Abstract: BACKGROUND: Preliminary work has found that homocysteine accelerates the aging of umbilical vein endothelial cells through reactive oxygen species. This experiment preliminarily studies the relevant mechanisms affecting endothelial cell aging.
OBJECTIVE: To investigate the effects of echinacoside on the expression of cytoplasmic polyadenylate binding protein 1 (CPEB1), sirtuin1 (SIRT1), P53 and P21 genes in human umbilical vein endothelial cells.
METHODS: Human umbilical vein endothelial cells were cultured in vitro. D-galactose 10 g/L was used to construct cell aging model. The proliferation viability of cells treated with echinacoside (0, 10, 20, 50, 100, 200, 300, 400, 500 mg/L) for 12 and 24 hours was analyzed by CCK-8 assay, and the optimal mass concentration and intervention time of echinacoside were screened. Human umbilical vein endothelial cells were divided into normal control group, D-galactose group and echinacoside groups (100, 200, and 300 mg/L). β-Galactosidase staining was used to detect the positive staining rate of cells after echinacoside pre-intervention for 12 hours. Real time RT-PCR and western-blot assay were used to detect the mRNA expression levels of CPEB1, Sirt1, P53 and P21 and the protein expression levels of CPEB1, Sirt1 and P21 in human umbilical vein endothelial cells after echinacoside pretreatment for 12 hours. 
RESULTS AND CONCLUSION: (1) The results of CCK-8 assay showed that the survival rate of echinacoside group at 12 hours was significantly higher than that at 24 hours. Compared with the D-galactose group, cell viability increased in the echinacoside 100, 200, and 300 mg/L groups to varying degrees (P < 0.000 1), and there was a significant difference among the three groups (P < 0.01). (2) Compared with the normal control group, the D-galactose group had the highest positive rate of β-galactosidase staining (P < 0.0001). 100, 200, and 300 mg/L echinacoside pretreatment for 12 hours could reduce the positive rate of β-galactosidase staining to a certain extent (P < 0.000 1). (3) Compared with the normal control group, the mRNA expression levels of CPEB1, P53 and P21 were increased (P < 0.000 1), and the expression level of SIRT1 mRNA was decreased (P < 0.000 1) in the D-galactose group. Compared with the D-galactose group, 100, 200, and 300 mg/L echinacoside pretreatment for 12 hours decreased the mRNA expression levels of CPEB1, P53, and P21 (P < 0.000 1), but increased Sirt1 mRNA expression (P < 0.000 1). Western blot assay results were consistent with real-time fluorescence quantitative polymerase chain reaction results. (4) These findings indicate that echinacoside may delay D-galactose-induced human umbilical vein endothelial cell senescence by down-regulating CPEB1 and P53/P21 expression levels and up-regulating Sirt1 expression levels.

Key words: echinoside, human umbilical vein endothelial cells, aging, CPEB1, Sirt1, P53, P21