| Objective:Atherosclerosis is a major cause of cardiovascular and cerebrovascular diseases harming to human health. Oxidative damage to the vascluar endothelium is the pathological basis of the occurrence and development of atherosclerosis. In recent years, oxidized low density lipoprotein (OX-LDL) induced oxidative damage has been concerned by many scholars.α-Lipoic Acid (LA) is known as a "universal antioxidant". In this study, we investigated the protective effect and possible mechanisms of LA in OX-LDL-induced oxidative injury to vascular endothelium in vitro, which aims to provide the basis of experiment and theory for the use of LA in atherogenic therapeutics and prevention.Methods:1.The oxidative injury model of human umbilical vein endothelial cells (HUVEC) in vitro:low-density lipoprotein (LDL) was isolated from the fresh human blood by two-step discontinuous density gradient ultracentrifugation. OX-LDL was prepared by Cu2+ and LDL incubated at 37℃for 24h.The degree of LDL oxidation was measured by MDA analysis. HUVEC were incubated with different concentrations of OX-LDL (100,200,300μg/mL) for 24h,48h. Endothelial cell survival rate were determined by MTT analyses; cell supernatants were taken to measure the content of MDA; and the changes in cell morphology were observed in each group.2. Protective effects of LA on HUVEC by oxidative damage in vitro:(1) Experiment of LA incubation with HUVEC:HUVEC were divided into control group and LA groups at different concentrations (0.1,0.2, 0.5mmol/L). Different concentrations of LA with HUVEC were incubated for 24h; MTT assayed the OD values in each group. (2) Protective effects of LA to HUVEC by oxidative damage in vitro:HUVEC were divided into control group, LDL group, OX-LDL group and the group of OX-LDL with different dose of LA 0.1,0.2,0.5mM respectively. In 96 or 6-well plates with 90% of cell fusion, the different concentrations of LA were pre-incubated with the cells 30min prior to an addition of OX-LDL 180μg/mL. The changes of cell survival rate by LA were determined by MTT analysis. Cell supernatants were taken to measure the content of MDA or LDH. After 24h, cells were collected for western blot, RT-PCR anlysis detecting LOX-1, Bcl-2, CRP protein expression and LOX-1, CRP mRNA expression.Results:1. Different concentrations of OX-LDL with HUVEC were incubated for 24h,48h, compared to the control group, OD values of LA groups were significantly reduced (P<0.01). After treated with the OX-LDL (100μg/mL,200μg/mL,300μg/mL) for 24h,48h, cell survival rate were 80.4±5.23%,55.3±3.28%,28.8±2.34% and 55.7±4.89%,21.8±3.01%, 14.4±1.93% respectively. The content of MDA of cell supernatant for 200μg/mL OX-LDL group (6.9±0.67nmol/mL) and 300μg/mL OX-LDL group (7.14±0.74nmol/mL) were significantly higher than control group (3.4±0.99nmol/mL)(P<0.01). Compared with groups incubated with OX-LDL for 24h, cell survival rate for 48h groups was much lower (P<0.01).Cell deformation was observed under the microscope in 100μg/mL OX-LDL group, but the outline of cell morphology were clear. In 200μg/mL OX-LDL group and 300μg/mL OX-LDL group, cells became round and non-adherent.2. (1)Effect of LA to HUVEC:compared with control group, when the concentration range of LA is at 0.1mmol/L-0.5mmol/L, cell survival rate didn't change (P>0.05). (2) Protective effects of LA to HUVEC by oxidative damage in vitro:LA enhanced the cell survival rate and increased the content of MDA or LDH significantly contrast to the control group (P<0.01).HUVEC were incubated with 180μg/mL OX-LDL for 24h, comparing with the OX-LDL group, LOX-1 mRNA and protein expression were significantly elevated in HUVEC, while Bcl-2 protein expression was lowered. LA can significantly reduce LOX-1 mRNA and protein over-expression, and increase Bcl-2 protein expression induced by OX-LDL in dose-denpendent manner. Conclusion:1.100-300μg/mL OX-LDL induced direct cytotoxicity in HUVEC in a concentration and time-dependent manner.2. LA could increase cell viability of HUVEC induced by OX-LDL, and reduce the content of MDA and LDH in dose-dependent manner.3. LA could up-regulate Bcl-2 protein expression, down-regulate LOX-1 protein and mRNA expression in HUVEC. Therefore, we suppose that LA may protect HUVEC from injury of OX-LDL through its anti-apoptosis and antioxidant effect in preventing the occurrence of AS.4. We couldn't detect the expression of CRP protein and mRNA in endothelial cell by OX-LDL.
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