Mechanisms Of Selenium And Vitamin C Inhibition Of VSMC Injury Induced By Triol

Atherosclerosis (AS) is one of the most serious diseases in human, and the cholesterol oxidation products (Ch-Ox) is currently considered as a key event in the pathogenesis of it. The researches on the cytotoxicity of Ch-Ox and its effect on the pathogenesis, the prevention and cure of AS are one of the frontier fields in human health. The potential preventive and therapeutical effects of vitamin C and the essential trace element selenium on AS are concerned, but the mechanisms of them remain elusive. In this paper, the effect of vitamin C on the growth of vascular smooth muscle cell (VSMC), the Triol (cholestane-3β-5α-6β-triol)-induced cellular injury and the inhibition mechanisms of the supplement of Se and vitamin C were investigated. The main results are as follows: 1) The effects of Vc on the proliferation and apoptosis of VSMC were investigated. The results indicated that low dose of Vc promoted the cell growth, while high dose of it inhibited the cell viability significantly, even induced cell apoptosis. Vc at high concentration (≥500μM) induced VSMC oxidative damage due to its oxidant properties. Significant decreases in activities of GPx and SOD were observed after the treatment of high dose of Vc. The intracellular accumulation of ROS, the increase of [Ca2+]i and the down-regulation of the mRNA level of bcl-2 were involved in VSMC apoptosis induced by Vc. 2) The cytotoxicity of Triol to VSMC and the protections by Na₂SeO₃ and Vc were investigated. The results showed Triol induced oxidative damage in VSMC. After the treatment of Triol, significant decreases in cell survival and activities of GPx, SOD and an increase in the content of MDA were observed. Na₂SeO₃ and Vc protected VSMC against damages induced by Triol in different mechanisms. In Se-supplementation group, intracellular activities of GPx and SOD were enhanced, and the level of lipid peroxidation was decreased. As a consequence, the cell survival was increased significantly. The longer the pretreated time was, the stronger the protection was. Vc protects VSMC against damages induced by Triol in a dose-dependent manner. Hydroperoxy phospholipids, hydroperoxy cholesteryl esters and cholesterol hydroperoxide were effectively reduced by Vc. Pre-incubation with Vc, the intracellular activities of GPx and SOD were maintained, the level of lipid peroxidatioin was decreased, and the cell survival was enhanced significantly. The protections against damages induced by Triol could be enhanced by co-preincubation with Na2SeO3 and Vc. 3) The membrane damage of Triol on VSMC and the protections by Na2SeO3 and Vc were investigated. The severe damage of cell membrane induced by Triol treatment was observed. The information of FTIR spectra in Triol-treated VSMC indicated the damage of Triol on the structures of membrane surface and transmembrane proteins. The membrane fluidity was decreased in Triol-treated VSMC. After the treatment of Triol, the roughness and the pore diameter of cell membrane were increased markedly. So the release of LDH was increased. The abnormal change in cell membrane induced by Triol can be inhibited effectively by Na2SeO3 and Vc. 4) The effects of Triol on the apoptosis of VSMC were investigated. The results suggested Triol induced VSMC apoptosis in a dose-dependent manner. The supplements of Na2SeO3 and Vc protected markedly against apoptosis induced by Triol in VSMC. Several fluorescence probes-DCFH-DA, Fura-2/AM and Rh-123 were used to detect the levels of intracellular ROS, [Ca2+]i and the mitochondria membrane potential (?ψm), respectively. The results showed Triol induced the burst of intracellular ROS, the increase of [Ca2+]i, which caused by the influx of extracelluar Ca2+ and the release of Ca2+ from the intracellular Ca2+ pool, and the dissipation of ?ψm, then mediates increased triggers the cells a start-up of the apoptosis program. Triol could induce the cells apoptosis which was closely accompanied with down-regulation of the levels of bcl-2 and c-myc mRNA. These results suggested that cell apoptosis induced by Triol was mediated by mitochondrialpathways. Selenium supplement as a micronutrition could keep the antioxidant capacity of VSMCs, decrease the generation of ROS induced by Triol, and consequently protect VSMCs partly from the damage, by stabilizing the membranes of cell and organelles such as mitochondria and sarcoplasmic reticulum, keeping the intracellular Ca2+ homeostasis, and maintaining the levels of mRNA of protooncogene c-myc and bcl-2. And the longer the pretreated time was, the stronger the protection was. Pre-incubation with Vc, the antioxidative capacity was restored, the intracellular level of ROS and [Ca2+]i were decreased and the levels of the antiapoptotic mediator bcl-2 mRNA (but not c-myc) were maintained. The protections against cell apoptosis induced by Triol could be enhanced by co-preincubation with Na2SeO3 and Vc.