Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, 14 Bobrzynskiego Str., 30-348 Krakow, Poland; Faculty of Chemistry, Jagiellonian University, 2 Gronostajowa Str., 30-387 Krakow, Poland.
Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, 14 Bobrzynskiego Str., 30-348 Krakow, Poland.
Biochim Biophys Acta Mol Cell Res. 2021 Feb;1868(2):118911. doi: 10.1016/j.bbamcr.2020.118911. Epub 2020 Nov 21.
In this work, the effect of an early oxidative stress on human endothelial cells induced by menadione was studied using a combined methodology of label-free Raman imaging and fluorescence staining. Menadione-induced ROS-dependent endothelial inflammation in human aorta endothelial cells (HAEC) was studied with focus on changes in cytochrome, proteins, nucleic acids and lipids content and their distribution in cells. Fluorescence staining (ICAM-1, VCAM-1, vWF, LipidTox, MitoRos and DCF) was used to confirm endothelial inflammation and ROS generation. The results showed that short time, exposure to menadione did not cause their apoptosis or necrosis (Annexin V Apoptosis Detection Kit) within the 3 h timescale of measurement. On the other hand, 3 h of incubation, did result in endothelial inflammation (ICAM-1, VCAM-1, vWF) that was associated with an increased ROS formation (MitoRos and DCF) suggesting the oxidative stress-mediated inflammation. Chemometric analysis of spectral data enabled the determination of spectroscopic markers of menadione-induced oxidative stress-mediated endothelial inflammation including a decrease of the bands intensity of cytochrome (604, 750, 1128, 1315 and 1585 cm), nucleic acids bands (785 cm), proteins (1005 cm) and increased intensity of lipid bands (722, 1085, 1265, 1303, 1445 and 1660 cm), without changes in the spectroscopic signature of the cell nucleus. In conclusion, oxidative stress resulting in endothelial inflammation was featured by significant alterations in the number of biochemical changes in mitochondria and other cellular compartments detected by Raman spectroscopy. Most of these, coexisted with results from fluorescence imaging, and most importantly occurred earlier than the detection of increased ROS or markers of endothelial inflammation.
在这项工作中，使用无标记拉曼成像和荧光染色的组合方法研究了甲萘醌诱导的人类内皮细胞早期氧化应激对人类内皮细胞的影响。研究了甲萘醌诱导的 ROS 依赖性人主动脉内皮细胞 (HAEC) 内皮炎症，重点关注细胞内细胞色素、蛋白质、核酸和脂质含量及其分布的变化。荧光染色（ICAM-1、VCAM-1、vWF、LipidTox、MitoRos 和 DCF）用于确认内皮炎症和 ROS 生成。结果表明，在 3 h 的测量时间范围内，短时间暴露于甲萘醌不会导致细胞凋亡或坏死（Annexin V Apoptosis Detection Kit）。另一方面，孵育 3 h 确实导致内皮炎症（ICAM-1、VCAM-1、vWF），这与 ROS 形成增加（MitoRos 和 DCF）相关，表明氧化应激介导的炎症。光谱数据的化学计量分析能够确定甲萘醌诱导的氧化应激介导的内皮炎症的光谱标志物，包括细胞色素（604、750、1128、1315 和 1585 cm）、核酸带（785 cm）、蛋白质（1005 cm）的强度降低和脂质带（722、1085、1265、1303、1445 和 1660 cm）的强度增加，而细胞核对光谱特征没有变化。总之，氧化应激导致内皮炎症的特征是通过拉曼光谱检测到的线粒体和其他细胞区室中生化变化的数量发生重大变化。其中大多数与荧光成像结果并存，最重要的是，发生在检测到 ROS 增加或内皮炎症标志物之前。
