Влияние водорастворимых производных фуллерена С60 на уровень окислительного стресса и на транскрипционную активность генов, регулирующих про- и антиоксидантную активность эмбриональных фибробластов легких человека, культивируемых in vitro

Abstract

The paper considers the effect of two water-soluble C60 fullerene derivatives modified with amino acid residues on the level of reactive oxygen species (ROS) in cultured human cells and on the transcriptional activity of genes that regulate pro- and antioxidant activity of cells. C60 fullerene derivatives with attached serine and phenibut amino acid residues were synthesized from C60Cl6 chlorofullerenes. Human embryonic lung fibroblast cultures were used in in vitro experiments. Various concentrations of fullerene derivatives were added to the culture medium, the cells were cultured in the presence of compounds from 1 to 72 hours. The level of ROS was determined using the dye 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), which is oxidized by free radicals to form fluorescent 2,7-dichlorofluorescein (DCF), by fluorescence microscopy, flow cytometry, and using a plate reader. The level of protein expression was determined by flow cytometry using specific antibodies. The level of gene expression was assessed by real-time polymerase chain reaction. It was shown that when the studied compounds are introduced into the cellular environment, they intensively absorb reactive oxygen species due to double conjugated bonds in the framework. At the same time, fullerene derivatives contribute to the development of secondary oxidative stress in cells 24 hours after administration. This effect occurs due to the activation of the NOX4 enzyme. In the cells of human embryonic lung fibroblasts, which were injected with the studied C60 fullerene derivatives, a correlation was found between the average value of the NOX4 enzyme protein and the level of reactive oxygen species. The lack of activation of the antioxidant transcription factor NRF2 upon incubation of human embryonic lung fibroblasts in the presence of C60 fullerene derivatives contributes to the development of secondary oxidative stress in cells.