Raman micro-spectroscopy monitoring of cytochrome c redox state in Candida utilis under low-temperature plasma-induced oxidative stress

Oxidative stress may result in different modes of cell death, such as necrosis, apoptosis and necroptosis. Currently, researchers are still striving to develop efficient tools/methods to distinguish the cell death modes in direct and label-free ways. In this study, we attempted to employ Raman micro-spectroscopy to observe the molecular changes in Candida utilis cells under oxidative stress induced by low-temperature plasma (LTP) and explore the spectroscopic biomarkers for the modes of cell death under oxidative stress. In the research, we confirmed that LTP could impose oxidative stress to the yeast cells, and recorded the changes of Raman signals of cytochrome c in the cells under the LTP oxidative stress. Subsequently, we identified the biochemical and morphological characteristic features corresponding to different modes of cell death. Interestingly, we found that LTP under certain conditions could induced the oxidative stress which caused the yeast cell death mainly by means of necroptosis, which was verified by Annexin V/PI, HMGB1 location assay and immunoprecipitation assay of RIP1/RIP3 necrosome. Correspondingly, we also showed that the LTP induced the necroptosis associated with the increase of cytoplasmic Ca2+ and mitochondrial ROS, a decrease of mitochondrial membrane potential, the release of oxidized cytochrome c from the mitochondrion to the cytoplasm, and the destruction of mitochondria in yeast cells. This work has therefore demonstrated that monitoring the redox state of cytochrome c using Raman micro-spectroscopy is very useful for distinguishing the modes of cell death and particularly may unveil the unique necroptosis process of cells under the extrinsic oxidative stress.