Hydralazine Revives Cellular and Ocular Lens Health-Span by Ameliorating the Aging and Oxidative-Dependent Loss of the Nrf2-Activated Cellular Stress Response

A major hallmark of aging-associated diseases is the inability to evoke cellular defense responses. Transcriptional protein Nrf2 (nuclear factor erythroid-derived 2-related factor) plays a pivotal role in the oxidative stress response, cellular homeostasis, and health span. Nrf2’s activation has been identified as a therapeutic target to restore antioxidant defense in aging. Here, we demonstrated that FDA-approved drug, hydralazine (Hyd), was a reactivator of the Nrf2/ARE (antioxidant response element) pathway in various ages and types of mouse (m) or human (h) lens epithelial cells (LECs) and mice lenses in-vitro/in-vivo. This led to Hyd-driven abatement of carbonyls, reduced reactive oxygen species (ROS), and reduced 4-HNE/MDA-adducts with cytoprotection, and extended lens healthspan by delaying/preventing lens opacity against aging/oxidative stress. We elucidated that Hyd activated the protective signaling by inducing Nrf2 to traverse from the cytoplasm to the nucleus and potentiated the ARE response by direct interaction of Nrf2 and ARE sequences of the promoter. Loss-of-function study and cotreatment of Hyd and antioxidant, N-acetyl cysteine (NAC) or Peroxiredoxin (Prdx)6, specified that Nrf2/ARE-driven increase in the promoter activity was Hyd-dependent. Our study provides proof-of concept evidence and, thereby, paves the way to repurposing Hyd as a therapeutic agent to delay/prevent aging and oxidative-related disorders.


Introduction
Preservation of redox homeostasis is central to maintaining cellular integrity and physiological function. An imbalance between oxidation and reduction phenomenon can lead to cell/tissue damage [1]. This imbalance can emerge from endogenous and exogenous stresses as well as deterioration of the cellular antioxidant defense pathway during aging [2][3][4], which is accompanied by cellular damage and cell death. Indeed, disruption of antioxidant response and, thereby, increased oxidative load has been shown to be a major cause for various aging-related pathologies, including blinding diseases [2,[5][6][7][8][9]. To cope with the pathogenic effects generated by oxidative stressors, cells have evolved antioxidant defense mechanisms to encounter these stresses to maintain redox homeostasis [2,10,11]. Studies have shown that cellular oxidant/antioxidant equilibrium is maintained in a dynamical way via threshold regulation of cellular antioxidants' expression [12]. Exposure of mammalian cells to cellular and environmental stressors generally initiates antioxidant transcriptional responses, which are mainly up-regulated through a master transcription factor, Nrf2 (nuclear factor erythroid-derived 2-related factor) [13,14]. This biological phenomenon results in the coordinated increased expression of phase II antioxidant genes, such as glutathione S-transferase (GSTπ), catalase (Cat), glutathione-peroxidase (GPxs), heme oxygenase 1 (HO1), glutamate-cysteine ligase subunits (GCLC and GCLM), NAD(P)H, quinone oxidoreductase 1 (NQO-1), and peroxiredoxins (Prdxs). However, when these All animal studies followed the recommendations set forth in the "Statement for the Use of Animals in Ophthalmic and Visual Research" by the Association for Research in Vision and Ophthalmology and were approved by the Institutional Animal Care and Use Committee (IACUC), University of Nebraska Medical Center (UNMC), Omaha, NE. LECs were isolated from C57BL/6 mice and maintained in DMEM with 10% FBS, as described earlier [51,74,76,79]. Like SRA-hLECs, mLECs were cultured in 100 mm plate and next-day treated with Hyd (diluted in the complete culture media) at 70% confluency.
C57BL/6 mice of 6 months (M), 16M, and 22 M were purchased from Charles River Laboratories (Wilmington, MA, USA) and were maintained at a stable temperature and humidity under specific pathogen-free conditions in a cleaned animal facility, as described earlier [3,31]. Primary mLECs were isolated from lenses and cultured in collagen IV precoated dishes containing complete DMEM media (15% FBS). At 90-100% confluency, cells were harvested and used for the study.

Cell Viability Assay
Cell growth or H 2 O 2 -mediated toxicity was analyzed using MTS assay. In brief, cell viability of Hyd (Millipore Sigma, Rockville, MD, USA)-treated or untreated LECs facing H 2 O 2 -induced oxidative stress was evaluated using a colorimetric MTS assay (Promega, Madison, WI, USA) according to the manufacturer's method and as described previously [6,80,81]. This assay of cell proliferation/viability uses 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2 to (4-sulphophenyl) 2H-tetrazolium salt. At the end of predefined incubation, MTS dye was added. After 2 h of incubation, the absorbance was read at 490 nm with a plate reader (DTX 880, Multimode detector, Spectra MAX Gemini, San Jose, CA, USA). The data obtained were normalized with absorbance of the untreated control(s).

Measurement of Reactive Oxygen Species (ROS)
Intracellular ROS levels of LECs facing H 2 O 2 -driven stress in absence or presence of Hyd were assessed with H 2 -DCF-DA (2 -7 -dichlorodihydrofluorescein diacetate) dye, a non-fluorescent fluorescein derivative as described in our published report [3,31]. On the day of experiment, the medium was discarded, and cell culture wells were washed with PBS and incubated in the same buffer containing 10 µM of H 2 -DCF-DA dye for 30 min at 37 • C. After diffusion into the cells, H 2 -DCF-DA is deacetylated by cellular esterase into a non-fluorescent compound that was later oxidized by ROS into 2 -7 -dichloroflurescein (DCF). ROS levels were quantified at Ex485 nm/Em530 with a Spectra Max Gemini EM (Molecular Devices, San Jose, CA, USA). To measure the intracellular redox state levels of lenses isolated from Hyd-administered C57BL/6 mice eye, lenses were carefully isolated and immediately frozen at −80 • C. The intracellular levels of ROS were quantified by H 2 -DCF-DA according to our published protocol [2,3,31]. Briefly, lenses were homogenized (100 mg/mL) in freshly prepared homogenization buffer (50 mM phosphate buffer containing 0.5 mM PMSF, 1 mM EDTA, 1 µM pepstatin, 80 mg/L trypsin inhibitor, pH 7.4). The same amount of homogenate was added to 96-well cell culture plates. To measure the levels of ROS, H 2 -DCF-DA (30 µM final concentration) dye was added and incubated for 30 min. Intracellular fluorescence was detected at O.D., Ex485 nm/Em530, with a Spectra Max Gemini EM (Molecular Devices, San Jose, CA, USA).

Measurement of the Oxidative Effect by Lipid Peroxidation Assay
Lipid peroxidation (LPO) assay is based on the reaction of N-methyl-2-phenylindole, a chromogenic reagent with MDA (malondialdehyde) and 4-HNE (4-hydroxyalkenals) at 45 • C. Either MDA or 4-HNE reacts with two molecules of N-methyl-2-phenylindole to yield a stable chromophore with maximal absorbance at 586 nm. LPO assay in LECs/lenses exposed with H 2 O 2 in absence or presence of Hyd was carried out according to manufacturer's instructions (Lipid Peroxidation Microplate Assay Kit; Oxford Biomedical Research, Rochester Hills, MI, USA) and our published report [11,80]. Total cell lysate was prepared from LECs/lenses, and equal amounts of protein were used for the assay. Absorbance was measured at 586 nm using a plate reader (DTX 880, Multimode detector, Spectra MAX Gemini, San Jose, CA, USA).

Protein Carbonyl Assay
The protein carbonyl content was measured in LECs/lenses exposed to H 2 O 2 in absence or presence of Hyd using Protein Carbonyl Content Assay Kit (MAK094, Sigma-Aldrich, Saint Louis, MO, USA) following the company's protocol. The protein content of LECs (samples) were determined using the BCA assay. Samples were treated with 10% streptozocin solution to remove interfering nucleic acid. Purified water served as background control. Dinitrophenylhydrazine (DNPH) solution was added to each sample and incubated for 10 min, and then 87% TCA solution was added to the sample. Supernatant was removed by high centrifugation, and ice-cold acetone was added to each pellet and was placed for 30 s in a sonication bath and then further incubated at −20 • C for 5 min. Carefully, acetone was removed from each pellet by high centrifugation, and 6M guanidine solution was added to the pellet and sonicated. An amount of 100 µL of each sample was transferred to the 96-well plate, and reading was measured at 375 nm (absorbance) with a plate reader (DTX 880, Multimode detector, Spectra MAX Gemini, San Jose, CA, USA).

Measurement of 8-Hydroxydeoxyguanosine
DNA is a biologically significant target of oxidative stress. The generation of 8-Hydroxydeoxyguanosine (8-OHdG) is a ubiquitous marker of oxidative stress among numerous types of oxidative DNA damage. We measured the levels of 8-OHdG in LECs/lenses exposed to H 2 O 2 with or without Hyd treatment using The OxiSelect™ Oxidative DNA Damage Elisa kit (STA-320, Cell Biolabs, INC., San Diego, CA, USA). To prepare a standard curve, different concentrations (0-20 ng/mL) of 8-OHdG standard was prepared by diluting in assay diluent as mentioned in the company's protocol. In brief, genomic DNA was isolated from LECs/lenses treated with H 2 O 2 in absence or presence of Hyd and converted to single stranded DNA. DNA samples were digested with nuclease P1 (Catalog No. N8630, Millipore Sigma, Rockville, MD, USA) followed by the incubation with 10 units of alkaline phosphatase (Catalog No. P5931, Millipore Sigma, Rockville, MD, USA) at 37 • C for 1 h in 100 mM Tris, pH 7.5. The 8-OHdG standards (50 µL) or samples (50 µL) were added to the well of the 8-OHdG conjugate coated plate and incubated at room temperature (RT) for 10 min with shaking. Thereafter, diluted anti-8-OHdG (50 µL) antibody was added to each well and incubated for 1 h on an orbital shaker at RT. The wells were washed, and diluted secondary antibody-enzyme conjugate (100 µL) was added and incubated on a shaker for 1 h at RT. After washing the wells, 8-OHdG activity was recorded by the addition of 100 µL warm substrate solution. Enzymatic reaction was stopped by the addition of 100 µL of stop solution, and absorbance was measured at 450 nm with a microplate reader (DTX 880, Multimode Detector, Molecular device, San Jose, CA, USA).

