Manoharan Renuka Ramalingam, Sedlářová Michaela, Pospíšil Pavel, Prasad Ankush
Department of Biophysics, Faculty of Science, Palacký University, Šlechtitelů 27, 783 71 Olomouc, Czech Republic.
Department of Botany, Faculty of Science, Palacký University, Šlechtitelů 27, 783 71 Olomouc, Czech Republic.
Biochim Biophys Acta Gen Subj. 2023 May;1867(5):130324. doi: 10.1016/j.bbagen.2023.130324. Epub 2023 Feb 10.
Reactive oxygen species play a key role in cellular homeostasis and redox signaling at physiological levels, where excessive production affects the function and integrity of macromolecules, specifically proteins. Therefore, it is important to define radical-mediated proteotoxic stress in macrophages and identify target protein to prevent tissue dysfunction. A well employed, THP-1 cell line was utilized as in vitro model to study immune response and herein we employ immuno-spin trapping technique to investigate radical-mediated protein oxidation in macrophages. Hydroxyl radical formation along macrophage differentiation was confirmed by electron paramagnetic resonance along with confocal laser scanning microscopy using hydroxyphenyl fluorescein. Lipid peroxidation product, malondialdehyde, generated under experimental conditions as detected using swallow-tailed perylene derivative fluorescence observed by confocal laser scanning microscopy and high-performance liquid chromatography, respectively. The results obtained from this study warrant further corroboration and study of specific proteins involved in the macrophage activation and their role in inflammations.
活性氧在生理水平的细胞内稳态和氧化还原信号传导中起关键作用，在该水平上，过量产生会影响大分子尤其是蛋白质的功能和完整性。因此，定义巨噬细胞中自由基介导的蛋白质毒性应激并鉴定靶蛋白以防止组织功能障碍非常重要。一种常用的THP-1细胞系被用作体外模型来研究免疫反应，在此我们采用免疫自旋捕获技术来研究巨噬细胞中自由基介导的蛋白质氧化。通过电子顺磁共振以及使用羟基苯基荧光素的共聚焦激光扫描显微镜证实了巨噬细胞分化过程中羟基自由基的形成。分别使用共聚焦激光扫描显微镜和高效液相色谱观察到的燕尾苝衍生物荧光检测到实验条件下产生的脂质过氧化产物丙二醛。本研究获得的结果有待进一步证实，并对参与巨噬细胞活化的特定蛋白质及其在炎症中的作用进行研究。
