Caruso Giuseppe, Fresta Claudia G, Martinez-Becerra Francisco, Antonio Lopalco, Johnson Ryan T, de Campos Richard P S, Siegel Joseph M, Wijesinghe Manjula B, Lazzarino Giuseppe, Lunte Susan M
Ralph N. Adams Institute for Bioanalytical Chemistry, University of Kansas, Lawrence, KS, USA.
Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS, USA.
Mol Cell Biochem. 2017 Jul;431(1-2):197-210. doi: 10.1007/s11010-017-2991-3. Epub 2017 Mar 13.
Excess nitric oxide (NO) production occurs in several pathological states, including neurodegeneration, ischemia, and inflammation, and is generally accompanied by increased oxidative/nitrosative stress. Carnosine [β-alanine-histidine (β-Ala-His)] has been reported to decrease oxidative/nitrosative stress-associated cell damage by reducing the amount of NO produced. In this study, we evaluated the effect of carnosine on NO production by murine RAW 264.7 macrophages stimulated with lipopolysaccharides + interferon-γ. Intracellular NO and intracellular and extracellular nitrite were measured by microchip electrophoresis with laser-induced fluorescence and by the Griess assay, respectively. Results showed that carnosine causes an apparent suppression of total NO production by stimulated macrophages accompanied by an unexpected simultaneous drastic increase in its intracellular low toxicity endproduct, nitrite, with no inhibition of inducible nitric oxide synthase (iNOS). ESI-MS and NMR spectroscopy in a cell-free system showed the formation of multiple adducts (at different ratios) of carnosine-NO and carnosine-nitrite, involving both constituent amino acids (β-Ala and His) of carnosine, thus providing a possible mechanism for the changes in free NO and nitrite in the presence of carnosine. In stimulated macrophages, the addition of carnosine was also characterized by changes in the expression of macrophage activation markers and a decrease in the release of IL-6, suggesting that carnosine might alter M1/M2 macrophage ratio. These results provide evidence for previously unknown properties of carnosine that modulate the NO/nitrite ratio of stimulated macrophages. This modulation is also accompanied by changes in the release of pro-inflammatory molecules, and does not involve the inhibition of iNOS activity.
在包括神经退行性变、局部缺血和炎症在内的多种病理状态下，会产生过量的一氧化氮（NO），并且通常伴随着氧化/亚硝化应激的增加。据报道，肌肽[β-丙氨酸-组氨酸（β-Ala-His）]可通过减少NO的产生量来减轻与氧化/亚硝化应激相关的细胞损伤。在本研究中，我们评估了肌肽对脂多糖+干扰素-γ刺激的小鼠RAW 264.7巨噬细胞产生NO的影响。分别通过激光诱导荧光微芯片电泳和格里斯试剂比色法测量细胞内NO以及细胞内和细胞外亚硝酸盐。结果表明，肌肽可明显抑制受刺激巨噬细胞产生的总NO，同时其细胞内低毒性终产物亚硝酸盐意外地同时急剧增加，而对诱导型一氧化氮合酶（iNOS）没有抑制作用。在无细胞体系中，电喷雾电离质谱（ESI-MS）和核磁共振波谱（NMR）显示形成了多种肌肽-NO和肌肽-亚硝酸盐加合物（比例不同），涉及肌肽的两种组成氨基酸（β-丙氨酸和组氨酸），从而为存在肌肽时游离NO和亚硝酸盐的变化提供了一种可能的机制。在受刺激的巨噬细胞中，添加肌肽还表现为巨噬细胞活化标志物表达的变化以及IL-6释放的减少，这表明肌肽可能会改变M1/M2巨噬细胞比例。这些结果为肌肽调节受刺激巨噬细胞的NO/亚硝酸盐比例的先前未知特性提供了证据。这种调节还伴随着促炎分子释放的变化，并且不涉及iNOS活性的抑制。