Protein Isolation and Expression Analysis
Protein expression was examined by Western blot analysis as described previously [3,7,18,20,23]. In brief, SRA-hLECs or mLECs/lenses treated with Hyd for different time periods were processed for protein extraction using ice-cold radioimmune precipitation buffer (RIPA buffer). Cellular extract containing equal amounts of protein (measured by the Bradford method) from each sample was run on SDS-PAGE gel and transferred to the PVDF membrane. After blocking the membrane at room temperature with 5% milk, the membranes were probed with Anti-Nrf2 (ab62352, Abcam, Waltham, MA, USA) and Anti-Prdx6 antibodies (Ab Frontier, Seoul, Republic of Korea). Tubulin (ab44928, Abcam, Waltham, MA, USA) and LaminB1 (ab133741, Abcam) were used as internal control. Following incubation with the primary antibody, PVDF membranes were incubated with secondary antibody (sc-2357 and sc-516102, Santa Cruz Biotechnology, Dallas, TX, USA), and protein bands on membrane were visualized by incubating the membrane with luminol reagent (sc-2048; Santa Cruz Biotechnology, Dallas, TX, USA). Protein band images were recorded with a FUJIFILM-LAS-4000 luminescent image analyzer (FUJIFILM Medical Systems Inc., Hanover Park, IL, USA).

Quantitative Real-Time PCR
The relative levels of Nrf2 and its target genes mRNA were measured by quantitative real-time PCR (RT-qPCR). Total RNA was isolated from Hyd-treated SRA-hLECs as well as mLECs/lenses derived from C57BL/6 mice by using the single-step guanidine thiocyanate/phenol/chloroform extraction method (Trizol Reagent, Invitrogen, Waltham, MA, USA), as described previously [2,19,31]. cDNA synthesis was conducted with 5 µg of total RNA using Superscript II RNAase H-reverse-transcriptase. RT-qPCR reactions were prepared using SYBR Green Master Mix (Roche Diagnostic Corporation, Indianapolis, IN, USA), specific primers (as shown in Table 1), and with diluted cDNA in a Roche ® LC480 Sequence detector system (Roche Diagnostic Corporation, Indianapolis, IN, USA). RT-qPCR was performed using program condition of 5 min hot start at 95 • C, followed by 45 cycles for 10 s (seconds) at 95 • C, 30 s at 60 • C, and 10 s at 72 • C. Data were analyzed by LightCycler480 software by Roche. All data analysis was performed in triplicate. The relative values of target genes were normalized to the control using corresponding β-actin as an internal control.

) Activity
Phospholipase A 2 (PLA 2 ) activity was carried out in accordance with the manufacturer's protocol (EnzChek Phospholipase A2 kit; E10217, Invitrogen, Waltham, MA, USA) and our published protocol [3,81,83]. Briefly, SRA-hLECs and mLECs were treated with different concentrations of Hyd for 24 h. Thereafter, total protein was isolated and quantified by BCA protein assay (ThermoFisher Scientific, Waltham, MA, USA). To prepare a standard curve, different concentrations (0-10 Units/mL) of PLA 2 was made by diluting PLA 2 stock solution (500 Units/mL) with 1 × reaction buffer up to 50 µL. For sample preparation, an equal amount of protein was diluted with 1 × PLA 2 reaction buffer to make volume up to 50 µL. The reaction was started by adding 50 µL of the substrate-liposome mix to each microplate well consisting of control, standard, and the samples with 100 µL total reaction volume. The fluorescence units were measured at optical density (O.D.), Ex485 nm/Em535 nm, using a microplate reader (DTX 880, Multimode Detector, Molecular device, San Jose, CA, USA).

Estimation of Glutathione (GSH) Peroxidase Activity
Glutathione (GSH) peroxidase activity was quantified according to company's protocol (Glutathione Peroxidase activity kit, Cat No. ADI-900-158, Enzo Life Sciences, Farmingdale, NY, USA) and our published protocol [3,81,83]. Total cell lysate was prepared from Hydtreated or untreated SRA-hLECs or mLECs. The equal amount of quantified protein was subjected to quantify the enzymatic activity. To set up the reaction, 20 µL of standard (glutathione peroxidase) or samples or control, and 140 µL of 1 × assay buffer, with 20 µL of 10× reaction mix were added to each well of a 96-well plate. An amount of 20 µL of cumene hydroperoxide was quickly added to each well to initiate the reaction. Thereafter, O.D. was measured at absorbance 340 nm every 1 min up to 15 min period. Blank O.D. was subtracted from the standard as well as sample O.D. to obtain the net rate of absorbance at 340 nm for the GSH peroxidase activity (DTX 880, Multimode Detector, Molecular device, San Jose, CA, USA).

Quantitation of Superoxide Dismutase (SOD) Activity
Superoxide Dismutase (SOD) enzymatic activity was measured as mentioned in the manufacturer's protocol (Superoxide Dismutase (SOD) Assay, Kit, LS-K224, LifeSpan BioSciences, Inc., Seattle, WA, USA) [3]. To prepare a standard curve, SOD enzyme was diluted using diluent at different concentrations (0-3 U/mL). Standards were prepared and 20 µL was added to a 96-well microplate. For samples, an equal amount of Hyd-treated or untreated SRA-hLECs and mLECs cellular protein and dilutions were added to separate wells of a 96-well plate with 20 µL volume. An amount of 160 µL of working reagent (mixture of 160 µL assay buffer, 5 µL xanthine, and 5 µL WST-1) was added to the standard, samples, and control wells. The contents were mixed by tapping the plate, 20 µL of diluted XO enzyme (1:20 diluted in diluent) was added, and immediately the O.D. was measured at 440 nm and 0 min (OD 0 ) with DTX 880 Multimode Detector (SpectraMAX Gemini, San Jose, CA, USA). The plate was incubated at room temperature in the dark for 60 min, and, again, the O.D. was measured at 440 nm (OD 60 ). Each standard and sample well were calculated using the following formula, ∆OD 60 = OD 60 − OD 0 , and was presented in percentage.

Quantitation of Catalase Activity
Catalase activity was determined following the manufacturer's instructions (Catalase Assay, Kit, LS-K245, LifeSpan BioSciences, Inc., Seattle, WA, USA) [3]. To prepare a catalase standard curve, 10 µL of different concentrations of H 2 O 2 (0, 120 µM, 240 µM and 400 µM) of each standard and 90 µL of assay buffer were added into separate wells of a 96-well plate and proceeded for detection. For samples, 10 µL of an equal amount of total protein isolated from Hyd-treated SRA-hLECs and mLECs were separately added into wells of a 96-well plate. To initiate the catalase reaction, 90 µL of freshly prepared 50 µM H 2 O 2 substrate (1 µL of the 4.8 mM H 2 O 2 with 95 µL assay buffer) was added to the blank, control, and sample wells. A 96-well plate was incubated for 30 min at room temperature. After quick mix, the plates were submitted for detection. For standard, samples, and controls detection, 100 µL of detection reagent (mixture of 102 µL assay buffer, 1 µL eye reagent, 1 µL HRP enzymes) was added to each reaction well of standard, samples, and controls. After 10 min incubation, optical density (O.D.) was measured at 570 nm with DTX 880 Multimode Detector (SpectraMAX Gemini, San Jose, CA, USA).

Isolation of Cytosol and Nuclear Protein Fractions
Cytosolic or nuclear fraction was isolated following our previously published methods [3,10,19,84]. Briefly, SRA-hLECs or mLECs were cultured in 100-mm plates overnight. Next-day cells were treated with Hyd for 24 h. For cytosolic fraction, cells were washed thrice with chilled phosphate-buffered saline (pH 7.4) and collected by centrifugation. The pellet obtained was suspended in 5 pellet volumes of cytoplasmic extraction buffer (10 mM HEPES (adjusted pH at 7.9), 0.1 mM EDTA, 10 mM KCl, 0.4% (v/v) Nonidet P-40, 1 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor). After a brief incubation on ice, it was centrifuged at 10,000 rpm for 10 min. Supernatant comprising the cytosolic fraction was transferred to a fresh tube. The pellet obtained was lysed in nuclear extract buffer (20 mM HEPES (adjusted pH at 7.9), 1 mM EDTA, 0.4 M NaCl, 10% (v/v) glycerol, 1 mM DTT, 0.5 mM PMSF and protease inhibitor) and subjected to continuous vortexing at 4 • C for 2 h to obtain the nuclear protein fraction. Finally, the extract was spun at 14,000 rpm for 15 min to pellet the nuclei. After centrifugation, the nuclear extract was aliquoted, and aliquots were stored at −80 • C. Protein was estimated according to the Bradford protein assay method, and the extract was used for the present study.

Determination of Nrf2-DNA (ARE) Binding Activity
The nuclear fraction was isolated, as noted above in Section 2.14. Nrf2-DNA binding activity was measured using kit TransAM Nrf2 Transcription Factor Assay Kit (Cat. No. 50296, Active motif, Carlsbad, CA, USA) and as described earlier [2,10,31]. Nuclear extract (10 µg) from Hyd-treated or untreated SRA-hLECs and mLECs were added to a well coated with oligonucleotides containing ARE (antioxidant response element) sequences, or it is mutated at ARE consensus binding sequences to each sample well. For blanks, 10 µL of complete lysis buffer was used. The controls and samples wells were incubated at room temperature (RT) for 1 h. The 100 µL primary antibody (1:1000 in binding buffer) was added after washing the wells and incubated at RT for 1h. After three washings, 100 µL anti-rabbit HRP conjugated antibody (1:1000 dilution) was added and further incubated for 1h at RT. By the addition of 100 µL of developing solution to the wells, the reaction was developed. The reaction was stopped by adding 100 µL of stop solution, and a reading was taken at O.D. 450 nm using a microplate reader (DTX 880, Multimode Detector, Molecular device, San Jose, CA, USA).

Chromatin Immunoprecipitation (ChIP) Assay (In Vivo DNA Binding Assay)
ChIP analysis was conducted by using the ChIP-IT ® Express kit (Cat. No. 53008; Active Motif, Carlsbad, CA, USA) and ChIP-IT ® qPCR analysis kit (Cat. No. 53029; Active Motif, Carlsbad, CA, USA) following manufacturer's protocol and as described previously [2,3,19,80,85]. Briefly, cells were processed following the protocol, and the fixation reactions were halted by addition of Glycine Fix-Stop solution. Cells were collected, and the cell pellet was disrupted in 1 mL ice-cold lysis buffer using a Dounce homogenizer. Released nuclei were resuspended in shearing buffer after centrifugation. Chromatin was then disrupted to obtain 200-300 bp using an ultrasonic cell disruptor (Microson, Farmingdale, NY, USA). Chromatin samples were used to assess DNA and transcription factor(s) enrichment by using control IgG, antibody specific to Nrf2 (ab62352, Abcam), to pull down the specific DNA fragment and then followed by RT-qPCR reaction. RT-qPCR was performed at 2 min at 95 • C, 15 s at 95 • C, 20 s at 58 • C, and 20 s at 72 • C for 40 cycles in 20 µL reaction volume (RT-qPCR). The results derived from RT-qPCR are presented as histograms.

