The protective effects of 6-CySeCD with GPx activity against UVB-induced injury in HaCaT cells.

BACKGROUND: The generation of harmful reactive oxygen species (ROS) induced by UVB irradiation could induce cell apoptosis and change the cell cycle. 6A,6A'-dicyclohexylamine-6B,6B'-diselenide-bis-ß-cyclodextrin (6-CySeCD) is a novel glutathione peroxidase (GPx; EC 1.11.1.9) mimic. The aim of this study was to investigate the anti-oxidative effects of 6-CySeCD in cultured immortalised human keratinocyte cells (HaCaT). METHODS: HaCaT cells were treated with 30 mJ/cm(2) UVB to establish a damage model. The cultured HaCaT cells were randomly assigned to the control, UVB and treatment groups. The treatment group was incubated with 20 µmol/L of GPx mimics before UVB irradiation. Cell viability was detected by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, the level of lipid peroxidation was determined by the formation of malondialdehyde (MDA), DNA fragmentation was observed using agarose gel electrophoresis and the levels of intracellular ROS and cell cycle progression were measured by flow cytometry. RESULTS: The levels of cytotoxicity, intracellular ROS, lipid peroxidation and oxidative DNA damage significantly increased after UVB irradiation in the HaCaT cells. UVB irradiation caused pre-G1 -phase arrest in HaCaT cells and significantly reduced the number of HaCaT cells in the S phase. The GPx mimics 6-CySeCD and 2-phenyl-l,2-benzisoselenazol-3(2H)-one (ebselen) significantly blocked UVB-induced apoptosis and changed the cell cycle of the HaCaT cells. The blocked effect of pretreatment 6-CySeCD in UVB-irradiated HaCaT cells was better than that of pretreatment with ebselen. CONCLUSION: 6-CySeCD can relieve the damage induced by UVB irradiation in HaCaT cells.

A novel selenium and copper-containing peptide with both superoxide dismutase and glutathione peroxidase activities.

Superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase (CAT) play crucial roles in balancing the production and decomposition of reactive oxygen species (ROS) in living organisms. These enzymes act cooperatively and synergistically to scavenge ROS. In order to imitate the synergism of these enzymes, we designed and synthesized a novel 32-mer peptide (32P) on the basis of the previous 15-mer peptide with GPX activity and a 17-mer peptide with SOD activity. Upon the selenation and chelation of copper, the 32-mer peptide is converted to a new Se- and Cu-containing 32-mer peptide (Se-Cu-32P) and displays both SOD and GPX activities and its kinetics was studied. Moreover, the novel peptide was demonstrated to be able to better protect vero cells from the injury induced by xanthine oxidase (XOD)/xanthine/Fe2+ damage system than its parents. Thus, this bifunctional enzyme imitated the synergism of SOD and GPX and could be a better candidate of therapeutic medicine.

A trifunctional enzyme with glutathione S-transferase, glutathione peroxidase and superoxide dismutase activity.

Superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione S-transferase (GST) and glutathione reductase (GR) play crucial roles in balancing the production and decomposition of reactive oxygen species (ROS) in living organisms. These enzymes act cooperatively and synergistically to scavenge ROS, as not one of them can singlehandedly clear all forms of ROS. In order to imitate the synergy of the enzymes, we designed and generated a recombinant protein, which comprises of a Schistosoma japonicum GST (SjGST) and a bifunctional 35-mer peptide with SOD and GPX activities. The engineered protein demonstrated SOD, GPX and GST activities simultaneously. This trifunctional enzyme with SOD, GPX and GST activities is expected to be the best ROS scavenger.

A novel dicyclodextrinyl diselenide compound with glutathione peroxidase activity.

A 6A,6A'-dicyclohexylamine-6B,6B'-diselenide-bis-beta-cyclodextrin (6-CySeCD) was designed and synthesized to imitate the antioxidant enzyme glutathione peroxidase (GPX). In this novel GPX model, beta-cyclodextrin provided a hydrophobic environment for substrate binding within its cavity, and a cyclohexylamine group was incorporated into cyclodextrin in proximity to the catalytic selenium in order to increase the stability of the nucleophilic intermediate selenolate. 6-CySeCD exhibits better GPX activity than 6,6'-diselenide-bis-cyclodextrin (6-SeCD) and 2-phenyl-1,2-benzoisoselenazol-3(2H)-one (Ebselen) in the reduction of H(2)O(2), tert-butyl hydroperoxide and cumenyl hydroperoxide by glutathione, respectively. A ping-pong mechanism was observed in steady-state kinetic studies on 6-CySeCD-catalyzed reactions. The enzymatic properties showed that there are two major factors for improving the catalytic efficiency of GPX mimics. First, the substrate-binding site should match the size and shape of the substrate and second, incorporation of an imido-group increases the stability of selenolate in the catalytic cycle. More efficient antioxidant ability compared with 6-SeCD and Ebselen was also seen in the ferrous sulfate/ascorbate-induced mitochondria damage system, and this implies its prospective therapeutic application.

The effect of nano-TiO2 photocatalysis on the antioxidant activities of Cu, Zn-SOD at physiological pH.

Security issues of nanoparticles on biological toxicity and potential environmental risk have attracted more and more attention with the rapid development and wide applications of nanotechnology. In this work, we explored the effect and probable mechanism of nano-TiO2 on antioxidant activity of copper, zinc superoxide dismutase (Cu, Zn-SOD) under natural light and mixed light at physiological pH. Nano-TiO2 was prepared by sol-hydrothermal method, and then characterized by X-ray Diffraction (XRD) and Transmission electron micrographs (TEM). The Cu, Zn-SOD was purified by sephadex G75 chromatography and qualitatively analyzed by sodium dodecyl sulfate polypropylene amide gel electrophoresis (SDS-PAGE). The effect and mechanism were elucidated base on Fourier Transform Infrared Spectrometer (FT-IR), Circular Dichroism (CD), zeta potential, and electron spin resonance (ESR) methods. Accompanying the results of FT-IR, CD and zeta potential, it could be concluded that nano-TiO2 had no effect on the antioxidant activity of Cu, Zn-SOD by comparing the relative activity under natural light at physiological pH. But the relative activity of Cu, Zn-SOD significantly decreased along with the increase of nano-TiO2 concentration under the mixed light. The results of ESR showed the cause of this phenomenon was the Cu(II) in the active site of Cu, Zn-SOD was reduced to Cu(I) by H2O2 and decreased the content of active Cu, Zn-SOD. The reduction can be inhibited by catalase. Excess O2·- produced by nano-TiO2 photocatalysis under mixed light accumulated a mass of H2O2 through disproportionation reaction in this experimental condition. The results show that nano-TiO2 cannot affect the antioxidant activity of Cu, Zn-SOD in daily life. The study on the effect of nano-TiO2 on Cu, Zn-SOD will provide a valid theory support for biological safety and the toxicological effect mechanism of nanomaterials on enzyme.