Figure 7.

(A) In vivo DNA binding assay disclosed that Hyd reinforced binding activity of Nrf2 in aging hLECs. Upper panel, schematic illustration of Prdx6 gene promoter with ARE site and ChIP primer locations. Lower panel, in vivo DNA-binding (ChIP) assay revealed that Hyd reinforced the binding activity of Nrf2 to its ARE sequences of the Prdx6 promoter in aging hLECs. ChIP assay was conducted using ChIP grade antibody specific to Nrf2. Immunoprecipitated DNA fragments isolated from vehicle control or Hyd-treated different ages of primary hLECs were purified and processed for ChIP-RT-qPCR analysis. The reduction in Nrf2 enrichment at ARE site in untreated LECs, while increased abundance of Nrf2 at the ARE site in response to Hyd treatment, could be significantly eminent. Young (23 y) vs. aged (55 y and 70 y) subjects and untreated vs. Hyd treated, ** p < 0.001. (B) Age-related reduction in transcriptional activity of Prdx6 promoter in hLECs of variable ages was restored by Hyd. Top panel, diagrammatic sketch of the 5′- constructs of hPrdx6 promoter (−918/+30 bps) linked to CAT reporter plasmid. Lower panel, CAT activity of Prdx6 promoter and CAT vector. The data represent the mean ± S.D. from three independent experiments. Younger age (22 y) vs. aging (57 y and 69 y) samples; untreated vs. Hyd-treated hLECs; ** p < 0.001. (C) Hyd-mediated Nrf2 activation of Prdx6 gene promoter was ROS independent. Hyd treatment significantly increased the Prdx6 promoter activity in a dose-dependent manner. The ARE-driven Prdx6 promoter activity was decreased in SRA-hLECs treated with TAT-HA-Prdx6 (10 µg/mL) protein or antioxidant molecules N-acetyl cysteine (NAC, 5 mM) alone for 24 h. At the same time, Prdx6 transcription increased in SRA-hLECs when cotreated with TAT-HA-Prdx6 protein or NAC and Hyd. The data represent the mean ± S.D. from three independent experiments. * p < 0.05; ** p < 0.001.