Preparation of Prdx6 Promoter-Fused to Chloramphenicol Acetyltransferase (CAT) Reporter Vector
Human genomic DNA was used to construct pCAT-hPrdx6 promoter plasmid, as reported previously in our published protocol [2,10,31]. In brief, isolated genomic DNA was subjected to genomic-PCR with region specific primers, and the 5 -flanking regions, ranging from −918 to +30 bp, were isolated by using Advantage ® Genomic PCR Kit (Cat. No. 639103 and 639104, Clontech Laboratories, Inc., Mountain View, CA, USA). The PCR product obtained was amplified and verified by sequencing as described previously [19,74]. To engineer pCAT-linked hPrdx6 promoter plasmid, the DNA fragment (−918 to +30 bp) was ligated into basic pCAT vector (Promega, Madison, WI, USA) at the SacI and XhoI sites. Primers used were as follows: forward primer 5 -GACAGAGTTGAGCTCCACACAG-3 and reverse primer 5 -CACGTCCTCGAGAAGCAGAC-3 . Expression and purification of recombinant protein TAT-HA-Prdx6 was prepared as described in our published report [83,86]. Hyd and N-acetyl-L-cysteine (NAC) were purchased from Sigma (Catalog No. A9165, Millipore Sigma, Rockville, MD, USA).

Nrf2 Knock down Experiment
To generate Nrf2-depleted SRA-hLECs, Nrf2 was knocked down using human Nrf2 specific shRNA (Sh-Nrf2, sc-37030-SH, SantaCruz Biotechnology, Dallas, TX, USA) plasmid. A scrambled shRNA was used as negative control (Sh-Control, sc-108060, SantaCruz Biotechnology, Dallas, TX, USA). SRA-hLECs were transfected with control shRNA or Nrf2 shRNA by using Neon TM Transfection System. After 2 days of transfection, transfectants were used for the study or selected with selection marker puromycin to make stable cell lines. SRA-hLECs were transfected by using the Neon Transfection System (Invitrogen, Waltham, MA, USA) as described in our published protocol [10,19,31,81].

Lens Organ Culture and H 2 O 2 Treatment
Hyd (left eye) or buffered saline (7.2) (right eye) was topically instilled in 16 or 22-M-old mice eyes for 7 or 15 days once daily (please see illustration in Results Section). At day 8 or 16, lenses were carefully isolated and were cultured in 48-well culture plates containing 199 media without any supplements. After 24 h, the clear lenses were subjected to H 2 O 2 -induced oxidative stress. The lenses were observed routinely. After 48 h, oxidative stress lenses were photographed using Nikon SMZ 745T microscope fitted with a Nikon camera and a computer loaded with an analysis software program [51,54,83,87]. To examine the effects of Hyd, the lenses were processed to measure levels of ROS, lipid peroxidation, protein carbonyls and oxidative DNA damage, and Nrf2 and antioxidant genes, as described above and in the Results and Legends Sections.

Statistical Analysis
All experiments were performed with at least three independent biological replicas. Results are expressed as the average mean ± standard deviation (S.D.). Statistical analyses were performed by Sigma plot. For post-hoc statistical tests, we performed Bonferroni and Holm-Sidak test using one-way ANOVA, these tests can be used for both comparisons versus a control group (compare several treatment groups to a single control group) and pairwise comparisons (comparison of the mean responses to the different treatment groups). Statistical significance between control and treatment groups was assessed, and p value < 0.05 and <0.001 was considered statistically significant.

Hyd Efficaciously Rescued Lens Epithelial Cells under Oxidative Stress
In addition to antihypertensive property, Hyd has been shown to exert its cytoprotective and antiaging effects by activating the Nrf2/ARE antioxidant pathway in various cell types [21,22,88,89]. Therefore, at first, we confirmed whether Hyd protects LECs against oxidative stress as reported previously for other cell types. As shown in Figure 1A, we treated LECs with different concentrations of Hyd for 24 h. We found that 20 µM of Hyd was nontoxic, and treated LECs survived better with an increase in cell growth. Interestingly, a quantitation of ROS using H 2 -DCF-DA dye revealed that Hyd treatment itself does not augment intracellular ROS levels in the cells, but it reduces the levels of ROS when examined after 8 h of treatment. The results indicated that Hyd does not act as a pro-oxidant. Next, to determine the functional significance of Hyd treatment, we assessed the survival levels of Hyd-treated or untreated LECs (SRA-hLECs or mLECs) using MTS assay against oxidative stress, as shown in Figure 1C-F. As expected, Hyd-treated LECs conferred resistance against H 2 O 2 -induced oxidative stress. Furthermore, assessment by H 2 -DCF-DA dye showed a significant reduction of ROS in these treated cells compared to untreated ones, suggesting that Hyd protected the LECs by reducing intracellular ROS. Our experiments also indicated that 20 µM could be a more effective concentration in defending LECs from oxidative damage. It is worth noting that LECs treated with Hyd displayed a significant reduction in the ROS levels of the cells untreated with H 2 O 2, indicating that Hyd is not a pro-oxidant. Based on these observations, we surmised that Hyd might provide its protective effect by activating the antioxidant pathway in LECs as reported previously in other model systems. Thus, we focused our present study to understand Hyd-mediated molecular event(s), which might be involved in the activation of Nrf2/ARE antioxidant pathway and cellular protection. The cell viability of LECs under H2O2induced oxidative stress was significantly improved with Hyd treatment. Cultured SRA-hLECs and mLECs were exposed to H2O2 in absence or presence of Hyd for 24 h as indicated in Figure 1, and cell viability was performed using MTS assay. Histograms represent the mean ± S.D. value of three independent experiments. * p < 0.05; ** p < 0.001. (E,F) Hyd defends LECs against H2O2-induced cell death by reducing the ROS levels. Hyd-treated and untreated SRA-hLECs and mLECs were exposed to H2O2. Then, 16 h later, ROS levels were quantified with H2-DCF-DA dye method. Histogram represents the mean ± S.D. of three independent experiments. * p < 0.05; ** p < 0.001.

Hyd Efficaciously Blunted Oxidative Stress-Driven Aberrant Cellular Levels of Lipid Peroxidation and Rescued Oxidative Protein and DNA
After confirming that Hyd protects LECs by mitigating ROS accumulation and considering the impact of oxidative-induced generation of toxic products-mediated aberrant modifications of proteins and DNA damage, we tested whether the Hyd halts oxidative -induced oxidative stress was significantly improved with Hyd treatment. Cultured SRA-hLECs and mLECs were exposed to H 2 O 2 in absence or presence of Hyd for 24 h as indicated in Figure 1, and cell viability was performed using MTS assay. Histograms represent the mean ± S.D. value of three independent experiments. * p < 0.05; ** p < 0.001. (E,F) Hyd defends LECs against H 2 O 2 -induced cell death by reducing the ROS levels. Hyd-treated and untreated SRA-hLECs and mLECs were exposed to H 2 O 2 . Then, 16 h later, ROS levels were quantified with H 2 -DCF-DA dye method. Histogram represents the mean ± S.D. of three independent experiments. * p < 0.05; ** p < 0.001.

Hyd Efficaciously Blunted Oxidative Stress-Driven Aberrant Cellular Levels of Lipid Peroxidation and Rescued Oxidative Protein and DNA
After confirming that Hyd protects LECs by mitigating ROS accumulation and considering the impact of oxidative-induced generation of toxic products-mediated aberrant modifications of proteins and DNA damage, we tested whether the Hyd halts oxidative DNA damage or attenuates the levels of oxidative stress-induced formation of toxic adducts, such as MDA (malondialdehyde)/4-HNE (4-hydroxyalkenals) or protein carbonylation.
To achieve this end, we exposed Hyd-pretreated or untreated LECs to H 2 O 2 for 24 h, and the levels of MDA+4-HNE, protein carbonyls, as well as DNA damage, were quantified. We observed that H 2 O 2 -treated LECs displayed a significant increase in MDA plus 4-HNE levels (Figure 2A,B; gray vs. red bar), which were significantly reduced by Hyd treatment (Figure 2A; SRA-hLECs, red vs. light orange-red bars and Figure 2B; mLECs, red vs. bluered bars). Next, we quantified levels of carbonyl groups in Hyd-treated and untreated LECs using a 2,4-DNPH (dinitrophenylhydrazine) assay. We found that H 2 O 2 -induced increase in protein carbonyl content ( Figure 2C,D; gray vs. red bar) could be significantly reduced in Hyd-treated LECs ( Figure 2C; SRA-hLECs, red vs. light orange-red bars and Figure 2D; mLECs, red vs. blue-red bars). Furthermore, the formation of 8-OHdG is a ubiquitous marker of oxidative stress among various types of oxidative DNA damage. We, therefore, examined whether Hyd can reduce the levels of 8-OHdG formation in LECs facing oxidative stress. To this end, we found that LECs treated with H 2 O 2 alone displayed an increased amount of 8-OHdG ( Figure 2E,F; gray vs. red bar) compared to Hyd-treated LECs, showing a significant reduction in the levels of 8-OHdG ( Figure 2E; SRA-hLECs, red vs. light orange-red bars and Figure 2F; mLECs, red vs. blue-red bars). Thus, the results revealed that Hyd has the ability to protect DNA from oxidative stress. DNA damage or attenuates the levels of oxidative stress-induced formation of toxic adducts, such as MDA (malondialdehyde)/4-HNE (4-hydroxyalkenals) or protein carbonylation. To achieve this end, we exposed Hyd-pretreated or untreated LECs to H2O2 for 24 h, and the levels of MDA+4-HNE, protein carbonyls, as well as DNA damage, were quantified. We observed that H2O2-treated LECs displayed a significant increase in MDA plus 4-HNE levels (Figure 2A,B; gray vs. red bar), which were significantly reduced by Hyd treatment (Figure 2A; SRA-hLECs, red vs. light orange-red bars and Figure 2B; mLECs, red vs. blue-red bars). Next, we quantified levels of carbonyl groups in Hyd-treated and untreated LECs using a 2,4-DNPH (dinitrophenylhydrazine) assay. We found that H2O2induced increase in protein carbonyl content ( Figure 2C,D; gray vs. red bar) could be significantly reduced in Hyd-treated LECs ( Figure 2C; SRA-hLECs, red vs. light orange-red bars and Figure 2D; mLECs, red vs. blue-red bars). Furthermore, the formation of 8-OHdG is a ubiquitous marker of oxidative stress among various types of oxidative DNA damage. We, therefore, examined whether Hyd can reduce the levels of 8-OHdG formation in LECs facing oxidative stress. To this end, we found that LECs treated with H2O2 alone displayed an increased amount of 8-OHdG ( Figure 2E,F; gray vs. red bar) compared to Hyd-treated LECs, showing a significant reduction in the levels of 8-OHdG ( Figure 2E; SRA-hLECs, red vs. light orange-red bars and Figure 2F; mLECs, red vs. blue-red bars). Thus, the results revealed that Hyd has the ability to protect DNA from oxidative stress. Hyd-treated or untreated LECs were exposed to H2O2 and then processed for LPO assay. A significant reduction of MDA + 4-HAE levels were observed in Hyd-treated LECs exposed to H2O2 compared to untreated control. Histogram represents the mean ± S.D. of three independent experiments. ** p < 0.001. (C,D) Protein carbonyls were measured using 2-DNPH assay in LECs treated with Hyd or H2O2 or Hyd plus H2O2. Hyd treatment significantly lowered the protein carbonyl levels raised by H2O2 exposure. Histogram represents the mean ± S.D. of three independent experiments. ** p < 0.001. (E,F) Hyd treatment blunted oxidative stress-induced DNA damage in LECs. LECs exposed to H2O2 alone or cotreated with Hyd for 24 h were subjected to measure the 8-OHdG levels. Data showed that Hyd attenuated oxidative DNA damage in LECs induced by H2O2 exposure. Histogram represents the mean ± S.D. of three independent experiments. * p < 0.05; ** p < 0.001. Hyd-treated or untreated LECs were exposed to H 2 O 2 and then processed for LPO assay. A significant reduction of MDA + 4-HAE levels were observed in Hyd-treated LECs exposed to H 2 O 2 compared to untreated control. Histogram represents the mean ± S.D. of three independent experiments. ** p < 0.001. (

