| Objective:Chinese Herbs Possessed of Activating Blood or clearing heat Function are two conventional medicines used for diabetic vascular complication, slvia and panax notoginseng is the representative of the activating-blood herb, coptis chinensis is that of the clearing-heat herb. In this study the main component of these three kinds of herbs were applied, to investigate the protective effect of them on the secret live function of the vascular endothelial cells (VEC), Vasomotion and Fibrinolytic System, by ways of detecting the effect of compatibility of the three components on the active substance secretlived by VEC and activation of NF-κB in High-gulcose injured Human Umbilical Vein Endothelial Cells (HUVEC), explore the mechanism that Chinese medicine protects VEC. It is supposed to provide some basic research for the therapy of diabetic vascular complication in Traditional Chinese Medicine.Methods:(1) High-glucose model:HUVEC was cultured for6,12,24,36,48hours respectively in three kinds of medium, the concentration of which were 5.5,15, 30mmol/L. MTT was used to detect the proliferation activity to select the best condition for the model.(2) Concentration of the components:Every component was used to interfere the HUVEC in several concentrations for 48 hours, and the most adequate concentration would be select by ways of MTT。The concentration of D were 0,0.02, 0.04,0.08,0.1,0.2,0.4,0.5, lmg/ml, that of S were0.05,0.1,0.2,0.4mg/ml, that of H were 0.02,0.05,0.1,0.2,0.5,1,2,5,10,20mg/L.(3) Effect of the components compatibility on the endothelial proliferation activation:The effective of these components was used by the form of compatibility, that is, there would be 7 intervention groups:D, S, H, DS, DH, SH, DSH, to culture the cells for 48 hours, then MTT would be used. (4) The effect on the secretlive function of VEC and related regulation:The compatibility cultured the cells for 48 hours, the level of nitric oxide (NO) in the supernatant of cell culture was measured by nitrate reductase method. The activity of eNOS was measured by chemical chromatometry. The concentration of t-PA, PAI-1 and PGI2was measured by double antibody scandwith ELISA methods. The activity of ET-1, ANG-II and TXB2 were measured by Radioimmunoassay. The expression of ET-1mRNA and eNOSmRNA was measured by RT-PCR (reversed transcription polymerase chain reaction). The expression of NF-κB P65 was detected by western blot.Results:(1) The best condition for high-glucose model:There were significant different between distinct glucose concentration group. The HG1 group (15mmol/l glucose) and the HG2 (30mmol/l glucose) both decreased significantly compared with control (ZC,5.5mmol/l glucose) (P=0.006 or P=0.000), declined about 5.522%,12.835% respectively.(2) The concentration of the components:Salvianolic acid B could promote the activity of the VECs, while the concentration was 0.02mg/ml-0.1mg/ml. Panax notoginsenosides could promote the activity of the VECs, while the concentration was 50mg/ml-400mg/ml. Coptis alkaloids could inhibit the proliferation of the cells, and the effect is associated with component concentration.(3) Effects of the compatibility on proliferation activity:The components and Their compatibility could promote the activity of the cells, the action intensity were: DSH>D>S>DS>H>DH>SH, DSH group was promoted about 36.997%.(4) Effects of compatibility on secretlive function and regulation:compatibility of component:Well the expression of the ET-1mRNA of the cells significantly increased (P<0.05), and the case of the eNOSmRNA is opposite to that of ET-1mRNA. The expression of the NF-κB was elevated, in the supernatant of high-glucose cell culture, the concentration of TXB2, ANG-II, ET-1, PAI-1 and iNOS increased, well the concentration of the NO, TNOS, PGI2 and t-PA decreased. Herb components and their compatibility was able to promote proliferation of ECs effectively, and regulate the secretlive function of ECs, inhibit the expression of ET-1mRNA and the activation of the NF-κB pathway, improve the expression of eNOSmRNA, reduce the concentration of TXB2, ANG-Ⅱ, ET-1, PAI-land iNOS, promote the concentration of NO, TNOS, PGI2 and t-PA. Several components have different action point during the regulation process.Conclusion:High glucose could injure the ECs, and by means of inducing the activation of the NF-κB nuclear translocation, high glucose could lead to endothelial dysfunction, especially on the Fibrinolytic System and Vasomotion. The prescribe HYJD could effectively suppress the activation of the NF-κB pathway, regulate the endothelial secretion, improve the antithrombotic capability. The NF-κB pathway might be an important target point for the prescribe HYJD protecting Vascular Endothelium and preventing diabetic complication. |
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