Abstract
Purpose: :
Loss of antioxidant expression has been implicated in cataractogenesis. Sulforaphane (SFN) protects cells by upregulating phase 2 antioxidant proteins. Using lens epithelial cells (LECs) from rat and human lenses coupled with peroxiredoxin 6-depleted (Prdx6-/-) from mouse, we demonstrated that LECs treated with SFN gained resistance against UV-B-induced damage. Resistance was associated with transcription factor Nrf2-mediated enhanced Prdx6 transcription and its electron donor GlutathioneS-transferase π (GSTπ).
Methods: :
Cultured LECs were treated with different concentrations (0, 500nM, 1, 2, 4 or 8 μM) of SFN for variable times, and exposed to UV-B (100 to 400J/m2). MTS and TUNEL assays evaluated cell viability and defined cell death. H2DCFH-DA dyes measured ROS levels. Western and real-time PCR analyses were used to examine SFN-dependent expression of Nrf2 protein and mRNA, and its correlation with Prdx6 and GSTπ expression. Human Prdx6 promoter (-918/+30nt) containing antioxidant response element (ARE, -357/-349) and its mutant at the ARE linked to CAT were prepared. Gel-shift assay, Site-Directed Mutagenesis and CAT-ELISA were used to characterize ARE/Nrf2 activity. ERRβ, an inhibitor of Nrf2 and siRNA specific to Nrf2, was used to validate results. Shumiya Cataract Rats (SCR) were fed a diet containing broccoli sprout powder including high-dose of SFN.
Results: :
Cell viability and TUNEL assay revealed that SFN protected LECs against UVB-induced toxicity. Cells displayed SFN concentration-dependent enhanced expression of Prdx6 and GSTπ mRNA and protein, and reduced expression of ROS. Transactivation assay with Prdx6 promoter with Nrf2 site showed increased promoter activity in treated cells, which was correlated with level of Nrf2 expression and nuclear localization. Cells with Nrf2 knockdown, disruption of Nrf2 site (ARE), or ERRβ treatment did not respond to SFN. In gel-shift assay, Nrf2 bound to Nrf2 site (-357/-349) in Prdx6 promoter, and SFN augmented binding affinity, validating the SFN-mediated Nrf2-depedent regulation of Prdx6. Cataractogenesis was delayed when the SFN-containing diet was supplied.
Conclusions: :
SFN protected LECs and delayed cataractogenesis in SCR, and exercised its activity by enhancing Prdx6 expression through Nrf2 activation. This study may provide a basis for new dietary strategies to delay oxidative injury.
Keywords: cataract • antioxidants • oxidation/oxidative or free radical damage