Enhanced Nrf2 Activation Research Articles

Oxidative and endoplasmic reticulum stress in diabetes-related hearing loss: Protective effects of thioredoxin

Diabetes mellitus (DM) induces complex physiological changes in the inner ear environment. This study investigates the roles of oxidative stress (OS) and endoplasmic reticulum stress (ERS) in diabetic hearing loss and explores the potential of thioredoxin (Trx) in regulating OS, ERS, and apoptosis-related factors to mitigate the progression of hearing impairment. We conducted auditory and serological assessments in 63 patients with type 2 diabetes and 30 healthy controls. Type 2 diabetes models were induced in wild-type and Trx transgenic (Tg) mice, with auditory brainstem response (ABR) used to evaluate hearing changes. Cochlear tissues were isolated to analyse markers of apoptosis, OS, and ERS. Both patients with diabetes and mouse models exhibited hearing loss, alongside increased serum levels of Trx1, TXNIP, and AOPP, indicating oxidative damage. H&E and succinate dehydrogenase (SDH) staining revealed varying degrees of hair cell loss from the base to the apex of the cochlea in diabetic mice, with decreased expression of the hair cell protein prestin gene. Notably, Tg mice showed significant delay in hearing loss progression. In vitro, advanced glycation end-products (AGEs) induced OS and ERS in cochlear-like HEI-OC1 cells, while Trx overexpression enhanced Nrf2 activity, alleviating AGE-induced cellular stress. In conclusion, Trx exhibits protective effects against diabetes-related hearing loss, potentially by enhancing Nrf2/HO-1/SOD2 function to reduce OS and ERS.

TBHQ mitigates fatty liver ischemia–reperfusion injury by activating Nrf2 to attenuate hepatocyte mitochondrial damage and macrophage STING activation

BackgroundLiver ischemia–reperfusion (IR) injury is an inevitable pathophysiological process in various liver surgeries. Previous studies have found that IR injury is exacerbated in fatty liver due to significant hepatocellular damage and macrophage inflammatory activation, though the underlying mechanisms are not fully understood. In this study, we aim to explore the role and mechanism of Nrf2 (Nuclear factor erythroid 2-related factor 2) signaling in regulating hepatocellular damage and macrophage immune response in fatty liver IR injury. MethodsThe study used high-fat diet-induced fatty liver mice to establish an IR model, alongside an in vitro co-culture system of primary hepatocytes and macrophages. This approach was used to examine mitochondrial dysfunction, oxidative stress, mitochondrial DNA (mtDNA) release, and activation of macrophage STING (Stimulator of interferon genes) signaling. We also conducted recovery verification using H-151 (a STING inhibitor) and tBHQ (an Nrf2 activator). ResultsCompared to the control group, mice on a high-fat diet demonstrated more severe liver IR injury, as evidenced by increased histological damage, elevated liver enzyme levels, and heightened inflammatory markers. The HFD group showed significant oxidative stress and mitochondrial dysfunction and damage post-IR, as indicated by elevated levels of ROS and lipid peroxidation markers, and decreased antioxidant enzyme activity. Elevated mtDNA release from hepatocytes post-IR activated macrophage STING signaling, worsening inflammation and liver damage. However, STING signaling inhibition with H-151 in vivo or employing STING knockout macrophages significantly reduced these injuries. In-depth mechanism studies have found that the transfer of Nrf2 protein into the nucleus of liver cells after IR in fatty liver is reduced. Pre-treatment with tBHQ ameliorated liver oxidative stress, mitochondrial damage and suppressed the macrophage STING signaling activation. ConclusionsOur study reveals a novel mechanism where the interaction between hepatocellular damage and macrophage inflammation intensifies liver IR injury in fatty liver. Enhancing Nrf2 activation to protect mitochondrial from oxidative stress damage and inhibiting macrophage STING signaling activation emerge as promising strategies for clinical intervention in fatty liver IR injury.

