another possible pathway by which tetracyclines may function as anti-inflammatory compounds.
在此，我们研究了四环素碱和一种半合成四环素衍生物强力霉素对体外培养的J774巨噬细胞中诱导型一氧化氮合酶的诱导作用，进而对脂多糖诱导产生一氧化氮（NO）的影响。用四环素碱（6.25 - 250微摩尔）或强力霉素（5 - 50微摩尔）处理J774细胞系，可剂量依赖性地降低脂多糖刺激（1微克/毫升）的诱导型一氧化氮合酶活性，进而减少亚硝酸盐的形成。例如，250微摩尔的四环素碱抑制率为70%，50微摩尔的强力霉素抑制率为68%。四环素的抑制作用既不是由于通过比色法3 - [4,5 - 二甲基噻唑 - 2 - 基] - 2,5 - 二苯基四氮唑溴盐（MTT）还原试验所研究的细胞活力降低，也不是由于对总蛋白合成的非特异性抑制，而是由于细胞中诱导型一氧化氮合酶蛋白含量的特异性降低，这通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）和蛋白质印迹法检测诱导型一氧化氮合酶表达的显著降低得到证实。然而，在脂多糖刺激的巨噬细胞系中未发现四环素对诱导型一氧化氮合酶mRNA积累有影响，这表明四环素对NO合成的抑制作用涉及转录后事件。当在脂多糖处理6小时后应用四环素时，其对脂多糖刺激的亚硝酸盐积累的减少作用明显较小，而在脂多糖处理12小时后则无此作用，这表明四环素改变了诱导型一氧化氮合酶激活过程中mRNA转录后发生的早期事件。本研究结果表明，调节NO合成是四环素可能作为抗炎化合物发挥作用的另一条可能途径。
