HUVECs Injury Induced By Divicine And The Potential Mechanism

Background:Divicine,the aglycone of vicine,which is an alkaloid glycoside found mainly in fava beans,and is toxic in individuals who have a hereditary deficiency of the enzyme glucose-6-phosphate dehydrogenase(G6PD).This deficiency causes a shortage of glutathione(GSH)in erythrocytes and induces the oxidative injury and acute haemolytic anaemia.To date,most of the studies focus on the oxidative effects of divicine on erythrocyte.Endothelial cells are monolayer of cells,covers the entire luminal surface of blood vessels and are susceptible to various physical and chemical factors in blood and thus participate in the occurrence and development of cardiovascular diseases.Since divicine might be present in the circulation after vicine intake,it would be possible that endothelial cells might be damaged as well.In the present work,the toxic effect of divicine on human umbilical cord vein endothelial cells(HUVECs)and its potential mechanisms were studied,so as to explore whether oxidative damage of endothelial cells is more likely to occur in patients with G6PD deficiency.Methods:(1)HPLC was used to compare the yield of divicine preparation by acid hydrolysis method and enzyme hydrolysis method.(2)CCK-8 assay was used to determine the viability of HUVECs treated with divicine at different concentrations(0,18.5,28.1,47.1,85.1μM)for various duration of time(6,12,24,48h).(3)Morphological changes of HUVECs were observed by inverted phase contrast microscope and transmission electron microscope after treatment with divicine.(4)Spectrophotometric method was used to detect the concentrations of GSH in HUVECs treated with different concentrations of divicine for 24h.(5)DCFH-DA was used as an indicator of intracellular reactive oxygen species(ROS)determined by flow cytometry.HPLC was used to determine the malondialdehyde(MDA)content of HUVECs treated with different concentrations of divicine for 24h.(6)HUVECs apoptosis was determined by using flow cytometry after treatment with different concentrations of divicine for 24h.Calcein-Am assay was carried out to determine the content of intracellular labile iron pool(LIP)after the endothelial cells were treated with different concentrations of divicine for 24h.(7)Inhibitory effects of different concentrations of DFO(25,50,100μM)on HUVECs injury induced by divicine were determined.Results:(1)Hydrolysed product of vicine-divicine was detected by HPLC.The concentration of divicine(y)was linear with vicine(x)between 25 to 800μM:y=0.3805x+9.0356(R~2=0.9987).(2)Incubation of HUVECs with 18.5～85.1μM divicine resuled in the decrease of cell viability in dose-and time-dependent manner for 6～48h.The cell viability decreased significantly after treatment with 28.1μM and 47.1μM divicine for 12-48h,compared with that of control(P<0.05).Incubation of HUVECs with 85.1μM divicine resulted in significant decrease of cell viability after 6～48h(P<0.05).(3)HUVECs exhihited polygonal,when confluency was reached,they exhibited a cobblestone pattern.After incubation of HUVECs with 28.1～47.1μM divicine for 24h,the cytoplasm exhibited plenty of granulation,and intercellular space was increased.Incubation of HUVECs with 85.1μM divicine for 24 resulted in a decrease of adherent cells with cells rounded and suspended.Transmission electron micrograph(TEM)of HUVECs showed various organelles with double nuclear membrane and obvious nuclear pores.The mitochondria showed typical tubular cristae.85.1μM divicine treatment caused the decrease of electron density of cytoplasm with the swelling of cellular apparatus.The mitochondria cristae collapsed and became discontinuous and the nuclear envelope also collapsed.(4)GSH concentrations were 8.32±1.42,7.41±2.51,5.16±1.13 and 2.85±1.38μM/g protein after treatment with 18.5～85.1μM divicine for 24 hours,respectively.Compared with the control group(11.64±3.71μM/g protein),the GSH content in HUVECs significantly decreased after treatment with18.5～85.1μM divicine for 24h(P<0.05).(5)The fluorescence intensity of intracellular ROS was 1.07±0.02,1.08±0.01,1.15±0.01 and1.37±0.11,respectively,after treatment of HUVECs with 18.5～85.1μM divicine for 24h,while the fluorescence intensity of the control group was 1.00±0.02.Incubation of HUVECs with47.1～85.1μM divicine cause significant increase of intracellular ROS(P<0.05).The concentrations of MDA were 0.32±0.07,0.34±0.02,0.39±0.06,and 0.53±0.08μM/g protein after incubation of HUVECs with 18.5～85.1μM divicine for 24h.MDA contents increased significantly after treatment with 28.1～85.1μM divicine compared with that of control(0.21±0.10μM/g protein)(P<0.05).(6)The apoptosis rate was 1.03±0.55%,1.20±0.20%,2.33±0.12%and 3.40±0.24%,respectively,after treatment with 18.5～85.1μM divicine for 24h,and the apoptosis rate of the control group was 0.63±0.21%.Apoptosis rate of HUVECs significantly increased after treatment with47.1～85.1μM divicine for 24h(P<0.05).The fluorescence intensity of intracellular LIP was1.00±0.32,0.92±0.29,1.57±0.32,3.36±1.12,and 5.03±1.11,respectively,after incubation of HUVECs with 0～85.1μM divicine for 24h.The content of LIP was significantly higher in 47.1and 85.1μM divicine group than that in control group(p<0.05).(7)25μM～100μM DFO treatment could inhibit the decrease of cell viability induced by 85.1μM divince in a dose-dependent manner(P<0.05).In addition,incubation of HUVECs with 100μM DFO for 24h could significantly inhibited the increase of intracellular ROS,LIP and MDA levels and the apoptosis rate induced by divicine(P<0.05).After treatment with 100μM DFO,HUVECs showed increase adherent cells under microscope,and double nuclear membrane became clear observed under electron micrograph.Conclusion:Divicine was toxic to HUVECs by increasing intracellular oxidative stress and lipid peroxidation,which could be inhibited by iron chelating agent DFO,suggesting that iron-dependent lipid peroxidation might be involved in divicine induced endothelial cells injury.