Effects Of Spirulina Kinase On The Cell Vitality And Release Of T-PA, PAI-1 And VWF In Cultured Human Umbilical Vein Endothelial Cells

Objective:Through culturing human umbilical vein endothelial cells (HUVEC) in vitro, establishing adrenaline (Adr) damage model, and Heparin (Hep) as the positive control group, to study the effect of spirulina kinase (SPK) on the cell vigor and secretion of t-PA, PAI-1 and vWF in cultured human umbilical vein endothelial cell, discussion of the effect on cardiovascular system, for the development of new thrombolysis drμg provide theory basis.Methods:1. Culturing HUVEC in vitro, through MTT colorimetric method detectes the effect of SPK and Adr and determine the concentration of the experimental group and damage model group on HUVEC vigor.2. Culturing HUVEC in vitro, randomly divided into seven group:control group (10% tire bovine serum), the adrenaline damage model group (100μg/ml), heparin control group (5 mg/L) and SPK control group (SPK 1 mg/ml), SPK low-dose group (SPK 0.5 mg/ml+Adr 100μg/ml), SPK medium -dose group (SPK 1 mg/ml +Adr 100μg/ml) and SPK high -dose group (SPK 2 mg/ml+Adr 100μg/ml). Incubating 12h,24h and 48h, testing the cell vitality by MTT colorimetric method and collects cell culture supernatant fluid, by ELISA method determines the vWF,t-PA and PAI-1.Results:1. The effect of SPK on the HUVEC vigor:Compared with the control group, within certain concentration range SPK can promote HUVEC growth. When the concentration of SPK<2 mg/ml, cell vitality show a concentration dependence rise When concentrations reach 2 mg/ml, the cell vitality maximize. And then, as SPK concentration increase,the cell vitality began to fall.2. The effect of Adr on cell vitality. Compared with the control group, Adr can inhibit the growth of HUVEC. Dosing 24h, the concentration of Adr< 50μg/ml is no apparent to the cell vitality(p>0.05). 100μg/ml, it has obvious inhibition on the cell vitality (p>0.05). And cellular injury rate reaches 32.92%, so choose this concentration to drug concentration for damage model group stimulate concentration.3. The effect of SPK on the HUVEC vigor stimulated by Adr:Compared with the control group,dosing 12h, there is no diffenence for each group of the cell vitality which is no significant differences(p>0.05). Dosing 24h and 48h, each group of the cell vitality are obviously decline(P<0.05). The cell vitality of Adr model group reduce the most; Compared with Adr model group,dosing 12h, SPK high-dose group obviously rise(P<0.05). Dosing 24h and 48h, heparin group, SPK groups rise significantly (P<0.05). And dosing 24h, the cell vitality of SPK groups are the dose-response rise.4. The effect of SPK on the HUVEC secreting t-PA:Compared with the control group, if SPK affect singly, there were no influence on HUVEC releasing t-pa.(p>0.05). Dosing 12h,24h and 48h, in Adr model group, heparin group and each group of SPK, the t-pa content in supernatant fluid are increased, in addition to dosing 12h, heparin group and SPK low-dose group had no significant difference (p>0.05), other groups are statistically significant difference (P<0.05). Compared with Adr model, dosing 24h and 48h, in SPK groups,HUVEC secrete t-pa show a dose-response reduced.5. The effect of SPK on the HUVEC secreting PAI-1. Compared with the control group, if SPK affect singly, there were no influence on HUVEC releasing PAI-1.(p>0.05) Dosing 24h and 48h, in Adr model group, the content of PAI-1 increased significantly (p< 0.05). Compared with Adr model group, dosing 12h, the secretion of PAI-1 of heparin group reduce obviously (p>0.05), and SPK groups have no obvious changes (p> 0.05); Dosing 24h, heparin group, SPK groups all can restrain the ascendant trend (p<0.05) (besides 24h, SPK low-dose group (p>0.05)), and show the dose-response inhibition. 6. The effect of SPK on the HUVEC secreting vWF. Compared with the control group, if SPK affect singly, there were no influence on HUVEC releasing vWF(p>0.05). Dosing 12h, there was no obvious difference of vWF on Adr group, Hep group and SPK groups(p>0.05). Adr model group, SPK low-dose group and SPK middle group of dosing 24h and Adr model group, heparin group and SPK groups of dosing 48h, vWF content in the supernatant fluid increased significantly (p<0.05). Compared with Adr damage model, dosing 12h, SPK high-dose group can effectively restrain the HUVEC release vWF (p<0.05); Dosing 24h and 48h, heparin group, SPK groups can restrain HUVE release vWF, except SPK low-dose group of 24h, Significantly different (p<0.05); And dosing 24h, SPK groups is the dose-response inhibition in relationships.Conclusion:Spirulina not only can improve HUVEC cells vigor separately, but also can antagonist adrenaline stimulation on HUVEC injury. SPK can protect HUVEC, reduce the release of t-PA,vWF and PAI-1 of HUVEC by adrenaline damage, thus improve t-pa/PAI-1 ratio, stable vWF secretion, make endothelial surface increased fibrinolytic activity, strengthen the antithrombotic function.