Wang Juanjuan, Tian Jihua, Kang Jing, Yang Jia, Chang Sijia, Ji He, Huang Taiping, Fan Weiping, Guo Jinli, Wang Yanhong
Objective To explore the injury effect and its possible mechanism of amiodarone on human umbilical vein endothelial cells (HUVECs). Methods After 3 generations of cultivation, the HUVECs were seeded in 96-well plates and incubated with amiodarone (0, 10, 20, 30, and 60-μmol/L) for 24-hours. The cell viability was detected using cell counting kit 8 (CCK-8) assay and the relative viability of cells incubated with different concentrations of amiodarone were calculated by taking the cell viability of the 0 μmol/L group as 100%. The concentration of amiodarone at which cell viability was reduced to 70% was selected for subsequent experiments. The effect of amiodarone of this concentration on the activity of HUVECs after action for different time (6, 12, 24, 36, and 48-hours) was detected using the CCK-8 assay. HUVECs cultured with amiodarone of this concentration were set as the experimental groups and those without amiodarone were set as the control group. Apoptosis rate of HUVECs was detected by Annexin V-FITC/P flow cytometry; the protein and mRNA expression levels of B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), Caspase-3, interleukin 10 (IL-10), IL-1β, IL-6, and tumor necrosis factor alpha (TNF-α) were detected using western blotting and real-time fluorescence quantitative polymerase chain reaction, respectively; the reactive oxygen species (ROS) was detected by DCFH-DA fluorescence probe assay; the superoxide dismutase (SOD) activity was detected by water-soluble tetrazolium-1 assay; the reduced glutathione (GSH) content was detected by microplate assay. Results The viabilities of HUVECs incubated with amiodarone at concentration of 10, 20, 30, and 60-μmol/L for 24-hours were (88.82±2.64)%, (74.96±1.75)%, (64.95±2.10)%, and (18.57±0.65)%, respectively; differences were all significant (all P<0.01) between each experiment group and control group, as well as between each experiment group. Amiodarone at a concentration of 30-μmol/L was used for subsequent experiments. After incubating with 30-μmol/L amiodarone for 6, 12, 24, 36, and 48-hours, the viabilities of HUVECs were (90.19±1.88)%, (82.81±2.51)%, (75.33±1.37)%, (65.76±1.85)%, and (47.01±3.29)%, respectively; differences were all significant (all P<0.01) between each experiment group and control group, as well as between each experiment group. Compared with the control group, the apoptosis rate of cells in the experimental group was significantly higher (48.59% vs. 16.34%, P<0.01), the protein and mRNA expression levels of pro-apoptotic proteins Bax and caspase-3, and pro-inflammatory factors IL-1β, IL-6, and TNF-α were higher (all P<0.01), whereas the protein and mRNA expression levels of anti-apoptotic protein Bcl-2 and anti-inflammatory factor IL-10 were lower (P<0.05, P<0.01). Conclusions Amiodarone can cause HUVECs injury, which would be enhanced with the increase of concentration and action time of amiodarone. Amiodarone may cause HUVECs injury by inducing apoptosis, inflammatory response, and oxidative stress.
