Relationship between expression of selenocysteine synthase and endothelial cell line ECV-304 injury induced by hydrogen peroxide

Abstract

Objective To observe the influence of selenocysteine synthase（SEPSECS） on injury of human umbilical vein endothelial cell line EVC-304 induced by hydrogen peroxide （H2O2）. Methods Transfection was conducted to transfect EVC-304 which was maintained in vitro. The cells were divided into four groups： control group, SEPSECS over-expression group, empty vector group and SEPSECS silenced expression group, then Real-time PCR and Western blotting were performed to detected SEPSECS mRNA and protein expression , respectively. Flow cytometry（FCM） was performed to detect cell cycle. Different concentrations of H2O2, which including 0, 200, 400, 600, 800, 1 000 μmol/L, were used to treat EVC-304 . Then malonaldehyde （MDA）, superoxide dismutase（SOD） secreted by the cells which were treated with H2O2 for 6 h, were checked by MDA or SOD kit. Results The SEPSECS mRNA expressions of control, SEPSECS silenced expression, empty vector and SEPSECS over-expression groups were 1.03 ± 0.24, 0.43 ± 0.11, 0.98 ± 0.27 and 1.61 ± 0.13, respectively. The protein expressions of control, SEPSECS silenced expression, empty vector and SEPSECS over-expression groups were 1.00 ± 0.26, 0.51 ± 0.10, 1.12 ± 0.38 and 1.51 ± 0.20, respectively. There was a significant difference between control and SEPSECS silenced expression groups （all P〈0.05）, at the same time , this phenomenon was also observed between empty vector and SEPSECS over-expression groups （all P〈0.05）. The level of MDA was increased along with the H2O2 concentration. Besides, cell cycle was also tested, however, significant difference was not observed（all P 〉 0.05）. Meanwhile, MDA of SEPSECS silenced expression groups[（15.8 ± 0.5）,（19.6 ± 1.5）μmol/L] were significantly higher than control groups[（12.4 ± 0.1）,（17.1 ± 0.5）μmol/L, all P 〈 0.05], on the other hand, MDA of SEPSECS over-expression groups[（10.8 ± 0.4）,（14.2 ± 1.1）μmol/L] were lower than empty vector groups [（12.7 ± 0.7）,（16.2 ± 1.1）μmol/L, all P 〈 0.05], when the H2O2 concentration was 800 or 1 000μmol/L. The level of SOD was decreased along with the H2O2 concentration. SOD of SEPSECS silenced expression groups[（7.7 ± 0.4）,（2.4 ± 0.3）μmol/L] were lower than control groups[（10.0 ± 1.0）,（6.0 ± 0.6）μmol/L, all P 〈 0.05], on the contrary, SOD of SEPSECS over-expression groups[（11.3 ± 0.6）,（12.7 ± 1.6）μmol/L] were higher than empty vector groups[（9.2 ± 0.6）,（6.7 ± 0.2）μmol/L, all P 〈 0.05], when the H2O2 concentration was 800 or 1 000μmol/L. Conclusion Expression of SEPSECS has a significant protective role on damaged EVC-304 which was induced by H2O2. Key words: Selenocysteine synthase;  Hydrogen peroxide;  Cell culture techniques