Atomic force microscopy reveals new biophysical markers for monitoring subcellular changes in oxidative injury: Neuroprotective effects of quercetin at the nanoscale

Oxidative stress has been recognised as an important pathological mechanism underlying the development of neurodegenerative diseases. The biomarkers for assessing the degree of oxidative stress have been attracting much interest because of their potential clinical relevance in understanding the cellular effects of free radicals and evaluation of the efficacy of drug treatment. Here, an interdisciplinary approach using atomic force microscopy (AFM) and cellular and biological molecular methods were used to investigate oxidative damage in P19 neurons and to reveal the underlying mechanism of protective action of quercetin. Biological methods demonstrated the oxidative damage of P19 neurons and showed that quercetin improved neuronal survival by preventing H2O2-induced p53 and Bcl-2 down-regulation and modulated Akt and ERK1/2 signalling pathways. For the first time, AFM was employed to evaluate morphologically (roughness, height, Feret dimension) and nanomechanical (elasticity) properties in H2O2-induced neuronal damage. The AFM analysis revealed that quercetin suppressed H2O2-provoked changes in cell membrane elasticity and morphological properties, thus confirming its neuroprotective activity. The obtained results indicate the potential of AFM-measured parameters as a biophysical markers of oxidative stress-induced neurodegeneration. In general, our study suggests that AFM can be used as a highly valuable tool in other biomedical applications aimed at screening and monitoring of drug-induced effects at cellular level.

氧化应激（Oxidative stress）已被证实是神经退行性疾病发生发展的重要病理机制。用于评估氧化应激程度的生物标志物因在阐释自由基的细胞效应以及评价药物治疗疗效方面具备潜在临床价值，而受到广泛关注。本研究采用原子力显微镜（Atomic Force Microscopy, AFM）结合细胞与生物分子学方法的跨学科策略，对P19神经元的氧化损伤展开研究，并阐释槲皮素发挥神经保护作用的潜在机制。生物学实验结果证实了P19神经元发生氧化损伤，同时表明槲皮素可通过阻断H₂O₂诱导的p53与Bcl-2表达下调，调节Akt及ERK1/2信号通路，从而提升神经元存活率。本研究首次利用AFM对H₂O₂诱导的神经元损伤进行形态学（粗糙度、高度、费雷特直径）与纳米力学（弹性）特性评估。AFM分析结果显示，槲皮素可抑制H₂O₂引发的细胞膜弹性与形态学特性改变，进而证实其神经保护活性。本研究结果表明，AFM检测得到的参数有望作为氧化应激诱导神经退行性病变的生物物理标志物。总体而言，本研究提示AFM可作为极具应用价值的工具，应用于其他旨在细胞水平筛选与监测药物诱导效应的生物医学研究中。