Mechanisms Of La³⁺ Inhibition Of ECV-304 Apoptosis Induced By Cholestane-3β, 5α, 6β-Triol

Oxysterol plays an important role in the pathogenesis of atherosclerosis. The early study showed the potential preventive and therapeutical effects of lanthanum ion(La³⁺)on atherosclerosis, but the related mechanism remains unclear.In this paper, the effect of La³⁺ on the apoptosis of human umbilical vein endothelial cell (ECV-304) induced by oxysterol cholestane-3β,5α,6β-triol (Triol) and the related mechanisms were investigated. The main results are as follows:(1) Cell viability was evaluated using MTT assay. Apoptosis was detected by flow cytometric analysis and fluorescent microscope. The results indicated Triol at concentration of 15⁵0μM was highly toxic to the ECV-304 cell and induced cells apoptosis. As shown by fluorescent staining, nuclei in Triol-treated cells were breaked into pieces and apoptotic body were formed that is typical in cell apoptosis. La³⁺ (10-6 mol/L) alone enhanced the cell activity. The cell viability was increased and the apoptosis rate was decreased when cell were pretreated with La³⁺ before exposured to Triol.(2) Fluorescence probe DCFH-DA and Rh-123 were used to detect the levels of intracellular ROS and the mitochondria membrane potential (Δψm). The results showed that Triol induced the burst of intracellular ROS, and the dissipation ofΔψm, then mediates increased triggers the cells a start-up of the apoptosis program. Pre-incubation with La³⁺ decreased the generation of ROS induced by Triol, and consequently protect ECV-304 partly from the apoptosis, by stabilizing the membranes of mitochondria.(3) The level of the phosphorylated ERK, p65 and phosphorylated I-κB in the cells were evaluated by Western Blotting. The results showed that Triol induced the increase of the level of the phosphorylated ERK, p65 and phosphorylated I-κB, which was reversed by pre-treatment with La³⁺. Furthermore, MEK inhibitor PD98059 inhibited the increase of p65 induced by Triol. These results suggested that signal of Triol damnified cells transferred to the inner of cell by MAPK signal gateway. The level of its core molecule–the phosphorylated ERK increased, and the activated ERK stimulated I-κB phosphorylation and made it activated, then p65 was released. p65 entried the cell, and accelerated the expression of deceased genic, thus give rise to the decease of cell. La³⁺ prevented signal of Triol damnified cells from transfering from outer cell to inner cell temporally by inhibiting the transduction of MAPK signal pathway. If NF-κB(p65)and I-κB could not be stimulate, the deceased signal can not transfer to the inner of cell core, and the expression of special genic was inhibited. As a result, the deceasing of cell was inhibited.