Hyd Activated the Nrf2/ARE-Driven Luciferase Activity in LECs
Previously, we have reported that Nrf2/ARE activation-dependent increased antioxidant gene transcription is directly related to expression and activities of antioxi-dants [2,10,19,21,31]. Given the results of Figure 1 showing the reduction of ROS levels in Hyd-treated cells, we surmised that Hyd's cytoprotective property should be attributed to activation of Nrf2/ARE transcription. To determine whether Hyd activates the Nrf2/ARE pathway, we performed Hyd-dependent Nrf2/ARE transactivation experiments. Toward this, we made use of pRBGP2-3x-ARE-LUC containing three ARE sites or its corresponding mutants pRBGP4-3xmut ARE-LUC (a kind gift from Hozumi Motohashi, Japan) [82]. As shown in Figure 3, SRA-hLECs or mLECs or different ages of primary hLECs were transfected with pRBGP2-3x-ARE-LUC or pRBGP4-3xmut ARE-LUC along with Renilla, pRL-TK vector (Promega, Madison, WI, USA). Transfectants treated with different concentrations of Hyd for 24 h were assayed for luciferase activity using Dual-Glo luciferase assay system (Promega, Madison, WI, USA). Data analysis disclosed that Hyd significantly up-regulated the Nrf2/ARE-mediated transcription in a dose-dependent manner, as shown in Figure 3A,B. However, in aging hLECs, 20 µM of Hyd could significantly reactivate the Nrf2/ARE-driven transcription under the same experimental conditions as shown in Figure 3C, suggesting that aging LECs required higher concentration of Hyd to reinforce Nrf2/ARE-mediated transcription. The results emphasized that the Nrf2/ARE-mediated signaling pathway can be reactivated by Hyd in aging LECs. Additionally, the results opened the door to design further experiments to define Hyd-mediated molecular mechanisms involved in the regulation of the antioxidant genes bearing ARE sites, such as Prdx6.

Hyd Activated the Nrf2/ARE-Driven Luciferase Activity in LECs
Previously, we have reported that Nrf2/ARE activation-dependent increased antio idant gene transcription is directly related to expression and activities of antioxidan [2,10,19,21,31]. Given the results of Figure 1 showing the reduction of ROS levels in Hy treated cells, we surmised that Hyd's cytoprotective property should be attributed to a tivation of Nrf2/ARE transcription. To determine whether Hyd activates the Nrf2/AR pathway, we performed Hyd-dependent Nrf2/ARE transactivation experiments. Towar this, we made use of pRBGP2-3x-ARE-LUC containing three ARE sites or its correspon ing mutants pRBGP4-3xmut ARE-LUC (a kind gift from Hozumi Motohashi, Japan) [82 As shown in Figure 3, SRA-hLECs or mLECs or different ages of primary hLECs we transfected with pRBGP2-3x-ARE-LUC or pRBGP4-3xmut ARE-LUC along with Renill pRL-TK vector (Promega, Madison, WI, USA). Transfectants treated with different co centrations of Hyd for 24 h were assayed for luciferase activity using Dual-Glo lucifera assay system (Promega, Madison, WI, USA). Data analysis disclosed that Hyd signi cantly up-regulated the Nrf2/ARE-mediated transcription in a dose-dependent manne as shown in Figure 3A,B. However, in aging hLECs, 20 µM of Hyd could significant reactivate the Nrf2/ARE-driven transcription under the same experimental conditions shown in Figure 3C, suggesting that aging LECs required higher concentration of Hyd reinforce Nrf2/ARE-mediated transcription. The results emphasized that the Nrf2/AR mediated signaling pathway can be reactivated by Hyd in aging LECs. Additionally, th results opened the door to design further experiments to define Hyd-mediated molecul mechanisms involved in the regulation of the antioxidant genes bearing ARE sites, suc as Prdx6.

Hyd Amplified the Antioxidants Expression via Activating Nrf2/ARE Pathways in LECs
The results derived from the above experiments implied that Hyd can reactivate Nrf2/ARE-mediated transcription in general. This argues that Hyd may exert its protective function by up-regulating Nrf2/antioxidant expression in LECs. To examine this, we used multiple LECs types, primary hLECs, SRA-hLECs, and mLECs. At first, we quantified Nrf2 and its target gene, Prdx6, at protein levels in Hyd treated/untreated LECs. Immunoblot analysis revealed that the cellular extracts of LECs treated with different concentrations of Hyd (0, 5, 10, and 20 µM for 24 h) had a concentration-dependent effect(s) on the increase in Nrf2 and Prdx6 protein expression ( Figure 4A,B). However, unexpectedly, 5 µM of Hyd could not induce Nrf2 and Prdx6 expression in mLECs as observed in hLECs, suggesting that mLECs may be less responsive due to their limited permeability to Hyd, or Hyd may have cell/tissue specificity. Furthermore, because Nrf2 up-regulates antioxidant genes as well as its own expression [90] through remodeling of transcriptional machinery, we next examined the relative expression levels of mRNA (transcripts) of Nrf2 ( Figure 4C) and its major target antioxidant genes such as Prdx6, NQO1, HO1, GCLC, and GCLM by RT-qPCR in SRA-hLECs and mLECs treated with Hyd ( Figure 4D-H). Quantitation of these genes by RT-qPCR demonstrated a dose-dependent increase in Nrf2 and its downstream target genes at 10 and 20 µM of Hyd.
transfected LECs were treated with different concentrations of Hyd for 24 h, as indicated. Relative LUC activity was observed. The data represent the mean ± S.D. from three independent experiments. ** p < 0.001. (C) Progressive decline in Nrf2/ARE-mediated transcription with advancing age was reactivated by Hyd treatment. Primary hLECs of different age groups were transiently transfected with pRBGP2-3xARE-LUC plasmid. At 14 h of post-transfection, hLECs were treated with Hyd (20 µM) for 24 h as indicated, and relative LUC activity was quantified. The data represent the mean ± S.D. values obtained from three independent experiments. ** p < 0.001.

Hyd Amplified the Antioxidants Expression via Activating Nrf2/ARE Pathways in LECs
The results derived from the above experiments implied that Hyd can reactivate Nrf2/ARE-mediated transcription in general. This argues that Hyd may exert its protective function by up-regulating Nrf2/antioxidant expression in LECs. To examine this, we used multiple LECs types, primary hLECs, SRA-hLECs, and mLECs. At first, we quantified Nrf2 and its target gene, Prdx6, at protein levels in Hyd treated/untreated LECs. Immunoblot analysis revealed that the cellular extracts of LECs treated with different concentrations of Hyd (0, 5, 10, and 20 µM for 24 h) had a concentration-dependent effect(s) on the increase in Nrf2 and Prdx6 protein expression ( Figure 4A,B). However, unexpectedly, 5 µM of Hyd could not induce Nrf2 and Prdx6 expression in mLECs as observed in hLECs, suggesting that mLECs may be less responsive due to their limited permeability to Hyd, or Hyd may have cell/tissue specificity. Furthermore, because Nrf2 up-regulates antioxidant genes as well as its own expression [90] through remodeling of transcriptional machinery, we next examined the relative expression levels of mRNA (transcripts) of Nrf2 ( Figure 4C) and its major target antioxidant genes such as Prdx6, NQO1, HO1, GCLC, and GCLM by RT-qPCR in SRA-hLECs and mLECs treated with Hyd ( Figure 4D-H). Quantitation of these genes by RT-qPCR demonstrated a dose-dependent increase in Nrf2 and its downstream target genes at 10 and 20 µM of Hyd.

