Neuroprotective effect of peroxiredoxin 6 against hypoxia-induced retinal ganglion cell damage

Abstract Background The ability to respond to changes in the extra-intracellular environment is prerequisite for cell survival. Cellular responses to the environment include elevating defense systems, such as the antioxidant defense system. Hypoxia-evoked reactive oxygen species (ROS)-driven oxidative stress is an underlying mechanism of retinal ganglion cell (RGC) death that leads to blinding disorders. The protein peroxiredoxin 6 (PRDX6) plays a pleiotropic role in negatively regulating death signaling in response to stressors, and thereby stabilizes cellular homeostasis. Results We have shown that RGCs exposed to hypoxia (1%) or hypoxia mimetic cobalt chloride display reduced expression of PRDX6 with higher ROS expression and activation of NF-κB. These cells undergo apoptosis, while cells with over-expression of PRDX6 demonstrate resistance against hypoxia-driven RGC death. The RGCs exposed to hypoxia either with 1% oxygen or cobalt chloride (0-400 μM), revealed ~30%-70% apoptotic cell death after 48 and 72 h of exposure. Western analysis and real-time PCR showed elevated expression of PRDX6 during hypoxia at 24 h, while PRDX6 protein and mRNA expression declined from 48 h onwards following hypoxia exposure. Concomitant with this, RGCs showed increased ROS expression and activation of NF-κB with IkB phosphorylation/degradation, as examined with H2DCF-DA and transactivation assays. These hypoxia-induced adverse reactions could be reversed by over-expression of PRDX6. Conclusion Because an abundance of PRDX6 in cells was able to attenuate hypoxia-induced RGC death, the protein could possibly be developed as a novel therapeutic agent acting to postpone RGC injury and delay the progression of glaucoma and other disorders caused by the increased-ROS-generated death signaling related to hypoxia.Peer Reviewe

Oxidative Stress in Cancer

Contingent upon concentration, reactive oxygen species (ROS) influence cancer evolution in apparently contradictory ways, either initiating/stimulating tumorigenesis and supporting transformation/proliferation of cancer cells or causing cell death. To accommodate high ROS levels, tumor cells modify sulfur-based metabolism, NADPH generation, and the activity of antioxidant transcription factors. During initiation, genetic changes enable cell survival under high ROS levels by activating antioxidant transcription factors or increasing NADPH via the pentose phosphate pathway (PPP). During progression and metastasis, tumor cells adapt to oxidative stress by increasing NADPH in various ways, including activation of AMPK, the PPP, and reductive glutamine and folate metabolism

Aerodynamic Heating Analysis of Re-entry Space Capsule Using Computational Fluid Dynamics

The present study deals with solving two-dimensional Reynolds Averaged Navier-Stokes equations for the Fire II re-entry capsule using Computational Fluid Dynamics (CFD). The primary goal is to model the aero thermodynamic flow characteristics around the capsule and estimate the surface heat flux distribution. Mach number value of 15.16 is chosen as a free stream condition corresponding to an altitude of 50 km. Taking advantage of the symmetry, only a quarter portion of the geometry is considered to generate the volume mesh for the simulation. The numerical models and convergence techniques that are implemented by the CFD solver are thoroughly described. Special attention has been paid to validate the code. The value of shock stand-off distance obtained by means of benchmark empirical formulation is compared to the CFD analysis. An additional comparison between the simulated results and the approximated engineering correlations of convective stagnation point heat fluxes is made to ensure the validity of the obtained results. Overall, a satisfactory agreement is observed between the estimated values by engineering correlations and those predicted by the numerical solver

Clock Protein Bmal1 and Nrf2 Cooperatively Control Aging or Oxidative Response and Redox Homeostasis by Regulating Rhythmic Expression of Prdx6

Many disorders of aging, including blinding-diseases, are associated with deficiency of brain and muscle arnt-like protein 1 (Bmal1) and, thereby, dysregulation of antioxidant-defense pathway. However, knowledge is limited regarding the role of Bmal1 regulation of antioxidant-pathway in the eye lens/lens epithelial cells (LECs) at the molecular level. We found that, in aging human (h)LECs, a progressive decline of nuclear factor erythroid 2-related factor 2 (Nrf2)/ARE (antioxidant response element)-mediated antioxidant genes was connected to Bmal1-deficiency, leading to accumulation of reactive oxygen species (ROS) and cell-death. Bmal1-depletion disrupted Nrf2 and expression of its target antioxidant genes, like Peroxiredoxin 6 (Prdx6). DNA binding and transcription assays showed that Bmal1 controlled expression by direct binding to E-Box in Prdx6 promoter to regulate its transcription. Mutation at E-Box or ARE reduced promoter activity, while disruption of both sites diminished the activity, suggesting that both sites were required for peak Prdx6-transcription. As in aging hLECs, ROS accumulation was increased in Bmal1-deficient cells and the cells were vulnerable to death. Intriguingly, Bmal1/Nrf2/Prdx6 and PhaseII antioxidants showed rhythmic expression in mouse lenses in vivo and were reciprocally linked to ROS levels. We propose that Bmal1 is pivotal for regulating oxidative responses. Findings also reveal a circadian control of antioxidant-pathway, which is important in combating lens/LECs damage induced by aging or oxidative stress

