Two different types of sets of lenses were used in the present study: Sets of healthy clear lenses from donors aged between 15–80 years were obtained from the eye bank of Shandong Eye Institute (Shandong, China) and the other sets of lenses were obtained from patients aged between 45–90 years who had undergone cataract surgery. These patients participated in the present study at the Department of Ophthalmology, Linyi People’s Hospital (Linyi, China). Patients with other systemic disease, including diabetic cataracts, were excluded in order to avoid misleading parameters. In total, ~120 samples with at least one sample from each subject were used. The Institutional Review Board of Linyi People’s Hospital approved the study and written informed consent was obtained from each participant.

HLECs were harvested and lysed with 200 μl immunoprecipitation assay buffer (Pierce IP Lysis Buffer; Thermo Fisher Scientific Inc., Rockford, IL, USA). The lysates were centrifuged at 13,500 × g for 15 min at 4°C and the protein content of the supernatant was determined using the Bradford method (14). The soluble proteins (10–20 μg) were loaded and separated by 10% SDS-PAGE (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and blotted onto a polyvinylidene fluoride membrane (Bio-Rad Laboratories, Inc.). Subsequently, the membranes were blocked with 5% non-fat dry milk powder solution for 1 h at room temperature prior to overnight incubation with Nrf2 and Keap1 rabbit polyclonal antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at 4°C. Following rinsing the membranes, the membranes were incubated with a secondary antibody for 1 h at room temperature and the bands were made more visible using enhanced chemiluminescence. The intensity of each band was normalized to that of β-actin and quantified using Image J analysis software (National Institutes of Health, Bethesda, MD, USA).

Total RNA was extracted from the HLECs using a Quick-RNA MicroPrep solution (Zymo Research, Orange, CA, USA) according to the manufacturer’s instructions. The purified total RNA was reverse transcribed using iScript Reverse Transcription Supermix for quantitative PCR (qPCR; Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. The reverse transcribed RNA was analyzed by qPCR using the SsoFast EvaGreen supermix (Bio-Rad). Roche’s ProbeFinder designed the optimal qPCR assay for the Nrf2 and Keap1 genes. The primer sequences used were as follows: Nrf2, forward 5′-ACACGGTCCACAGCTCATC-3′ and reverse 5′-TGCCTCCAAAGTATGTCAATCA-3′ with a product size of 96 bp; Keap1, forward 5′-GGGTCCCCTACAGCCAAG-3′ and reverse 5′-TGGGGTTCCAGAAGATAAGC-3′ with a product size of 66 bp and β-actin, forward 5′-CCAACCGCGAGAAGATGA-3′ and reverse 5′-CCAGAGGCGTACAGGGATAG-3′ with a product size of 97 bp. Each reaction was performed in triplicate and a standard curve was prepared using a serial dilution of a reference sample. The relative copy numbers were obtained from the standard curve and were normalized to the values obtained for β-actin as the internal control.

The genomic DNA of HLECs was subjected to bisulfite conversion using an EZ DNA Methylation-Direct™ kit (Zymo Research). HLECs from the area of capsulotomy were isolated from the center of the anterior surface of lenses and of age-related cataractous lenses under a dissection microscope (Carl Zeiss AG, Oberkochen, Germany). The bisulfite converted DNA was then used for bisulfite genomic DNA sequencing.

The bisulfite-modified DNA was amplified by bisulfite sequencing PCR using Platinum PCR SuperMix High Fidelity (Invitrogen Life Technologies, Carlsbad, CA, USA) with primers specific to the human Keap1 promoter region 1 with 330 bp (forward 5′-TTAGTTATTTAGGAGGTTGAGGTT-3′ and reverse 5′-AACCCCCCTTCTCACTA-3′) and region 2 with 448 bp (forward 5′-AGTGAGAAGGGGGGTTTGG-3′ and reverse 5′-CCAAAAATAAAATAAACACC-3′). The primers were designed using the Methyl Primer Express software from Applied Biosystems (Foster City, CA, USA). The PCR products were purified by gel extraction using the Zymoclean™ Gel DNA recovery kit (Zymo Research) and then cloned into the pCR4-TOPO vectors using a TOPO TA Cloning kit (Invitrogen Life Technologies). The recombinant plasmids were transformed into One Shot TOP10 chemically competent Escherichia coli (Invitrogen Life Technologies) using the regular chemical transformation method, according to the manufacturer’s instructions. Plasmid DNA was prepared from ~12 independent clones of each amplicon using a PureLink Quick Plasmid Miniprep kit (Invitrogen Life Technologies) and sequenced to determine the status of cytodine-phosphate-guanine methylation. The sequenced data of each clone was then compared with the in silico reference human Keap1 promoter bisulfite converted DNA sequence, derived using the Methyl Primer Express software (Applied Biosytems).