Hyd Treatment Enhanced the Enzymatic Activities of Antioxidant Genes
Considering our previous report that antioxidant genes expression and activities were deteriorated during aging and that expression or activity can be reactivated by Metformin [3], we are, therefore, inclined to know whether, similar to Metformin, Hyd can amplify the enzymatic activities of antioxidants, such as phospholipase A2 (PLA2; Figure  5A, SRA-hLECs; Figure 5B, mLECs), glutathione (GSH) peroxidase ( Figure 5C, SRA-hLECs; Figure 5D, mLECs), superoxide dismutase (SOD; Figure 5E, SRA-hLECs; Figure  5F, mLECs), and catalase ( Figure 5G, SRA-hLECs; Figure 5H, mLECs) enzymatic activities. Figure 5 shows a significant increase in enzymatic activities of all antioxidants examined in Hyd-treated LECs. However, we anticipate that Hyd may alter the antioxidants' conformation, resulting in an increase in antioxidant enzymatic activities. Taken together, our data indicated that Hyd treatment significantly increased the Nrf2's target antioxidant gene's expression as well as their enzymatic activities, as shown in Figure 5.

Hyd Triggered the Nuclear Accumulation of Nrf2 and Augmented the Nrf2 Activity in Concentration-Dependent Fashion
Because nuclear accumulation of Nrf2 is a prerequisite for its transcriptional activity, we next pursued determining the effect of Hyd on the cellular status of Nrf2. To this end, LECs were treated with Hyd for 24 h. Immunoblotting of nuclear and cytosolic fractions with antibody specific to Nrf2 showed that nuclear accumulation of Nrf2 was increased in Hyd-treated LECs, and accumulation was increased at 20 µM of Hyd ( Figure 6A,B, lower panel). However, unexpectedly, we observed a very small change (not significant change) in the abundance of Nrf2 in the cytosolic fraction of LECs. Having observed that Hyd facilitates Nrf2's nuclear accumulation, next we asked whether Hyd-induced increased enrichment of Nrf2 is functionally active. To examine this, we performed the Nrf2 transactivation assay. Equal amounts of nuclear fraction isolated from SRA-hLECs ( Figure  6C) and mLECs ( Figure 6D) were used to assess the Nrf2's activity by using Trans-Nrf2 transcription factor assay kit (Active Motif). Data of Figure 6C,D demonstrated that the DNA binding activity of the Nrf2 was increased with the increase in Hyd concentration,

Hyd Treatment Enhanced the Enzymatic Activities of Antioxidant Genes
Considering our previous report that antioxidant genes expression and activities were deteriorated during aging and that expression or activity can be reactivated by Metformin [3], we are, therefore, inclined to know whether, similar to Metformin, Hyd can amplify the enzymatic activities of antioxidants, such as phospholipase A 2 (PLA 2 ; Figure 5A, SRA-hLECs; Figure 5B, mLECs), glutathione (GSH) peroxidase ( Figure 5C, SRA-hLECs; Figure 5D, mLECs), superoxide dismutase (SOD; Figure 5E, SRA-hLECs; Figure 5F, mLECs), and catalase ( Figure 5G, SRA-hLECs; Figure 5H, mLECs) enzymatic activities. Figure 5 shows a significant increase in enzymatic activities of all antioxidants examined in Hyd-treated LECs. However, we anticipate that Hyd may alter the antioxidants' conformation, resulting in an increase in antioxidant enzymatic activities. Taken together, our data indicated that Hyd treatment significantly increased the Nrf2's target antioxidant gene's expression as well as their enzymatic activities, as shown in Figure 5. suggesting that accumulated Nrf2 in the nucleus was functionally active, and Hyd may act via activating the Nrf2/ARE pathway by enhancing Nrf2's nuclear accumulation.

Hyd Triggered the Nuclear Accumulation of Nrf2 and Augmented the Nrf2 Activity in Concentration-Dependent Fashion
Because nuclear accumulation of Nrf2 is a prerequisite for its transcriptional activity, we next pursued determining the effect of Hyd on the cellular status of Nrf2. To this end, LECs were treated with Hyd for 24 h. Immunoblotting of nuclear and cytosolic fractions with antibody specific to Nrf2 showed that nuclear accumulation of Nrf2 was increased in Hyd-treated LECs, and accumulation was increased at 20 µM of Hyd ( Figure 6A,B, lower panel). However, unexpectedly, we observed a very small change (not significant change) in the abundance of Nrf2 in the cytosolic fraction of LECs. Having observed that Hyd facilitates Nrf2's nuclear accumulation, next we asked whether Hyd-induced increased enrichment of Nrf2 is functionally active. To examine this, we performed the Nrf2 transactivation assay. Equal amounts of nuclear fraction isolated from SRA-hLECs ( Figure 6C) and mLECs ( Figure 6D) were used to assess the Nrf2's activity by using Trans-Nrf2 transcription factor assay kit (Active Motif). Data of Figure 6C,D demonstrated that the DNA binding activity of the Nrf2 was increased with the increase in Hyd concentration, suggesting that accumulated Nrf2 in the nucleus was functionally active, and Hyd may act via activating the Nrf2/ARE pathway by enhancing Nrf2' s nuclear accumulation.

In Vivo DNA Binding and Transactivation Assays Disclosed That Hyd Activated Antioxidant Transcription via Nrf2/ARE Mechanism
To test the ability of Hyd in activating/reactivating Nrf2/ARE binding in aging/aged hLECs, we performed the chromatin immunoprecipitation (ChIP) assay to examine the enrichment of Nrf2 on ARE sequences of the hPrdx6 gene promoter. Primary hLECs of variable ages treated with Hyd for 24 h were subjected to ChIP assay with antibody specific to Nrf2. As shown in Figure 7A, the Nrf2 enrichment at ARE sequences in Prdx6 promoter was significantly increased in Hyd-treated, aging primary hLECs. However, younger hLECs were relatively more responsive to Hyd treatment compared to aging hLECs. This indicated that the aging hLECs retained Nrf2 activity and can be reactivated by means of Hyd treatment. Furthermore, to examine whether Hyd-induced increased Nrf2 binding to the Prdx6 promoter was functional, we carried out transcription assay with the Prdx6 promoter-linked to CAT reporter plasmid by transfecting hLECs. We found that Hyd could significantly amplify the promoter activity from its basal activity levels in all aging cells ( Figure 7B). As expected, younger cells were more responsive than the aged hLECs as observed in the Nrf2/ARE binding experiments ( Figure 7A). Taken together, our in vivo DNA binding assay coupled with the transactivation assay demonstrated that Hyd exerts protective response through the Nrf2/ARE antioxidant activation pathway in LECs as reported for other cell/tissue types. It was interesting to observe that Hyd does not induce ROS production as shown in Figure 1. Furthermore, to rule out the ROS-dependent hormetic response by Hyd, we utilized antioxidant treatment, such TAT-HA-Prdx6 protein [5,6,54] and N-acetyl cysteines (NAC), SRA-hLECs transfected with the pCAT-hPrdx6 plasmid. Then, 48 h later, the transfectants were treated with different concentrations of Hyd or cotreated with TAT-HA-Prdx6 protein (10 µg/mL) or NAC (5 mM) for 24 h. The ARE-driven CAT activity was increased in response to Hyd treatment in a dose-dependent manner ( Figure 7C; gray vs. light-orange bars), while reduced activity was detected in cells transduced with TAT-HA-Prdx6 protein ( Figure 7C; gray vs. green bars) or treated with NAC ( Figure 7C; gray vs. blue bars). Nonetheless, SRA-hLECs were treated with TAT-HA-Prdx6 protein or NAC along with different concentrations of Hyd displayed increased Prdx6 promoter activity. These results indicated that Hyd-mediated Nrf2 activation was ROS independent.  h. Cytosol and nuclear protein extract were immunoblotted with antibody specific to Nrf2. Tubulin and lamin B1 were used as loading control. Numbers under each protein band demonstrate densitometry value. (C,D) Hyd-treated LECs displayed significantly enhanced Nrf2 activity. Nuclear extracts from SRA-hLECs (C) and mLECs (D) treated with different amounts of Hyd for 24 h were analyzed for Nrf2/ARE interaction by ELISA (enzyme-linked immunosorbent assay). An equal amount of nuclear protein was processed and assayed for Nrf2 activity using a commercially available kit (Active motif). The data represent the mean ± S.D values from three independent experiments. p values were determined Hyd-treated vs. untreated control. ** p < 0.001.

In Vivo DNA Binding and Transactivation Assays Disclosed that Hyd Activated Antioxidant Transcription via Nrf2/ARE Mechanism
To test the ability of Hyd in activating/reactivating Nrf2/ARE binding in aging/aged hLECs, we performed the chromatin immunoprecipitation (ChIP) assay to examine the enrichment of Nrf2 on ARE sequences of the hPrdx6 gene promoter. Primary hLECs of Cytosol and nuclear protein extract were immunoblotted with antibody specific to Nrf2. Tubulin and lamin B1 were used as loading control. Numbers under each protein band demonstrate densitometry value. (C,D) Hyd-treated LECs displayed significantly enhanced Nrf2 activity. Nuclear extracts from SRA-hLECs (C) and mLECs (D) treated with different amounts of Hyd for 24 h were analyzed for Nrf2/ARE interaction by ELISA (enzyme-linked immunosorbent assay). An equal amount of nuclear protein was processed and assayed for Nrf2 activity using a commercially available kit (Active motif). The data represent the mean ± S.D values from three independent experiments. p values were determined Hyd-treated vs. untreated control. ** p < 0.001. or NAC (5 mM) for 24 h. The ARE-driven CAT activity was increased in response to Hyd treatment in a dose-dependent manner ( Figure 7C; gray vs. light-orange bars), while reduced activity was detected in cells transduced with TAT-HA-Prdx6 protein ( Figure 7C; gray vs. green bars) or treated with NAC ( Figure 7C; gray vs. blue bars). Nonetheless, SRA-hLECs were treated with TAT-HA-Prdx6 protein or NAC along with different concentrations of Hyd displayed increased Prdx6 promoter activity. These results indicated that Hyd-mediated Nrf2 activation was ROS independent. 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 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) Hydmediated 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.

Hyd Failed to Rescue the Nrf2-Deficient SRA-hLECs against H 2 O 2 -Induced Toxicity
To examine the impact of the Hyd-induced amplification of the Nrf2 antioxidant protective pathway, we carried out Hyd-mediated cytoprotective assay using Nrf2-depleted SRA-hLECs facing oxidative stress. To achieve this, we employed ShNrf2 to knock down Nrf2 and generated Nrf2-depleted SRA-hLECs. Expression assays analyses, Western blot ( Figure 8A), and RT-qPCR ( Figure 8B) revealed that Nrf2 could be successfully knocked down. Results of experimentation revealed that Hyd triggered a remarkable increase in the expression of Nrf2 protein and mRNA in Sh-Control SRA-hLECs, as shown in Figure 8A,B, compared to Sh-Nrf2 SRA-hLECs. To examine the Hyd-mediated Nrf2 antioxidant-dependent protective effects, Sh-Control (Nrf2 expressing, control) and Sh-Nrf2 SRA-hLECs (Nrf2-depleted SRA-hLECs) were exposed to H 2 O 2 in the absence or presence of Hyd for 24 h. Cell viability (MTS assay) assessment disclosed that Hyd was significantly ineffective in protecting Nrf2-depleted SRA-hLECs ( Figure 8C), while it could protect transfectants containing Sh-Control, (Nrf2 expressing hLECs), suggesting that Hyd exerts its protective effects specifically via the activation of the Nrf2/ARE antioxidant pathway.

