The Effect Of Homocysteine On The Expression Of Lectin-like Oxidized Low Density Lipoprotein Receptor-1 In Cultured Human Umbilical Vein Endothelial Cells

ObjectiveHyperhomocysteinemia has been identified as a new independent risk factor for atherosclerosis in rescent years. Up to now, the mechanism in atherogenesis of homocysteine is not clear. Coronary heart disease harms severely human health and life. So it has important clinical significance to investigate the mechanism in atherogenesis of homocysteine. Oxidized low density lipoprotein( OxLDL) play a key role in the pathogenesis of endothelial dysfunction and atherosclerosis. Lec-tin - like oxidized low density lipoprotein receptor -1 ( LOX - 1) is a major special receptor for OxLDL in endothelial cells. Its inducible expression is involved in OxLDL - mediated multiple roles in atherogenesis . In this study, we examined the expression of LOX - 1 mRNA and protein in human umbilical vein endothelial cells to investigate the possible molecular mechanism of homocysteine in atherogenesis.Methods1. Culture of cells: Human umbilical vein endothelial cells ( ECV304 ) were secondary cultured regularly in 1640 supplemented with 10%FBS, in 5% CO₂ at 37℃ and saturate humidity.2. Groups of experiment; HUVECs were cultured in 25cm² culture bottles. When cells were at subconfluence, they were preincubated in fresh medium without FBS for 24h, and then were divided into following groups at random;(1) Groups of different concentration of homocysteine: Cells were incubated in medium without FBS in the absence or presence of homocysteine(0. 01, 0.05, 0.1, 0. 5, 1, 5mM) for 24h. (2) Groups of different time of lmM homocysteine : Cells were incubated in medium without FBS in the presence of homocys-teine(lmM) for different time(0, 6, 12, 24, 48h).3. LOX - 1 mRNA was assessed by RT - PCR: (1) Total RNA were extracted by TRIzol. (2)RNA were reversely transcribed into cDNA. (3)Reversed -transcribed products were amplified with Taq DNA polymerase. (4) PCR products were separated by agarose gel electrophoresis. The density of the amplified PCR fragments were analyzed by the gel documentation system and were expressed as ratios of LOX - 1 to the β - actin band .4. The protein expression of LOX - 1 was assessed by Western blot analysis: The proteins extracted were separated by 10%SDS -PAGE eletrophoresis, then were transferred to membranes, blocked with milk and incubated with primary LOX -1 antibody and secondary antibody. The expression levels were assessed by semi — quantitative analysis through photographic analysis system.5. Statistical analysis: All values were presented as the mean ± standard deviation. Comparisons among groups were made using the one - way ANOVA, Differences were considered to be statistically significant at a value of P <0.05.Results1. Effect of homocysteine on the expression of LOX -1 mRNA: ( 1) Groups of different concentration of homocysteine: The expression of LOX -1 mRNA was weak in control group. The difference of the expression of LOX - 1 mRNA between 0. OlmM and control group wasn't significant(P >0.05) ;The expression of 0.05 5mM groups increased significantly ( P < 0.05 ) in concentrations - dependent manners. (2) Groups of different time of lmM homocystein; After treatment with lmM homocysteine for 6h,LOX - 1 protein expression was elevated(P<0.05) in HUVECs, reaching its maxium at 24 ⁴8h.2. Effect of Homocysteine on the expression of LOX - 1 protein; ( 1) Groups of different concentration of homocysteine: Incubation of HUVECs with...