The Effect of CCCP on Mitophagy in CD4<sup>+</sup>CD25<sup>+</sup> Regulatory T Cells in Human Peripheral Blood

Objective: To investigate the effects and mechanisms of different concentrations of CCCP on mitophagy in human peripheral blood regulatory T cells. Methods: Tregs were isolated, identified and then grouped, treating with CCCP at a concentration of 2.5 μM, 5 μM, 10 μM, 20 μM and 40 μM for 24h in an incubator. Flow cytometry detected the reactive oxygen species (ROS), mitochondrial membrane potential (MMP), mitochondrial quality, and fluorescence microscopy observed the co-localization of mitochondria and lysosomes in each group. Results: The purity of CD4+CD25+Tregs was (93.36 ± 1.87) %. With the increase of CCCP concentration, the level of ROS gradually increased, while the MMP decreased gradually. About the mitochondria and lysosome fusion, the fluorescence intensity of orange (yellow) was the highest when the concentration of CCCP was in the range of 5-10 μM while decreased with the CCCP concentration continually increasing. The mitochondrial quality decreased with the increase of CCCP concentration. However, there was no significant difference between groups C, D and E. The mitochondrial quality of groups F and G were significantly lower than that of group E. Conclusions: With the concentration of CCCP gradually increased, the level of ROS in Treg cells increased, and MMP decreased, which promoted the mitophagy, mitochondrial quality maintains homeostasis; When ROS accumulated, and MMP decreased significantly, the mitophagy was inhibited, and the mitochondrial quality was significantly decreased.