Fractionated low-dose ionizing radiation induces senescence of human cardiovascular endothelial cells through the mtROS-ERS-MQC axis

Objective To investigate the effects of fractionated low-dose ionizing radiation (LDIR) on cellular senescence and the expression levels of markers related to the mitochondrial reactive oxygen species (mtROS)–endoplasmic reticulum stress (ERS)–mitochondrial quality control (MQC) axis in human cardiovascular endothelial cells (EA.hy926). Methods EA.hy926 cells were irradiated five times at dose of 0 mGy, 25 mGy, 50 mGy, and 100 mGy respectively, and were cultured for an additional 48 h after the fifth X-ray irradiation. The senescence levels were assessed using senescence-associated β-galactosidase staining; protein levels associated with senescence, ERS, and MQC were analyzed by Western blot; and mtROS, mitochondrial Ca 2+ , mitochondrial membrane potential, and the degree of mitochondrial permeability transition pore were evaluated by fluorescence staining. Results Compared with the control group, the percentage of SA-β-gal-positive cells in the 50 mGy×5 group (1.85 ± 0.09) and the 100 mGy×5 group (2.60 ± 0.20) were significantly increased ( F = 107.50, P < 0.01); the protein levels of senescence-associated P53, P21 and P16 were all significantly increased in the 100 mGy×5 group (1.31 ± 0.08, 1.54 ± 0.04, 1.68 ± 0.06; F = 26.68, 10.30, 19.23; P <0. 05); the protein levels of ERS-related markers PERK and GRP78 were both significantly increased in the 100 mGy×5 group (1.71 ± 0.34, 1.31 ± 0.06; F = 7.81,15.27; P < 0.05). In contrast to the control group, mtROS levels in the 50 mGy×5 group (2.03 ± 0.18) and the 100 mGy×5 group (3.02 ± 0.22) were significantly elevated ( F = 117.90, P < 0. 01) and the protein levels of MQC-related PGC-1α, MFN2, MFN1, DRP1, Parkin and PINK1 were all significantly increased in the 100 mGy×5 group ( F = 5.64∼17.48; P < 0.05). Relative to untreated controls, mitochondrial Ca 2+ levels in all dose groups were significantly increased ( F = 821.40, P <0.01); mitochondrial membrane potential in all dose groups were significantly decreased ( F =58.30, P <0.01); the opening degree of mitochondrial permeable transition pore in the 50 mGy and 100 mGy groups were significantly increased ( F = 10.58, P < 0.01). The percentage of SA-β-gal-positive cells were positively correlated with indicators of mtROS, ERS, mitochondrial dysfunction and MQC system dysregulation ( P < 0.05). Conclusion Fractionated LDIR can induce senescence in human cardiovascular endothelial cells, leading to alterations in markers associated with the mtROS-ERS-MQC axis.