Initially, the protein levels of Nrf2 and Keap1 from human clear lenses of different ages (15, 25, 35, 45, 55, 65, 70, 75 and 80 years) were examined. An age-dependent decrease in the level of Nrf2 was detected by protein immunoblot analysis (Fig. 1). A significant decrease in the protein level of Nrf2 was detected in lenses from the group aged between 65 and 80 years compared with that observed in the group aged between 15 and 45 years. However, a contrast in results was observed in the Keap1 immunoblots. A significant increase in the protein level of Keap1 was detected in lenses of group aged between 65 and 80 years compared with the group aged between 15 and 45 years (Fig. 1). An increase in the protein level of Keap1 may explain the decrease in the level of Nrf2, as Keap1 is a regulator protein, which binds to Nrf2 and targets it for proteasomal degradation. Therefore, the mechanism underlying this marked increase in the protein levels of Keap1 with increasing age was next investigated.

Following the results of the protein blotting, the mRNA and gene expression levels of Nrf2 and Keap1 were examined in human clear lenses from different age groups (15–80 years). qPCR was performed to analyze or quantify the level of mRNA transcription, the results of which reflected the protein immunoblotting results. A gradual decrease was observed in the level of Nrf2 gene expression as age increased (Fig. 2). In addition, a significant decrease in the expression of Nrf2 was observed in lenses from the group aged between 65 and 80 years compared with those <40 years old. However, a gradual increase was observed in the gene expression of Keap1 in lenses from the group aged between 15 and 65 years, while a significant increase was observed after 65 years (Fig. 2). This result suggested that the marked increase in the gene expression of Keap1 with increasing age may have been regulated by epigenetic modification, including DNA methylation or demethylation.

The present study aimed to investigate whether DNA methylation was involved in Keap1 gene expression. Consequently, the gene specific DNA methylation in the promoter region of the Keap1 gene was examined by implementing the genomic DNA Bisulfite sequencing method. The keap1 promoter region was analyzed using two sets of primers as region 1 (−430 to −110) and region 2 (−115 to +335; Fig. 3A). The clear lenses from different age groups (15–80 years) and the age-related cataract lenses from patients aged between 45 and 90 years were selected for the investigation. Complete methylation was identified in the clear lenses from the group aged between 15 and 45 years (Fig. 3B). However, gradual demethylation was observed in the group aged between 45 and 65 years (Fig. 3C) and significant demethylation was found in lenses from the group aged between 65 and 80 years (Fig. 3D). Notably, similar significant demethylation was observed in the age-related cataract lenses from patients aged between 45 and 90 years (Fig. 3E). These results demonstrated that gradual demethylation occurred throughout aging, which was due to the exposure of oxidative stress in the cells. Depending on the level of exposure, demethylation of the Keap1 promoter DNA or increase in the gene expression of Keap1 occurred and suppressed the protein levels of Nrf2. This led to the failure of the antioxidant system in the lenses and ultimately to cataract formation. The methylation study revealed the percentage of demethylation in different age groups, which was estimated as 3% (age 15–45 years), 16% (age 45–65 years), 39% (age 65–80 years) and 42% in cataract lenses from patients aged between 45 and 90 years (Fig. 4). This result clearly indicated that demethylation of the Keap1 gene led to lens oxidation or cataract formation, which was observed in aging lenses. The percentage of demethylation in lenses from the group aged between 65 and 80 years (39%) was almost equal to the percentage observed in cataract lenses (42%). Therefore, due to Keap1 demethylation, the aged lenses were susceptible to cataract formation.