Hyd Failed to Rescue the Nrf2-Deficient SRA-hLECs against H2O2-Induced Toxicity
To examine the impact of the Hyd-induced amplification of the Nrf2 antioxidant protective pathway, we carried out Hyd-mediated cytoprotective assay using Nrf2-depleted SRA-hLECs facing oxidative stress. To achieve this, we employed ShNrf2 to knock down Nrf2 and generated Nrf2-depleted SRA-hLECs. Expression assays analyses, Western blot ( Figure 8A), and RT-qPCR ( Figure 8B) revealed that Nrf2 could be successfully knocked down. Results of experimentation revealed that Hyd triggered a remarkable increase in the expression of Nrf2 protein and mRNA in Sh-Control SRA-hLECs, as shown in Figure  8A,B, compared to Sh-Nrf2 SRA-hLECs. To examine the Hyd-mediated Nrf2 antioxidantdependent protective effects, Sh-Control (Nrf2 expressing, control) and Sh-Nrf2 SRA-hLECs (Nrf2-depleted SRA-hLECs) were exposed to H2O2 in the absence or presence of Hyd for 24 h. Cell viability (MTS assay) assessment disclosed that Hyd was significantly ineffective in protecting Nrf2-depleted SRA-hLECs ( Figure 8C), while it could protect transfectants containing Sh-Control, (Nrf2 expressing hLECs), suggesting that Hyd exerts its protective effects specifically via the activation of the Nrf2/ARE antioxidant pathway.   Figure 8A,B exposed to H 2 O 2 in absence or presence of Hyd as indicated. Hydtreated transfectants containing shRNA specific to Nrf2 demonstrate a significant increase in cell death compared to ShControl counterpart as evidenced by MTS assay. All histogram data represent the mean ± S.D. values obtained from three independent experiments. ** p < 0.001, ns; denotes nonsignificant.

Hyd Amplified Nrf2 and Its Target Expression and Protected the Primary mLECs against H 2 O 2 -Induced Death by Mitigating ROS Accumulation
Based on the above experiments, we sought to determine whether basal gene expression levels in aging primary mLECs can be reactivated by Hyd treatment and lead to cytoprotection. To achieve this, primary mLECs isolated from 6 month (M) and 22 M old C57BL/6 male mouse were treated with Hyd for 24 h. Data analysis disclosed that Hyd significantly reduced the ROS production in primary aging/aged mLECs as shown in Figure 9A. In a parallel experiment, RT-qPCR assay of the transcript levels revealed that Hyd significantly reactivated expression of Nrf2 and its target gene, Prdx6 ( Figure 9B,C), demonstrating that Hyd can restore the dysregulated functions of the antioxidant pathway. However, mLECs isolated from the younger mouse were more responsive to Hyd treatment compared to the aging mouse. Next, we examined Hyd's protective ability in defending primary LECs against H 2 O 2 -induced oxidative stress. Cell viability assessment using MTS assay ( Figure 9D) showed enhanced viability of mLECs against oxidative stress in the presence of Hyd versus vehicle control ( Figure 9D). Our findings underscore that FDAapproved drug Hyd should be a promising viable molecule to restore the dysregulated antioxidant cytoprotective response in aging eye lenses/LECs. nonsignificant.

Hyd Amplified Nrf2 and Its Target Expression and Protected the Primary mLECs against H2O2-Induced Death by Mitigating ROS Accumulation
Based on the above experiments, we sought to determine whether basal gene expression levels in aging primary mLECs can be reactivated by Hyd treatment and lead to cytoprotection. To achieve this, primary mLECs isolated from 6 month (M) and 22 M old C57BL/6 male mouse were treated with Hyd for 24 h. Data analysis disclosed that Hyd significantly reduced the ROS production in primary aging/aged mLECs as shown in Figure 9A. In a parallel experiment, RT-qPCR assay of the transcript levels revealed that Hyd significantly reactivated expression of Nrf2 and its target gene, Prdx6 ( Figure 9B,C), demonstrating that Hyd can restore the dysregulated functions of the antioxidant pathway. However, mLECs isolated from the younger mouse were more responsive to Hyd treatment compared to the aging mouse. Next, we examined Hyd's protective ability in defending primary LECs against H2O2-induced oxidative stress. Cell viability assessment using MTS assay ( Figure 9D) showed enhanced viability of mLECs against oxidative stress in the presence of Hyd versus vehicle control ( Figure 9D). Our findings underscore that FDA-approved drug Hyd should be a promising viable molecule to restore the dysregulated antioxidant cytoprotective response in aging eye lenses/LECs.   Table 1. All histograms represent the mean ± S.D. values from three independent experiments. ** p < 0.001. (D) Hyd was efficacious in defending both younger and aged mLECs from H 2 O 2 -induced cellular damage. Hyd treated or untreated primary mLECs were exposed to H 2 O 2 , and cell viability was assessed after 24 h. The histogram data represent the mean ± S.D. values from three independent experiments. * p < 0.05; ** p < 0.001.

Hyd, When Instilled Topically in Mouse Eye, Enhanced Nrf2/ARE-Mediated Antioxidant Gene In Vivo
It is evident from the above experiments that Hyd treatment activates the Nrf2/AREmediated protective pathway in vitro; we envisage that the results obtained from in vitro studies can be appraised if they could be reproducible in vivo. It is worth mentioning that to identify the aging-related adverse signaling linked molecular targets and to determine the protective molecular mechanism of small molecules, eye lenses/LECs have been suggested to be the best biological model system. In this study, we selected Hyd to be delivered in the form of eye drops, as this route of application will be acceptable to the subjects. In addition, we reason that this will avoid administration orally or by other routes, which may cause adverse effects in persons who do not have hypertension. Furthermore, distinct biological ages of organs have been identified, and aging of these organs have diverse genetic architectures and occurs at different rates [91,92]. In this regard, eye lenses are found to be aged faster than other organs [93], suggesting that aging is organ specific [94]. Backed by these studies, we think that the drug evaluated for its therapeutic potential to block eye lens opacity can be used for other organs' health restoration. Thus, by using C57BL/6 mice, which have been widely used for aging research [95], herein we evaluated efficacy of Hyd's cytoprotective effect and prevention of lens opacity in vivo/ex vivo. We have used 16 M old C57BL/6 mice (equivalent to~52 years of human age) [96] for topical instillation of Hyd (25, 50, and 100 µM/ 5µL). The left eye of the mice was selected for topical instillation of physiological saline (pH 7.2), whereas the right eye received Hyd for 7 days or 15 days once daily as indicated in Figure 10A. On the eighth or 16th day, lenses were isolated and subjected to total RNA ( Figure 10B-G) isolation to examine the Nrf2 and its major phase II antioxidant genes. RT-qPCR analysis revealed a dose-dependent increase in Nrf2's target genes such as Prdx6, NQO1, HO1, GCLC, and GCLM at 25 and 50 µM concentrations of Hyd. However, we observed that 100 µM concentration of Hyd was not as effective as 50 µM in up-regulating the antioxidant pathway. Nrf2 mRNA expression was significantly high at 50 µM compared to 25 and 100 µM. The results revealed that Hyd could internalize in the eye lens and be biologically active as reported in other model systems. Since the RNA translation product is responsible for biological activities of genes, we next examined the cellular levels of Nrf2 and Prdx6 protein. Under the same conditions of experiments, Western blot analysis of total extract isolated from the Hyd-treated and untreated lenses of 16 M old C57BL/6 mice eyes, as shown in Figure 10A, demonstrated a relative increased abundance in Nrf2 and its target gene Prdx6, and the significant increased abundance in Nrf2/Prdx6 was dose-dependent ( Figure 10H). Taken together, data revealed that 50 µM was the optimum concentration for stimulating the Nrf2/ARE signaling pathway in vivo.

