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The molecular chaperone sigma 1 receptor mediates rescue of retinal cone photoreceptor cells via modulation of NRF2
摘要： Sigma 1 receptor (Sig1R), a putative molecular chaperone, has emerged as a novel therapeutic target for retinal degenerative disease. Earlier studies showed that activation of Sig1R via the high-affinity ligand (+)-pentazocine ((+)-PTZ) induced profound rescue of cone photoreceptor cells in the rd10 mouse model of retinitis pigmentosa; however the mechanism of rescue is unknown. Improved cone function in (+)-PTZ-treated mice was accompanied by reduced oxidative stress and normalization of levels of NRF2, a transcription factor that activates antioxidant response elements (AREs) of hundreds of cytoprotective genes. Here, we tested the hypothesis that modulation of NRF2 is central to Sig1R-mediated cone rescue. Activation of Sig1R in 661W cone cells using (+)-PTZ induced dose-dependent increases in NRF2-ARE binding activity and NRF2 gene/protein expression, whereas silencing Sig1R significantly decreased NRF2 protein levels and increased oxidative stress, although (+)-PTZ did not disrupt NRF2-KEAP1 binding. In vivo studies were conducted to investigate whether, in the absence of NRF2, activation of Sig1R rescues cones. (+)-PTZ was administered systemically for several weeks to rd10/nrf2+/+ and rd10/nrf2-/- mice. Through post-natal day 42, cone function was significant in rd10/nrf2+/+, but minimal in rd10/nrf2-/- mice as indicated by electroretinographic recordings using natural noise stimuli, optical coherence tomography and retinal histological analyses. Immunodetection of cones was limited in (+)-PTZ-treated rd10/nrf2-/-, though considerable in (+)-PTZ-treated rd10/nrf2+/+mice. The data suggest that Sig1R-mediated cone rescue requires NRF2 and provide evidence for a previously-unrecognized relationship between these proteins.
关键词： retinitis pigmentosa,NRF2-KEAP1,retinal neuroprotection,retina,rd10 mouse,NRF2-Neh luciferase assay,oxidative stress
更新于2025-11-21 11:08:12
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Measuring the interaction of transcription factor Nrf2 with its negative regulator Keap1 in single live cells by an improved FRET/FLIM analysis
摘要： Transcription factor NF-E2 p45-related factor 2 (Nrf2) and its principal negative regulator, Kelch-like ECH-associated protein 1 (Keap1), comprise a molecular effector and sensor system that robustly responds to perturbations of the cellular redox homeostasis by orchestrating a comprehensive cytoprotective program. Under homeostatic conditions, Nrf2 is a short-lived protein, which is targeted for ubiquitination and proteasomal degradation. Upon encounter of electrophiles, oxidants or pro-inflammatory stimuli, the cysteine sensors in Keap1 are chemically modified, rendering Keap1 unable to target Nrf2 for degradation, and consequently leading to accumulation of the transcription factor and enhanced transcription of cytoprotective genes. Detailed understanding of the protein-protein interactions between Nrf2 and Keap1 has been achieved by use of various in vitro systems, but few assays are available to assess these interactions in the context of the living cell. We previously developed an imaging-based FLIM/FRET methodology to visualise and measure the interaction between Nrf2 and Keap1 in single cells. Here, our goal was to improve this methodology in order to increase throughput and precision, and decrease cell-to-cell variability. To eliminate the possibility of orientation bias, we incorporated a flexible linker between Keap1 and the FRET acceptor fluorescent protein tag. To ensure the correct image capture of Nrf2 fused to the FRET donor fluorescent protein tag, we matched the maturation time of the fluorescent tag to the half-life of the endogenous Nrf2, by using sfGFP as the FRET donor. Using a global binning approach increased the assay throughput, whereas including the measured Instrument Response Function in the analysis improved precision. The application of this methodology revealed a strong covariation of the results with the expression level of the acceptor. Taking the acceptor level into account circumvented cell-to-cell variability and enhanced sensitivity of the measurements of the Keap1-Nrf2 interaction in live cells.
关键词： FRET,live cell imaging,fluorescence lifetime,FLIM,sfGFP,protein-protein interaction,global binning,Keap1,Instrument Response Function,Nrf2
更新于2025-11-21 11:08:12
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SIRT6 protects retinal ganglion cells against hydrogen peroxide-induced apoptosis and oxidative stress by promoting Nrf2/ARE signaling via inhibition of Bach1
摘要： Oxidative stress-induced damage of retinal ganglion cells (RGCs) is a major contributor to retinal degenerative diseases, such as glaucoma. Sirtuin 6 (SIRT6) has emerged as a cytoprotective protein against various insults. However, whether SIRT6 exerts a protective effect against oxidative stress-damaged RGCs remains unknown. In this study, we aimed to investigate the potential role and regulatory mechanism of SIRT6 in hydrogen peroxide (H2O2)-induced oxidative damage of RGCs in vitro. We found that SIRT6 expression was significantly downregulated in RGCs with H2O2 treatment. Functional experiments showed that overexpression of SIRT6 improved survival and reduced apoptosis and the production of reactive oxygen species (ROS) in H2O2-treated RGCs. In contrast, SIRT6 knockdown had the opposite effect. Moreover, we found that SIRT6 overexpression promoted the nuclear accumulation of nuclear factor erythroid 2-related factor 2 (Nrf2) and increased the activity of antioxidant response element (ARE). In addition, we found that the promotional effect of SIRT6 on Nrf2/ARE signaling was associated with inhibition of BTB and CNC homology 1 (Bach1), an inhibitor of Nrf2. However, overexpression of Bach1 or inhibition of Nrf2/ARE signaling partially reversed the SIRT6-mediated protective effect. Taken together, these results demonstrate that SIRT6 protects RGCs from oxidative stress-induced damage by promoting the activation of Nrf2/ARE signaling via inhibition of Bach1, suggesting a potential role of SIRT6 in retinal degenerative diseases.
