Protective Mechanism Of Spirulina Kinase On Endothelial Cells Damaged By Active Oxygen

Objective:Through setting up the oxidative damage model of human umbilical vein endothelial cells (HUVEC) which make by Hydrogen peroxide (H2O2), to study the effect of Spirulina kinase (SPK) on the redox system of vascular endothelial cells, and the cell permeability, the cell adhesion, and speculated the relationship between the Spirulina kinase with atherosclerosis.Methods:1. Culturing HUVECS in vitro, through MTT colorimetric method detects the effect of SPK and H2O2 and determine the concentration of the experimental groups and damage model group.2. Setting up the oxidative damage model, and divided into six groups randomly:N, the normal group;D, the damage group (2mmol.L-1 H2O2);E, positive group (10μg.ml-1VitE+2mmol.L-1 H2O2);L, low-dose group (1mg.ml-1 SPK+2mmol.L-1 H2O2);M, medium-dose group (2mg.ml-1 SPK+2mmol.L1-1 H2O2);H, high-dose group (3mg.ml-1 SPK+2mmol.L-1H202);3.Determine NO, NOS, SOD, MDA, GSH-PX, LDH and permeability by different detection methods,ICAM-1 by ELISA method.Results:1. The effect of H2O2 on the HUVEC vigor:The slightly effect is found on the vigor when the concentration of H2O2 is lmmol/L, (P>0.05), and it is not change with time extension. The inhibitory rate of cell over 40% when the concentration of H2O2 is 2mmol/L in 4h, the effect will be maximized with time extension. When the concentration of H2O2 is 4mmol/L> 8mmol/L, it will make severely damaged to the cell, we can see the piece of cell fall off, broken is floating on the surface medium.2. The effect of SPK on the HUVEC vigor:As the concentration of SPK increase its effect on the cell vitality from promoting to suppress. Compared with the control group, the cell vitality became the strongest when the SPK concentration reaches 2mg/ml. The cell growth inhibition when the concentration of SPK over 6 mg/ml. Compared to damage group, the cell vitality of endothelial cells which incubation by SPK is enhanced, and the effect was dose dependent.3、The influence of SPK on endothelial cell monolayer permeabilityCompared with normal group, the cell monolayer permeability in the damage group increased more than 6 times, (362.49±26.94VS54.81±12.46). And compared with damage group, the cell monolayer permeability in the Vitamin E group reduced by more than 60%(133.52±36.61VS362.49±26.94). SPK group also showed obvious inhibitory effect, and this effect was dose dependent.4、The effect of SPK on the HUVEC secreting NO and NOS:Compared with normal group, the endothelial cells which damaged by H2O2 oxidation can make the NO quantity significantly lower, (P< 0.05); Compared with the damage group, Vitamin E and SPK can increase NO secretion, in addition to the low dose group,the increase have differences, The work of Vitamin E is stronger than SPK. In cell’s NOS quantity, damage group showed a trend of rise and decline in SPK group, but there were no significant differences (P> 0.05).5、The effect of SPK on the SOD,GSH-PX activity and MDA content in the cell.Compared with normal group, the activity of SOD and GSH-PX were significantly decreased in damage group, lipid peroxidation products MDA content increased obviously. The activity of SOD and GSH-PX were significantly higher in the Vitamin E group and SPK group, when compared with damage group, while the MDA content decreased obviously. So do the ratio of SOD/MDA.6、The effect of SPK on the ATP content in the cell and the leakage quantity of LDH.Compared with normal group, damage group of ATP content decreased obviously (P< 0.05), but the leakage quantity of LDH increased significantly; Compared with damage group, positive group and SPK group of ATP content were significantly increased, the leakage quantity of LDH are reduced, and the trend shows show dose effect relationship with the concentrations of SPK.7、The effect of SPK on the ICAM-1 content in the cell.The oxidation stimulation of H2O2 can make the endothelial cells secrete ICAM-1 up to 2-3 times that of normal cells. Compared with damage group, ICAM-1 content decreased obviously in Vitamin E group and SPK groups. And high levels of SPK effect more apparent than Vitamin E.Conclusion:SPK can resistance the deleterious effect of H2O2,can induced oxidative damage of HUVECS, improve HUVEC cell vitality, and has certain protective effect on HUVEC, reduction of H2O2 stimulated cell secretion of MDA, LDH, and make a promotion of the activity of SOD and GSH-PX, reduce the monolayer cell permeability, increase the NO content, reduce the number of cells secrete ICAM-1, antioxidant capacity, eventually improve the capacity of antioxidant in endothelial cells,against oxidative stress damage.