Topical Application of Hyd Reduced the ROS Generation and Augmented Nrf2 and Its Antioxidant Genes Expression in Aged Mouse Lenses In Vivo
Having observed that 50 µM of Hyd was optimal for Nrf2 antioxidant pathway activation and its biological function (Figures 8 and 10), we wanted to examine whether topical application of Hyd is biologically effective in aged mouse eye lenses (22 M old) (since aging-related changes or phenotypes can alter the effectivity of drug). To this end, at first, we quantified ROS levels using H 2 -DCF-DA dye method. Hyd-instilled eye lenses showed a significant reduction in ROS levels ( Figure 11B) when compared to control, salineinstilled eye lenses. Next, we examined whether Hyd activated the Nrf2-ARE-mediated antioxidant pathway in 22 M old mice (equivalent to~66 years of human age) [96]. Using the similar experimental protocol as noted in 16 M old mice (Figure 10), 22 M old mice received physiological saline (left eye) and 50 µM of Hyd (right eye) for 7 days daily. At the eighth day, total RNA and protein isolated from lenses were assessed for Nrf2 antioxidant genes expression. RT-qPCR and Western blot analyses revealed that Hyd reactivated and increased mRNA and protein expression of Nrf2 and its downstream antioxidant genes, as shown in Figures 10 and 11. Nevertheless, compared to 22 M old mice, 16 M old mice were more responsive to Hyd treatment. Taken together, data revealed that Hyd has the ability to revive the Nrf2 antioxidant pathway in aging lenses, when delivered topically (as eye drop) and, thereby, amplified the antioxidant defense even in aged lenses/LECs. These findings tempted us to perform further experimentation ( Figure 12) to investigate whether Hyd-treated eye lenses extend the healthspan of the lenses by preventing or delaying lens opacity against oxidative stress ex vivo. were used for the experiment. Hyd or buffered saline was instilled topically once daily for 7 days and 15 days as follows: Group 1, 2, and 3, vehicle control (left eye, buffered saline, pH 7.2) and Group 1, 2, and 3, Hyd treated (right eye, 25 µM/5 µL or 50 µM/5 µL or 100 µM/5 µL each eye, respectively). On day 8 or 16, mice were sacrificed, and total RNA (7 or 15-days treatment) and protein (15-days treatment) were isolated from the lenses. RT-qPCR (B-G) and immunoblotting (H) analyses were performed, and data were presented. (B-G) Hyd treatment significantly increased the expression of Nrf2 (B), and Nrf2 target antioxidant genes (C-G), as shown. The data presented as mean ± S.D. values obtained from three independent experiments. Control vs. Hyd-treated, * p < 0.05; ** p < 0.001. ns, not significant. (H) An equal amount of protein was loaded onto SDS-PAGE gel and immunoblotted with Nrf2, Prdx6, and β-actin antibodies, as shown. Nrf2 and Prdx6 protein bands were scanned and quantified, and the obtained values were normalized with corresponding β-actin band values. Relative densities were presented as histograms below the protein bands. * p < 0.05; ** p < 0.001. were used for the experiment. Hyd or buffered saline was instilled topically once daily for 7 days and 15 days as follows: Group 1, 2, and 3, vehicle control (left eye, buffered saline, pH 7.2) and Group 1, 2, and 3, Hyd treated (right eye, 25 µM/5 µL or 50 µM/5 µL or 100 µM/5 µL each eye, respectively). On day 8 or 16, mice were sacrificed, and total RNA (7 or 15-days treatment) and protein (15-days treatment) were isolated from the lenses. RT-qPCR (B-G) and immunoblotting (H) analyses were performed, and data were presented. (B-G) Hyd treatment significantly increased the expression of Nrf2 (B), and Nrf2 target antioxidant genes (C-G), as shown. The data presented as mean ± S.D. values obtained from three independent experiments. Control vs. Hyd-treated, * p < 0.05; ** p < 0.001. ns, not significant. (H) An equal amount of protein was loaded onto SDS-PAGE gel and immunoblotted with Nrf2, Prdx6, and β-actin antibodies, as shown. Nrf2 and Prdx6 protein bands were scanned and quantified, and the obtained values were normalized with corresponding β-actin band values. Relative densities were presented as histograms below the protein bands. * p < 0.05; ** p < 0.001. eighth day, total RNA and protein isolated from lenses were assessed for Nrf2 antioxidant genes expression. RT-qPCR and Western blot analyses revealed that Hyd reactivated and increased mRNA and protein expression of Nrf2 and its downstream antioxidant genes, as shown in Figures 10 and 11. Nevertheless, compared to 22 M old mice, 16 M old mice were more responsive to Hyd treatment. Taken together, data revealed that Hyd has the ability to revive the Nrf2 antioxidant pathway in aging lenses, when delivered topically (as eye drop) and, thereby, amplified the antioxidant defense even in aged lenses/LECs. These findings tempted us to perform further experimentation ( Figure 12) to investigate whether Hyd-treated eye lenses extend the healthspan of the lenses by preventing or delaying lens opacity against oxidative stress ex vivo.

Hyd's Topical Instillation in Mouse Eye Optimized H 2 O 2 -Induced ROS Levels and Extended Lenses Health Span by Preventing/Delaying Lens Opacity against Oxidative Stress Ex-Vivo
Hyd protects LECs against H 2 O 2 -induced oxidative damage in vitro; we intended to know whether topical application of Hyd in the eye lenses in vivo could delay onset of lens opacity against oxidative stress ex vivo. To examine this, we performed in vitro lens organ culture of eye lenses topically instilled with Hyd or physiological saline and were exposed to H 2 O 2, as shown in Figure 12. Physiological saline (left eye) and Hyd (right eye) were topically instilled in 16 M old C57BL/6 mice eyes for 7 days. At the eighth day, isolated lenses were cultured in vitro facing H 2 O 2 (100 µM)-induced stress. Then, 48 h later, the lenses were photographed and presented as shown in Figure 12B. In vivo Hydinstilled lenses showed a significant reduction in lens opacity (Figure 12(Bd)) compared to the physiological saline-instilled lenses (Figure 12(Bb)). Percentage of lens opacity was presented in the form of histograms ( Figure 12C). In the parallel experiments, Hyd-or physiological saline-instilled lenses in vivo were exposed to H 2 O 2 , and 48 h later the lenses were subjected to measure the levels of ROS ( Figure 12D), MDA ( Figure 12E), protein carbonyls ( Figure 12F), 8-OHdG ( Figure 12G), and mRNA ( Figure 12H). Data analysis revealed a significant reduction in ROS, MDA, protein carbonyls, and 8-OHdG levels and that levels of examined molecules were directly linked to Hyd-mediated increased expression of Nrf2 and its target genes such as Prdx6, NQO1, and HO1 ( Figure 12H; red bar vs. red-blue bar). Collectively, data demonstrated that Hyd could pass through the capsule of the aging lenses and internalize in LECs/lenses, as demonstrated by Hyd's biological activity. It was intriguing to observe that Hyd extended eye lens healthspan by delaying the onset of lens opacity by enhancing Nrf2/antioxidant defense response. Results from the current study provide the proof of concept that Hyd has the ability to delay/prevent age-related cataracts.