关键词： Nrf2,SIRT6,retinal ganglion cells,Bach1.
更新于2025-09-23 15:22:29
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Activation of Nrf2/HO-1 pathway protects retinal ganglion cells from a rat chronic ocular hypertension model of glaucoma
摘要： Objective The objective of this work was to find out the effects of nuclear factor erythroid 2-related factor/heme oxygenase-1 (Nrf2/HO-1) pathway on retinal ganglion cell (RGC) injury in glaucoma. Methods The chronic ocular hypertension (COH) rat models of glaucoma were constructed, and intraocular pressure (IOP) and RGC numbers were detected at different time points. Additionally, rats were divided into normal group (normal control rats), model group (COH model rats), and model + tBHQ group (COH model rats treated with Nrf activator, tBHQ). RGC apoptosis was detected by using TUNEL staining, and the expressions of Nrf2/HO-1 were detected by qRT-PCR and western blotting. Results COH model rats showed significant IOP elevation and the increased mRNA and protein expressions of Nrf2 and HO-1 from 1 to 6 weeks after operation, with the evidently decreased RGC numbers at 4 weeks and 6 weeks after operation (all P < 0.05). Besides, rats in the model group had increased apoptosis index (AI) of RGCs and the elevated mRNA and protein expressions of Nrf2/HO-1 with remarkably reduced RGC numbers when compared with normal control rats, but the model rats treated with tBHQ exhibited an apparent decrease in AI of RGCs, as well as remarkable increases in RGC numbers and the mRNA and protein expression of Nrf2/HO-1 (all P < 0.05). Conclusion Activation of Nrf2/HO-1 pathway significantly reduced the apoptosis and injury of RGCs in rats with chronic ocular hypertension (COH), thereby protecting RGCs in glaucoma, which could be a promising clinical target to prevent RGC degeneration in glaucoma.
关键词： Apoptosis,Nrf2/HO-1 pathway,Glaucoma,Retinal ganglion cells
更新于2025-09-23 15:22:29
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Nrf2 played an important role in radiation protection effect of low-level laser exposed on umbilical cord mesenchymal stem cell
摘要： To investigate the protective function of low-level laser irradiation (LLLI) against ionizing irradiation and explore the molecular mechanism of photomodulation of Nrf2 protein, the impact of LLLI (635 nm, 5.7 J/cm2) before 2 Gy gamma ray radiation of radio-sensitive tissue hematopoietic stem cells was evaluated. As a result, reduced levels of reactive oxygen species and increased expression of antioxidant enzymes were detected. Moreover, increased expression of Nrf2 was observed after LLLI, whereas brusatol pretreatment before LLLI abolished this effect. In vivo, transplantation of human umbilical cord mesenchymal stem cells (hUC-MSCs) was employed for therapy of hematopoietic function in an acute radiation sickness (H-ARS) mouse model, which was induced by 6-Gy ionizing irradiation; different hUC-MSC pretreatments including LLLI and Nrf2 RNAi were accounted for during experimental grouping. LLLI treatment of cells significantly increased the erythrocyte count and number of myelopoiesis clones (P < 0.05), but such improvements were reduced by Nrf2 RNAi pretreatment compared with cells transplanted without intervention. Therefore, LLLI may improve the radiation protection effect through molecular mechanisms related to the Nrf2 antioxidant pathway.
关键词： Radiation protection,Nrf2,LLLI
更新于2025-09-19 17:13:59
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Knockdown of FOXO6 inhibits high glucose–induced oxidative stress and apoptosis in retinal pigment epithelial cells
摘要： Oxidative stress and apoptosis in retinal pigment epithelium cells are involved in the pathogenesis of diabetic retinopathy (DR). Forkhead box class O 6 (FOXO6) is a member of the FOXO family that can regulate diabetes‐induced oxidative stress. However, the role of FOXO6 in DR has not been clarified. The aim of the present study was to investigate the effects of FOXO6 on high glucose (HG)‐induced oxidative stress and apoptosis in ARPE‐19 cells. The results showed that FOXO6 was overexpressed in clinical vitreous samples from DR patients and in HG‐induced ARPE‐19 cells. Knockdown of FOXO6 by small interfeing RNA targeting FOXO6 (si‐FOXO6) mitigated the HG‐induced the production of reactive oxygen species and malondialdehyde, as well as the inhibition of superoxide dismutase activity. Knockdown of FOXO6 reduced the rate of cell apoptosis in HG‐induced ARPE‐19 cells. The increase in bax expression and decrease in bcl‐2 expression caused by HG stimulation were reversed by si‐FOXO6 transfection. Furthermore, knockdown of FOXO6 enhanced the activation of Akt/Nrf2 pathway in HG‐stimulated ARPE‐19 cells. Taken together, suppression of FOXO6 protects ARPE‐19 cells from HG‐induced oxidative stress and apoptosis, which is in part mediated by the activation of Akt/Nrf2 pathway.
关键词： Akt/Nrf2 pathway,oxidative stress,retinal pigment epithelium cells,forkhead box class O 6,diabetic retinopathy,apoptosis
更新于2025-09-04 15:30:14