Moeslinger Thomas, Friedl Roswitha, Spieckermann Paul Gerhard
Institute of Physiology, Center for Physiology and Pathophysiology, Medical University of Vienna, Schwarzspanierstrasse 17, 1090 Vienna, Austria.
Life Sci. 2006 Jun 20;79(4):374-81. doi: 10.1016/j.lfs.2006.01.015. Epub 2006 Feb 13.
Azathioprine is used as an anti-inflammatory agent. Although there are numerous data demonstrating cytotoxic and immunosuppressive properties of azathioprine and its metabolite 6-mercaptopurine, the mechanism of the anti-inflammatory action of azathioprine has not yet been fully clarified. During our study, we investigated the effects of azathioprine on the inducible nitric oxide synthase (iNOS) in lipopolysaccharide stimulated murine macrophages (RAW 264.7) by measurement of iNOS protein (immunoblotting), iNOS mRNA (semiquantitative competitive RT-PCR), and NO production (nitrite levels). Azathioprine (0-210 muM) induces a concentration dependent inhibition of inducible nitric oxide synthesis (IC50: 33.5 muM). iNOS protein expression showed a concentration dependent reduction as revealed by immunoblotting when cells were incubated with increasing amounts of azathioprine. Azathioprine decreases iNOS mRNA levels as shown by semiquantitative competitive RT-PCR. In contrast, 6-mercaptopurine showed no inhibition of inducible nitric oxide synthesis. Azathioprine did not reduce iNOS mRNA stability after the addition of actinomycin D. Enzymatic activity assays with increasing concentrations of azathioprine (0-210 muM) showed no statistically significant inhibition of iNOS enzyme activity compared to cell lysates without azathioprine. Nuclear translocation of NF-kappaB p65 subunit and binding of NF-kappaB p50 subunit from nuclear extracts to a biotinylated-consensus sequence was unaffected by azathioprine treatment. iNOS inhibition by azathioprine was associated with a decreased expression of IRF-1 (interferon regulatory factor 1) and IFN-beta (beta-interferon) mRNA. Azathioprine induced iNOS inhibition seems to be associated with an action of the methylnitroimidazolyl substituent. This suggests a route to the rational design of nontoxic anti-inflammatory agents by replacing the 6-mercaptopurine component of azathioprine with other substituents. The inhibition of inducible nitric oxide synthesis might contribute to the anti-inflammatory activities of azathioprine.
硫唑嘌呤用作抗炎药。尽管有大量数据表明硫唑嘌呤及其代谢产物6-巯基嘌呤具有细胞毒性和免疫抑制特性，但硫唑嘌呤抗炎作用的机制尚未完全阐明。在我们的研究中，我们通过测量诱导型一氧化氮合酶（iNOS）蛋白（免疫印迹法）、iNOS mRNA（半定量竞争性逆转录聚合酶链反应）和一氧化氮生成（亚硝酸盐水平），研究了硫唑嘌呤对脂多糖刺激的小鼠巨噬细胞（RAW 264.7）中诱导型一氧化氮合酶的影响。硫唑嘌呤（0 - 210 μM）诱导诱导型一氧化氮合成的浓度依赖性抑制（半数抑制浓度：33.5 μM）。当细胞与越来越多的硫唑嘌呤孵育时，免疫印迹显示iNOS蛋白表达呈浓度依赖性降低。半定量竞争性逆转录聚合酶链反应显示硫唑嘌呤降低iNOS mRNA水平。相比之下，6-巯基嘌呤未显示对诱导型一氧化氮合成的抑制作用。加入放线菌素D后，硫唑嘌呤未降低iNOS mRNA稳定性。与不含硫唑嘌呤的细胞裂解物相比，用浓度递增的硫唑嘌呤（0 - 210 μM）进行的酶活性测定未显示对iNOS酶活性有统计学上显著的抑制作用。硫唑嘌呤处理未影响核因子κB p65亚基的核转位以及核提取物中核因子κB p50亚基与生物素化共有序列的结合。硫唑嘌呤对iNOS的抑制与干扰素调节因子1（IRF-1）和β-干扰素（IFN-β）mRNA表达降低有关。硫唑嘌呤诱导的iNOS抑制似乎与甲基硝基咪唑基取代基的作用有关。这提示了一条通过用其他取代基替代硫唑嘌呤的6-巯基嘌呤成分来合理设计无毒抗炎药的途径。诱导型一氧化氮合成的抑制可能有助于硫唑嘌呤的抗炎活性。
