Upregulation of Peroxiredoxin 6, an Antioxidant, in Human Lens Epithelial Cells and Rat Lenses Following UV–B Exposure: Cellular Protection and Prevention of Cataractogenesis

Abstract

Abstract: : Purpose: Exposure of solar ultraviolet (UV) radiation is one of the major factors of the induction of cataractogenesis. UV irradiation generates active oxygen species (ROS), thus induces oxidative stress. We have reported previously that peroxiredoxin6, so called anti–oxidant protein2 (AOP2) protects cells from H₂O₂ and/or hyperglycemia induced– apoptosis in human and mouse lens epithelial cells. In the present study, we demonstrated the efficacy of extrinsic supply of Prdx6 in the prevention of UV–B induced insults in in– vitro irradiation model with human lens epithelial cells (hLECs) and rat lenses. We also investigated the fate of Prdx6 in these models. Methods: For the transduction of Prdx6 and evaluation of its protective potential, we constructed Prdx 6 linked to TAT, transduction domain. A full length cDNA was cloned into NcoI and EcoRI sites of pTAT–HA prokaryotic vector and recombinant protein was purified using Ni–NTA agarose column . hLEC or lenses isolated from rats were cultured with or without purified recombinant TAT–HA–AOP2 and exposed to 10J/s/m² UVB for 50, 200 and 800sec or 50 min, respectively. H2–DCFH–DA dye method was applied to monitor ROS level in both groups. MTS assays were performed for cell viability, and MitoLight apoptosis assay was done to define apoptotic cell death. To evaluate protective effect of Prdx6, lenses cultured with/without TAT–HA–AOP2 were observed periodically for the development of opacity following the exposure of UV–B and photographed. Results: Real–time PCR analysis revealed increased expression of Prdx6 mRNA in hLECs or rat lenses at 4h, by contrast, reduced Prdx6 mRNA level was observed at 16 h following post–exposure with UV–B. hLECs exposed with UV–B for longer time (800 sec) revealed diminution of Prdx6 mRNA even at 4h. Extrinsic supply of TAT–HA–AOP2 (10 to 20µg/ml) to rat lenses or hLECs culture media could attenuate the UV–B induced lens opacity and apoptotic cell death. LECs exposed with UV–B showed higher level of ROS and that could be diminished by the extrinsic supply of TAT–HA–Prdx6 to the hLECs or rat lenes in vitro. Conclusions: The results implied that UV–B induced–damage to lenses or LECs is associated with oxidative stress. Since extrinsic supply of Prdx6 could attenuate the UV–B induced abuse, this molecule may have potential to prevent cataractogenesis.

Keywords: antioxidants • oxidation/oxidative or free radical damage • cataract