Obligatory Role of AMPK Activation and Antioxidant Defense Pathway in the Regulatory Effects of Metformin on Cellular Protection and Prevention of Lens Opacity

Increasing levels of oxidative-stress due to deterioration of the Nrf2 (NFE2-related factor)/ARE (antioxidant response element) pathway is found to be a primary cause of aging pathobiology. Metformin having anti-aging effects can delay/halt aging-related diseases. Herein, using lens epithelial cell lines (LECs) of human (h) or mouse (m) and aging h/m primary LECs along with lenses as model systems, we demonstrated that Metformin could correct deteriorated Bmal1/Nrf2/ARE pathway by reviving AMPK-activation, and transcriptional activities of Bmal1/Nrf2, resulting in increased antioxidants enzymatic activity and expression of Phase II enzymes. This ensued reactive oxygen species (ROS) mitigation with cytoprotection and prevention of lens opacity in response to aging/oxidative stress. It was intriguing to observe that Metformin internalized lens/LECs and upregulated OCTs (Organic Cation Transporters). Mechanistically, we found that Metformin evoked AMPK activation-dependent increase of Bmal1, Nrf2, and antioxidants transcription by promoting direct E-Box and ARE binding of Bmal1 and Nrf2 to the promoters. Loss-of-function and disruption of E-Box/ARE identified that Metformin acted by increasing Bmal1/Nrf2-mediated antioxidant expression. Data showed that AMPK-activation was a requisite for Bmal1/Nrf2-antioxidants-mediated defense, as pharmacologically inactivating AMPK impeded the Metformin’s effect. Collectively, the results for the first-time shed light on the hitherto incompletely uncovered crosstalk between the AMPK and Bmal1/Nrf2/antioxidants mediated by Metformin for blunting oxidative/aging-linked pathobiology

Sulforaphane-Induced Klf9/Prdx6 Axis Acts as a Molecular Switch to Control Redox Signaling and Determines Fate of Cells

Sulforaphane (SFN), an activator of transcription factor Nrf2 (NFE2-related factor), modulates antioxidant defense by Nrf2-mediated regulation of antioxidant genes like Peroxiredoxin 6 (Prdx6) and affects cellular homeostasis. We previously observed that dose levels of SFN are crucial in determining life or death of lens epithelial cells (LECs). Herein, we demonstrated that higher doses of SFN (>6 μM) activated death signaling by overstimulation of Nrf2/ARE (antioxidant response element)-mediated Kruppel-like factor (Klf9) repression of Prdx6 expression, which increased reactive oxygen species (ROS) load and cell death. Mechanistically, Klf9 bound to its repressive Klf9 binding elements (RKBE; 5-CA/GCCC-3) in the Prdx6 promoter, and repressed Prdx6 transcription. Under the condition of higher dose of SFN, excessive Nrf2 abundance caused death signaling by enforcing Klf9 activation through ARE (5-RTGAYnnnGC-3) in Klf9 promoter that suppress antioxidant genes such as Prdx6 via a Klf9-dependent fashion. Klf9-depletion showed that Klf9 independently caused ROS reduction and subsequent cell survival, demonstrating that Klf9 upregulation caused cell death. Our work revealed the molecular mechanism of dose-dependent altered activity of SFN in LECs, and demonstrated that SFN activity was linked to levels of Nrf2/Klf9/Prdx6 axis. We proposed that in the development of therapeutic interventions for aging/oxidative disorders, combinations of Klf9-ShRNA and Nrf2 inducers may prove to be a promising strategy

Switching of Redox Signaling by Prdx6 Expression Decides Cellular Fate by Hormetic Phenomena Involving Nrf2 and Reactive Oxygen Species

Changes in intracellular reactive oxygen species (ROS) levels due to remodeling of antioxidant defense can affect the status of biological homeostasis in aging/oxidative stress. Peroxiredoxin 6 (Prdx6), an antioxidant gene downstream target for the Nrf2 pathway, plays a role in regulating ROS homeostasis. Using aging human (h) lens epithelial cells (LECs) or Prdx6-deficient (Prdx6−/−) mouse (m) LECs, here we showed that dichlorofluorescein (DCF) oxidation or H2O2 were strictly controlled by Prdx6. We observed that a moderate degree of oxidative stress augmented Nrf2-mediated Prdx6 expression, while higher doses of H2O2 (≥100 µM) caused a dramatic loss of Prdx6 expression, resulting in increased DCF oxidation and H2O2 amplification and cell death. Mechanistically, at increased oxidative stress, Nrf2 upregulated transcriptional factor Klf9, and that Klf9 bound to the promoter and repressed the Prdx6 gene. Similarly, cells overexpressing Klf9 displayed Klf9-dependent Prdx6 suppression and DCF oxidation with H2O2 amplification, while ShKlf9 reversed the process. Our data revealed that H2O2 and DCF oxidation levels play a hormetical role, and the Nrf2-Klf9-Prdx6 pathway is pivotal for the phenomena under the conditions of oxidative load/aging. On the whole, the results demonstrate that oxidative hormetical response is essentially based on levels of oxidative triggering and the status of Klf9-Prdx6 pathway activation; thus, Klf9 can be considered as a therapeutic target for hormetic shifting of cellular defense to improve protective resilience to oxidative stress

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