Influence Of ADMA On The Expression Of LOX-1 Of HUVEC And The Effects Of PDTC

Influence of ADMA On the Expression of LOX-1 of HUVEC and the Effects of PDTCObjectiveEndothelial dysfunction is emerging as a common denominator for diverse cardiovascular abnormalities, such as atherosclerosis, hypertension, etc. inhibition of endothelial nitric oxide synthase ( eNOS) is one of the hallmarks of developing endothelial cell dysfunction. One of the mechanisms leading to endothelial dysfunction is the accumulation of an endogenous inhibitor of eNOS, asymmetric dimethylarginine (ADMA). It has been demonstrated that ADMA can increase expression of mRNA encoding lectin - like oxidize low - density lipoprotein receptor - 1 ( LOX - 1) , which is one of the important receptor of oxidized low - density lipoprotein in endothelial cells. And it was found that LOX — 1 promoter possesses have binding sites for numerous transcription factor, including NF - kB. This experiment interfere in HUVEC with ADMA ,and detect expression of NF — κBp65 protein and LOX — 1 mRNA. At the same time , approach the potential molecular mechanism in this process with special inhibitor of NF-kB,PDTC.Material and Methods1. Cell culture and groupHuman umbilical vein endothelial cells ( ECV304) were divided into seven groups : (1) normal control group;(2) ADMA 3μmol/l group;(3) ADMA 10μmol/l group;(4) ADMA 15μmol/l group;(5) ADMA 15,xmol/l + PDTC 50μmol/l group;(6) ADMA 15μmol/l + PDTC 100μmol/1 group;(7) ADMA 15|xmol/l +PDTC 150[imol/l group.2. Detect index(1) Analyse the quantitative diversity of LOX - 1 mRNA expression with demi - quantitative RT - PCR.(2) Measure the diversity of cell NF - kB p65 protein expression with Western blot.3. Statistic analysisAll data are expressed as means ± s. d. All statistical were performed by SPSS12.0 software. Statistical comparison of all numerical data between groups were performed by one -way ANOVA. A,P value <0.05 was taken as statistical significant.Result1. Morphology variance of experimentation cellsAll cells every group growed adherencely and were polygon, better photo-nasty , edge unsharpness, nucleus and particle were saw and look as stones to pave a road sample before inputing interact factors. There is no marked change in morphous after starvation 24 hours. Only the seventh group cells (ADMA 15 mol/1 + PDTC 150 mol/1 group) became round, edge sharpness, and some growed adherencely, the others fell off. There is no marked change in morphous in all cells of the other groups.2. Influence of ADMA on the expression of endothelial cell NF - KBp65 protein and LOX - 1 mRNAThe expression of NF - kB p65 protein and LOX - 1 mRNA is higher in ADMA group than the normal control group ( P <0. 01 ) . And there is dose dependent between the concentration of ADMA and the expression. There is positive correlation between the concentration of ADMA and the expression of NF -KBp65 protein. There is also positive correlation between the concentration of ADMA and the expression of LOX — lmRNA every group endothelial cells.3. Influence of PDTC on the expression of endothelial cell NF - KBp65 protein and LOX - 1 mRNAThe expression of NF - ?cBp65 protein and LOX - 1 mRNA obviously de-creased in the group, which the endothelial cells was treated by PDTC and ADMA for 24 hours than the 15junol/l ADMA group. 100|xmol/LPDTC can degrade the expression of both utmostly. The expressin of both was higher in the 150jxmol/lPDTC group than the lOOjxmol/LPDTC group.Conclusion1. ADMA can enhancement AS through activate human umbilical vein endothelial cells NF - kB , up - regulate the expression of LOX - 1, and damage endothelial function.2. Some density PDTC, the idio - inhibitor of NF - kB , can inhibit the influence of ADMA on expression of human umbilical vein endothelial cells NF -kB and LOX - 1 significantly, and can protect and improve endothelial cells during the AS process.