Changes of connexin 43 in irradiated human vascular endothelial cells and its influence on cell stiffness / 中华放射医学与防护杂志

ABSTRACT

Objective:To investigate the changes of connexin 43 (Cx43) in human umbilical vein endothelial cells (HUVEC) after X-ray irradiation and its influence on the stiffness of irradiated cells.Methods:Western blot was used to detect the expression of Cx43 in HUVEC cells at different time points (0, 6, 12, 24 and 48 h) after different doses of X-ray irradiation (0, 2.5, 5, 10 and 20 Gy), and the phosphorylation levels of three phosphorylation sites (Ser279/282, Ser368 and Tyr265) of Cx43 at different time points (3, 6, 24 and 48 h) after 0, 5 and 10 Gy irradiation. The distribution of Cx43 protein in the irradiated HUVEC cells was detected by immunofluorescence. The stiffness changes of cells were detected by atomic force microscopy (AFM) at the depths of 50, 100 and 200 nm.Results:The expression of Cx43 in HUVEC cells was reduced at 6, 12, 24 and 48 h after 10 Gy X-ray irradiation( t=3.262, 3.708, 3.686, 6.825, P<0.05)and this decrease had a dose dependent manner at 24 h after 2.5, 5, 10 and 20 Gy irradiation ( t=3.034, 10.720, 13.130, 13.650, P<0.05). At 24 h after 5, 10 and 20 Gy X-ray irradiation, the distribution of Cx43 in HUVEC cells was transported from intercellular gap junctions to nucleus and perinuclear region. At 24-48 h after irradiation, the phosphorylation level of Ser368 at Cx43 increased and in a dose dependent manner. At 24 h after irradiation, the stiffness of the irradiated cells decreased significantly under the conditions of 100 and 200 nm ( t=3.362, 5.122, P<0.05), and recovered with overexpression of Cx43 ( t=2.674, 4.398, P<0.05). Conclusions:X-ray irradiation leads to the phosphorylation of Ser368 at Cx43, which promotes the degradation and nucleus/perinuclear translocation of Cx43 and reduces the stiffness of HUVEC. Increasing the expression level of Cx43 is helpful to the stiffness recovery of irradiated vascular endothelial cells, suggesting that Cx43 may be a potential target for regulating radiation injury of vascular endothelial cells.