α-Lipoid Acid Prevents Endothelial Dysfunction in HUVEC-12 Via Up-regulation of Lysyl Oxidase
|School||Zunyi Medical College,|
|Keywords||lysyl oxidase homocysteine α-lipoid acid matrix metalloproteinase-2 endothelial function|
Objective:To examine the effect ofα-lipoid acid (ALA) on the expression of lysyl oxidase (LOX) and MMP-9 in human umbilical vein endothelial cells (HUVECs), and to investigate the possible mechanism of endothelial injury induced by homocysteine.Methods:1. HUVECs were cultured in vitro.2. Culture the HUVECs with medium containing the different concentrations of Hey, ALA for 24h, Survival of HUVECs were tested by MTT assay.3. ROS activity was assessed by a highly sensitive fluorescent assay.4. Total RNA was isolated and performed RT-PCR. Then photograph and analyze the band. Compare the LOX, MMP-2 toβ-actin for half-quantitative analysis.5. Total protein was isolated and performed Western blot. Compare the LOX toβ-actin for quantitative analysis.Results:1. Hey can inhibit the survival of HUVECs, and the effect is concentration-dependent after 24h incubation(p<0.01). Among the ALA groups of different concentrations, there was a significant increase (P<0.01 at 0.5mmol/L and 1.0mmol/L).2. Incubation of HUVECs with increasing concentrations of Hey resulted in a dose-dependent and significant increase in intracellular fluorescence intensity which meant an increased generation of ROS. While pre-incubated with Hey before ALA significantly reversed its effect on endothelial ROS generation (P<0.05).3. mRNA levels of LOX were down-regulated by Hey treatment in endothelial cells in a dose-dependent manner(P<0.01). The addition of MMP-9 expression was associated with a decrease in LOX expression. Inhibition of LOX activity either by Hey or by BAPN, the inhibitor of LOX, increased endothelial MMP-9 expression (P<0.01 at 0.05mmol/L). Western blot results suggest that Hey may down-regulate LOX expression in endothelial cells in a dose-dependent manner(P<0.01).4. ALA may up-regulate LOX expression, down-regulate MMP-2 expression in endot-helial cells after Hey treatment. The reduction of MMP-9 expression was associated with an increase in LOX expression. RT-PCR results suggest that pre-incubated with Hey before ALA significantly increased LOX mRNA expression and down-regulate MMP-2 expression. Western blot results suggest that pretreated with Hey before ALA markedly ameliorated the effect of Hey on LOX expression (P<0.01).Conclusion:1. ALA has protective effects on endothelial cells.2. Hey can increase ROS generation which leads to endothelial dysfunction. ALA can protect the vascular endothelium against the injury induced by Hey, keeping the HUVECs away from oxidative injury.3. Hey can decrease the level of LOX mRNA and up-regulate MMP-2 mRNA expres-sion in HUVECs. The results suggest that LOX inhibition in contributes to the endothelial dysfunction associated with hyperhomocysteinemia.4. Endothelial dysfunction induced by Hey is associated with a decrease of LOX expr-ession. ALA can increase the level of LOX mRNA and down-regulate MMP-2 mRNA expression in HUVECs. The results suggest that up-regulation the expression of LOX by ALA can contribute to the vascular beneficial actions of this drug and to the cardiovascular risk reduction achieved by this therapy.