Figure 1.

Aging human lens epithelial cells (hLECs) displayed increased levels of reactive oxygen species (ROS) load and progressive decline in nuclear factor erythroid 2-related factor 2 (Nrf2) target antioxidant genes, which were associated with a reduction in brain and muscle arnt-like protein 1 (Bmal1)-Clock expression. (A–I) Primary hLECs isolated from lenses of different ages were divided into three groups: Group 1 (young, 16–24 years, n = 6); Group 2 (middle age, 52–60 years, n = 8), and Group 3 (old, 64–78 years, n = 10). (A) Cells were cultured in 96-well plate (5000/well). Cultured cells were washed with phosphate buffered saline (PBS) and ROS levels were quantified by H2-DCF-DA dye assay as indicated. The data represent the mean ± S.D. from three independent experiments. Group 1 vs. 2 and 3; **p < 0.001. (B–I) Primary hLECs (directly detached from lenses to avoid cell culture effects) showed significant loss of Bmal1 (B), Clock (C), Nrf2 (D), Peroxiredoxin 6 (Prdx6) (E), NQO1 (F), HO1 (G), SOD1 (H), and SOD2 (I) mRNA expression, which was correlated with increased ROS (A) levels. Total RNA was extracted, as described in Materials and Methods, and then processed for real-time PCR analysis with corresponding specific primers. The data represent the mean ± S.D. from three independent experiments. Young (Group 1) vs. aging group samples; *p < 0.05, **p < 0.001. (J) Aging hLECs showed a significantly progressive loss of clock protein Bmal1, Nrf2, and Prdx6 expression. Cellular proteins were isolated from hLECs and human lenses of different ages, as described in Materials and Methods and as indicated. An equal amount of protein was loaded onto SDS-PAGE, and immunoblotted with an antibody specific to Bmal1 or Nrf2 or Prdx6. Membrane was probed with the Tubulin antibody as loading/internal control. Membrane was imaged and recorded with a FUJIFILM-LAS-4000 image analyzer.