Discussion
Increased oxidative load due to dysregulation of the Nrf2/ARE pathway is positively correlated with many pathologies and age-related degenerative diseases [97][98][99]. Recent studies reveal that activation of the Nrf2 antioxidant pathway is of great interest as a promising biological target for the pharmacological intervention to treat or prevent oxidative/age-related degenerative diseases [58,[98][99][100][101]. However, clinical trial approaches on antioxidants, such as N-acetyl cysteine (NAC), Vitamin C, or E have not shown encouraging outcomes [88,102,103]. The failure of effectivity of the antioxidants can be related to their direct stoichiometric quenching of ROS. This demonstrates that cellular oxidants act through the activation of aberrant cellular signaling leading to deranged cell physiology of cells. Because Nrf2 has been found to play a pivotal role in cytoprotection against oxidative stress and aging pathobiology, it is now considered as an attractive therapeutic molecule for drug target [36,[104][105][106][107]. Several compounds that target Nrf2/ARE have been tested in many oxidative-related pathobiologies; currently, at least one Nrf2 activator, dimethyl fumarate, has been approved by the US Food and Drug Administration (FDA) [108][109][110]. This suggests that cells are well equipped with a very responsive and sensitive Nrf2/ARE defense pathway [2,3,10,19]. Recent substantial studies have documented the comprehensive effect of Nrf2 activators to control or treat a variety of pathologies. In the view of the fact that the Nrf2 antioxidant pathway has been found to decline with aging, leading to increased oxidative stress-induced accumulation of protein adducts, such as carbonylation, it is a cause for onset of several disorders [22,89,[111][112][113][114][115][116][117][118][119]. We, therefore, intended to rediscover the existing FDA approved drug(s) having scavenging carbonyls and Nrf2 antioxidant activation properties. In this scenario, we selected Hyd, a clinically proven "broad spectrum" scavenger of lipid-derived electrophiles (LDEs) and a potential activator of the Nrf2/ARE pathway [22,67,69,88,89].
The protective efficacy of Hyd has never been evaluated in eye lens/LECs, where dysregulation of Nrf2-ARE with oxidative stress amplification has been found to be a cause for onset of cataractogenesis [30,99,118]. In this study, we showed for the first time that Hyd is remarkably efficacious at protecting aging mouse or human LECs against oxidative stress (Figures 1 and 9). We observed that application of Hyd significantly reduced the ROS levels with decreased MDA/4-HNE accumulation and DNA damage in LECs ( Figure 2). These results indicated that Hyd has a great potential to rescue LECs against oxidative stress and could be as effective as reported for other cell types [21,22,88,89]. Our work also showed that Hyd treatment dramatically reduced oxidative stress-induced protein carbonyl levels ( Figure 2). Protein carbonylation has been found to be a major culprit for initiation/progression of several aging pathobiologies, including cataractogenesis [113][114][115]118,120]. Furthermore, environmental and cellular stresses lead to the accumulation of ROS in a variety of organs, including the lens, and thereby increases the risk of disease onset. Oxidative stress is an initiator of lipid peroxidation (LPO) propagation, leading to aldehydes formation, such as MDA and 4-HNE, during aging. Both are electrophilic and can adduct to nucleophilic proteins, yielding to protein cross-linking, denaturation, and aggregation [112,119,121]. Degenerative diseases involving protein damage by electrophilic toxicants and oxidative stress are extensive, such as cardiovascular and neurological disorders as well as blinding diseases, including lens opacity [65,122,123]. Recent studies of Hyd's protective effectivity in animals and invertebrate models showed that Hyd has life and healthspan extending properties [21,22,69,88,89]. As whole, studies demonstrate that in LECs, Hyd exerts its cytoprotective activity by abating ROS-induced oxidative DNA and protein damage (Figure 2), as reported earlier in other model systems [21,22,70,89,124].
Moreover, the essential question raised by our data is how Hyd delivery to cells/tissues results in ROS mitigation. To understand the molecular mechanism(s) involved in Hydmitigated ROS levels, our data revealed that Hyd suppresses intracellular ROS levels. This rules out the involvement of a ROS-dependent hormetic mechanism [2,3,10] ( Figure 1E,F; gray vs. light orange bar) in induction of the antioxidant pathway [2]. Furthermore, Nrf2 is an authentic stress-responsive factor that transcriptionally activates antioxidant genes by binding to ARE sequences of the gene promoters [125]. Nrf2 has now been defined as a multiorgan protector, and it also blocks progression of aging/oxidative stress-associated disorders, including cataracts [36,98,99,125]. Based on these studies, including our own published reports (and as described in the Introduction Section), we surmised that protective activity exerted by Hyd could be attributed to its activation of the Nrf2/ARE antioxidant pathway. Indeed, our data derived from transactivation experimentation with engineered promoter plasmid containing ARE sites [3,82] revealed that Hyd treatment significantly increased the Nrf2/ARE-driven promoter activity [126]. Notably, the increased transcription was corroborated with increased expression of Nrf2 and major phase II antioxidant genes (such as Prdx6, NQO1, HO1, GCLC, and GCLM) (Figures 3 and 4). Surprisingly, we observed that the antioxidants examined showed significantly increased enzymatic activity in Hyd-treated LECs ( Figure 5). In this case, one possible mechanism for the observed increase in the antioxidant enzymes activities can be related to conformational changes of antioxidants. Nonetheless, how Hyd promoted enzymatic activity of antioxidants warrants further investigation. Taken together, our data disclose that Hyd acts via activating the Nrf2mediated antioxidant pathway. In this support, we found that Hyd treatment increased nuclear accumulation of Nrf2 and augmentation of its target antioxidant genes expression but without affecting cellular levels of Keap1. This molecular phenomenon suggests that Hyd-mediated nuclear accumulation of Nrf2 could be independent of Keap1 activity. We observed that Nrf2 in the nuclear extract of Hyd-treated LECs was transcriptionally active (Figures 6 and 7). These findings of our study suggest that Hyd may activate Nrf2 at posttranslational levels and rule out any role of ROS in regulation of Nrf2/Keap1-mediated up-regulation of antioxidant genes. However, we firmly believe that further investigation is required to unveil the molecular mechanism involved in Hyd-mediated activation of the Nrf2 pathway in lens/LECs. Nevertheless, recently, Dehghan and colleagues [21] have reported that Hyd activation of Nrf2 is a PKA-mediated mechanism. Additionally, it has been reported that CAMP/PKA and SIRT1 are upstream activators of the Nrf2/ARE antioxidant pathway in fasting mice and human hepatocytes [127]. Hyd belongs to the non-electrophilic activator of Nrf2, and it is possible that Hyd can disrupt interaction(s) between Nrf2 and its regulatory interacting protein, Keap1 [128]. Another possibility is that Hyd can target other signaling networks known to be involved in Nrf2-dependent pathways, such as the GSK-3β-TrCP, synoviolin, NF-κB, Notch, or AMP kinase pathways [129]. Alternatively, it has also been suggested that it can be attributed to Hyd's scavenging property. Hyd-trapped LDEs adducts on Keap1 may offer the protein vulnerability to Cul3-mediated degradation and thus promote Nrf2 release, thereby allowing Nrf2 to accumulate in the nucleus [130]. Although these studies support plausible mechanism(s) of Hyd-mediated regulation of the Nrf2/ARE antioxidant activation pathway, further work is warranted to explain the many Hyd-mediated beneficial effects in context to mammalian cell health.
Recently, a comprehensive body of experimental work reveals that the protective activity of Hyd is mainly attributed to its ability to amplify the Nrf2-mediated antioxidant pathway and its carbonyl scavenging property [21,67,69,70,124]. Our works also showed that Hyd protects LECs by activating the Nrf2 antioxidant pathway as well as by reducing the levels of carbonyl adducts (Figures 2, 4 and 5). In addition, our results demonstrated that Hyd can correct and reactivate the antioxidant pathway in aging LECs, suggesting that Hyd is a perfect viable reagent to rescue aging LECs against different kinds of oxidative stress-induced program cell death. Furthermore, emerging studies have shown that in lens/LECs, aging and oxidative stress can induce various types of program cell death, such as apoptosis, pyroptosis, and ferroptosis [6,51,131,132]. It has been established that these program cell deaths are linked to the onset of cataractogenesis [133][134][135][136]. It is worth mentioning that in vitro experiments conducted with Hyd at concentrations of 20 to 50 µM appear to be pharmacologically and physiologically relevant. Nonetheless, several in vitro studies have been conducted with higher doses of Hyd [71,124]. It has been found that at concentrations of 50 to 500 µmol/L, Hyd can exert its beneficial biological effects in endothelial and fibroblast cells [71,126]. Moreover, we observed that Hyd has some extent of similarity with metformin or resveratrol in providing a beneficial effect by activating the Nrf2 antioxidant pathway and mimicking dietary restriction [56,137]. Nonetheless, there are apparent differences between them regarding mode of action in enhancing cell/organ healthspan as well as lifespan [138][139][140]. Interestingly, compared to metformin's doses required for activating beneficial signaling, Hyd exerts its beneficial effects at significantly lower concentrations, reducing the chances of contraindications and off-target shooting effects. This gives the advantage over other Nrf2 inductors, which are particularly critical for long-term treatment of the drug for clinical settings and application.
One of the most important findings of our present study is that topical instillation of Hyd in mouse eyes could efficiently up-regulate the Nrf2 antioxidant pathway in the lenses/LECs (Figure 10), resulting in prevention of lens opacity ex vivo. Data showed that treated lenses displayed reduced ROS levels with a significant reduction in the levels of MDA/4-HNE and protein carbonyl ( Figure 12). These findings are in agreement with previously published studies, documenting that Hyd exerts its protective activity through activation of the antioxidant pathway and carbonyl adducts scavenging [21,22,69,88,124]. Furthermore, assessment of oxidative stress-induced lens opacity in relation to age-related cataracts has shown that LPO-driven cross-linking of proteins and proteins carbonylation are linked to cataract formation [118]. The aberrant levels of protein carbonyl and MDA and 4-HNE adducts have been proposed to be a marker for the protein redox status and onset of lens opacity [141,142]. Collectively, the results from our study showed that the Nrf2/ARE pathway is a safeguard of the lens, and its reactivation by means of topical application of Hyd in eyes can maintain lens clarity against oxidative stress. The results revealed that topically instilled 50 µM of Hyd in mouse eyes were significantly cytoprotective and extended lens' healthspan. Backed with these findings, we concluded that Hyd is a potent protector of the eye lens via two mechanisms, (i) reinforcement of the Nrf2/ARE protective pathway and (ii) blunting of oxidative damage of protein and DNA. Furthermore, Hyd has strong affinity for several cells and tissues [71], and it is possible that Hyd can reach millimolar active concentrations to eye lenses via passing through the lens capsule (being a small molecule) as evidenced by its biological beneficial activity (activation of the Nrf2/ARE pathway and reduction in the protein adducts). Thus, accumulative studies derived from a variety of diverse animal and cell culture-based experimental systems, including our own results and previous reports, strongly support the potential for the repurposing of Hyd to prevent/block aging-related pathologies responsible for disease conditions. It has been shown that Hyd, whether it is delivered in vitro or in vivo/ex vivo, is highly efficacious to delay or prevent oxidative and aging-connected pathological states [21,22,67,69,70,88,89,124,[143][144][145][146]. We think that due to the health promoting functions of Nrf2 [30,62,98,99,147], identification of Nrf2-activating small molecular compounds with a carbonyl scavenging property, such as Hyd, has a high potential for use as a therapeutic molecule to combat aging-associated disorders.

Conclusions
In conclusion, we reported for the first time, using in vitro and in vivo/ex vivo experiments with multiple LECs derived from mouse and human lenses along with eye lenses, that Hyd regulates redox signaling in favor of cell/organ health by activating the Nrf2 antioxidant pathway. Our data disclosed that Hyd halts ROS amplification-mediated protein or DNA damage, as well as suppresses the levels of carbonyl products, implicated for the onset of aging pathologies. This beneficial molecular event resulted in increased cellular protection and eye lens healthspan by preventing/delaying lens opacity. Furthermore, we established that the mechanism(s) driving the changes in antioxidant gene expression was related to Hyd-induced nuclear accumulation of Nrf2 and, thereby, functional enrichment of Nrf2 at ARE sequences of the antioxidant gene promoters, such as Prdx6; however, it will be necessary to unravel the exact molecular events and pathways by which Hyd promotes and regulates these changes with the involved mechanistic function of Hyd on the Nrf2 nuclear localization and antioxidant pathway activation. As Hyd is a proposed antiaging compound and effective at lower concentrations, thereby avoiding off-targets compared to other known Nrf2 activators, it is tempting to propose that Hyd deserves to be considered as a viable therapeutic molecule to ablate oxidative-/aging-associated pathogenesis and disease conditions ( Figure 13).
Prdx6; however, it will be necessary to unravel the exact molecular events and pathways by which Hyd promotes and regulates these changes with the involved mechanistic function of Hyd on the Nrf2 nuclear localization and antioxidant pathway activation. As Hyd is a proposed antiaging compound and effective at lower concentrations, thereby avoiding off-targets compared to other known Nrf2 activators, it is tempting to propose that Hyd deserves to be considered as a viable therapeutic molecule to ablate oxidative-/agingassociated pathogenesis and disease conditions ( Figure 13). Increased ROS levels result in the oxidative-driven lipid, protein, DNA, and cellular damage and in turn lead to lens opacity/cataracts (a disease state). (Right panel) Illustration of the molecular mechanism(s) of Hyd-mediated restoration of dysregulated the Nrf2-dependent antioxidant protective pathway, thereby leading to cellular protection and extension of lens healthspan and prevention of lens opacity in response to oxidative stress, in vitro and in vivo/ex vivo. Hereby, we propose that blinding diseases related to oxidative stress or aging can be delayed or prevented by means of Nrf2 activators, such as Hyd.
Author Contributions: D.P.S. and B.C. designed and conceived the experiments. D.P.S., B.C. and E.K. performed the experiments and D.P.S., B.C. and E.K. conducted data analysis. B.C. and D.P.S. wrote the main manuscript and E.K. and R.R.K. reviewed and provided comments to make editing. D.P.S. supervised the work and provided funding. D.P.S., B.C., E.K. and R.R.K. read and provided Figure 13. (Left panel) Schematic illustration displaying the effects of oxidative stress/aging on the dysregulation of Nrf2 pathways and their consequences at molecular and cellular levels. Aging lenses/LECs or lenses/LECs facing oxidative stress bear reduced levels of antioxidant expression due to dysregulated Nrf2 antioxidant pathways, which cause increased ROS and oxidative damage. Increased ROS levels result in the oxidative-driven lipid, protein, DNA, and cellular damage and in turn lead to lens opacity/cataracts (a disease state). (Right panel) Illustration of the molecular mechanism(s) of Hyd-mediated restoration of dysregulated the Nrf2-dependent antioxidant protective pathway, thereby leading to cellular protection and extension of lens healthspan and prevention of lens opacity in response to oxidative stress, in vitro and in vivo/ex vivo. Hereby, we propose that blinding diseases related to oxidative stress or aging can be delayed or prevented by means of Nrf2 activators, such as Hyd.
Author Contributions: D.P.S. and B.C. designed and conceived the experiments. D.P.S., B.C. and E.K. performed the experiments and D.P.S., B.C. and E.K. conducted data analysis. B.C. and D.P.S. wrote the main manuscript and E.K. and R.R.K. reviewed and provided comments to make editing. D.P.S. supervised the work and provided funding. D.P.S., B.C., E.K. and R.R.K. read and provided inputs on the paper and approved the final version. All authors have read and agreed to the published version of the manuscript.