| ObjectiveEndothelial cell damage and death may play an important role in the development of atherosclerosis(AS) and endothelial injury is the early pathological and physiological process of the cardiovascular disease(CVD). So mantaining the health of vascular endothelial cells is important for AS related diseases. Homocysteine(Hcy) could induce the injury of vascular endothelial cell, previous clinical and experimental studies have indicated that moderate increases in plasma Hcy concentration are casual risk factors for vascular disease including AS. Folic acid(FA) is an important factor of Hcy metabolites in the blood. Here, we hypothesize that folic acid alleviates Hcy induced vascular injure, and the mechanism should be disscussed. MethodsIn this study, HUVCE cells were incubated for 48 h with a variety of folic acid(0-1000 nmol/L), at first 24 h with a combination of 1000 ?mol/L Hcy or vehicle. Cell viability was detected by MTT, and cell growth inhibition rate was calculated. MDA in cells were detected by MDA assay kit. The activity of LDH in cell culture medium was detected by LDH Assay kit. The intracellular concentration of folate was examined by FA assay kit(chemiluminescent microparticle immunoassay). The intracellular concentrations of Hcy, SAM and SAH were examined by HPLC respectively. The mRNA expression levels of VEGF and MCP-1 were determined by RT-PCR. The protein expression levels of VEGF and MCP-1 were detected by Western Blot. Total antioxidant capacity in cells was detected by T-AOC assay kit. The activity of SOD in cells was detected by SOD assay kit. The activity of GSH-Px in cells was detected by GSH-Px assay kit. GSH in cells were detected by GSH assay kit. FCM was used to detect cell apoptosis rate of HUVEC. The mRNA expression levels of anti-apoptotic gene Bcl2 and pro-apoptotic genes TP53, Caspase-3, Caspase-4, Caspase-6 and Caspase-8 were determined by RT-PCR. Caspase-3/7 assay kit was used to detect the activity of Caspase-3/7 activity in cells. ResultsHcy decreased cell viability of HUVCE, compare with the cells incubate with 20 nmol/L folic acid without Hcy. The cells which incubate with 20 nmol/L folic acid without Hcy arranged tightness and neat, however, for the cells which incubate with Hcy, Hcy induced cells arranged loosen. Treatment with folic acid induced significant changes in morphology and cell numbers, especially after treatment with the highest concentration of folic acid. And then FA increased cell viability, and decreased cell growth inhibition rate, which incubate with FA. FA also reduced the degree of oxidative damage of HUVEC.Compared with Hcy+FA-N group, MDA and LDH activity were decreased in Hcy+FA-H group(P<0.05). FA increased the antioxidant capacity of cell. Compared with Hcy+FA-N group, T-AOC, SOD activity, intracellular GSH concentration and GSH-Px activity were increased in Hcy+FA-H group(P < 0.05). FA reduced intracellular Hcy, SAH concentrations, however, elevated intracellular concentration of SAM. FA increased the expressions of mRNA and protein of VEGF but reduced the expression of MCP-1. FA increased the expression of mRNA of Bcl2, reduced the expression of mRNA of TP53, Caspase-3, Caspase-4 and Caspase-8. And FA decreased the Caspase-3/7 activity of the cells. ConclusionsFolic acid amelioratives the oxidative damage induced by Hcy, increase cell activity, reduce growth inhibition rate, improve endothelial function, enhance the antioxidant capacity of cells and reduce the apoptosis rate. Folic acid attenuates the toxicity effects of homocysteine in human umbilical vein endothelial cells. Further observations indicated that this effect of folic acid was mediated by regulating the mRNA and protein expression of VEGF and MCP-1, and decreasing the activity of caspase-3/7, up-regulating Bcl2, down-regulating TP53, caspase-3, caspase-4 and caspase-8. In conclusion, suitable doses of supplemental folic acid may play a protective role in HUVEC cell damage induced by homocysteine. |
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