LXA4 protected mice from renal ischemia/reperfusion injury by promoting IRG1/Nrf2 and IRAK-M-TRAF6 signal pathways

Excessive inflammatory response and increased oxidative stress play an essential role in the pathophysiology of ischemia/reperfusion (I/R)-induced acute kidney injury (IRI-AKI). Emerging evidence suggests that lipoxin A4 (LXA4), as an endogenous negative regulator in inflammation, can ameliorate several I/R injuries. However, the mechanisms and effects of LXA4 on IRI-AKI remain unknown. In this study, A bilateral renal I/R mouse model was used to evaluate the role of LXA4 in wild-type, IRG1 knockout, and IRAK-M knockout mice. Our results showed that LXA4, as well as 5-LOX and ALXR, were quickly induced, and subsequently decreased by renal I/R. LXA4 pretreatment improved renal I/R-induced renal function impairment and renal damage and inhibited inflammatory responses and oxidative stresses in mice kidneys. Notably, LXA4 inhibited I/R-induced the activation of TLR4 signal pathway including decreased phosphorylation of TAK1, p36, and p65, but did not affect TLR4 and p-IRAK-1. The analysis of transcriptomic sequencing data and immunoblotting suggested that innate immune signal molecules interleukin-1 receptor-associated kinase-M (IRAK-M) and immunoresponsive gene 1 (IRG1) might be the key targets of LXA4. Further, the knockout of IRG1 or IRAK-M abolished the beneficial effects of LXA4 on IRI-AKI. In addition, IRG1 deficiency reversed the up-regulation of IRAK-M by LXA4, while IRAK-M knockout had no impact on the IRG1 expression, indicating that IRAK-M is a downstream molecule of IRG1. Mechanistically, we found that LXA4-promoted IRG1-itaconate not only enhanced Nrf2 activation and increased HO-1 and NQO1, but also upregulated IRAK-M, which interacted with TRAF6 by competing with IRAK-1, resulting in deactivation of TLR4 downstream signal in IRI-AKI. These data suggested that LXA4 protected against IRI-AKI via promoting IRG1/Itaconate-Nrf2 and IRAK-M-TRAF6 signaling pathways, providing the rationale for a novel strategy for preventing and treating IRI-AKI.

Phloretin-induced STAT3 inhibition suppresses pancreatic cancer growth and progression via enhancing Nrf2 activity

BackgroundPancreatic ductal adenocarcinoma (PDAC) is a malignant pancreatic tumor charactered by a rapid progression and high lethal rate. Hyperactivation of STAT3 signaling exerts a vital effect on the growth and progression of PDAC. While dietary flavonoid phloretin has anti-inflammatory and antioxidant activities, it remains unclear whether phloretin has anti-tumor effects on PDAC. PurposeThe focus of the present study is to elucidate the effects of phloretin on PDAC and investigate its underlying molecular mechanisms. Study design and methodsEffect of phloretin were assessed in the pancreatic cancer cells (PCCs) by colony formation assay, real-time cell analysis, flow cytometry, Immunofluorescence staining, and cell migration assay. The expressions of mRNA and protein were respectively analyzed by quantitative PCR and Western blotting. A xenograft model was used to appraise the antitumor efficacy of phloretin. ResultsPhloretin treatment significantly restrained cell viability and metastasis, induced DNA injury and ROS accumulation, and triggered mitochondrial-dependent apoptosis in PCCs. Mechanistically, phloretin exhibits anti-tumor potential via inactivating STAT3 signaling and enhancing Nrf2 activity. STAT3 overexpression and Nrf2 silencing partially relieved phloretin-induced inhibition on cell growth and metastasis in PCCs. Phloretin remarkably blocked pancreatic tumor growth and metastasis in vivo. ConclusionsPhloretin suppresses pancreatic cancer growth and progression through inhibition of STAT3 mediated by enhancing Nrf2 activity. Phloretin may serve as a promising therapeutic agent for PDAC.

FOXP1 regulates oxidative stress, SIRT1 expression, and resistance to chemotherapies in acute myeloid leukemia cells.

Transcription factor Forkhead box P1 (FOXP1) belongs to the same protein family as the FOXOs that are well-known regulators of murine hematopoietic stem progenitor cell (HSPC) maintenance by dampening oxidative stress. FOXP1 and FOXOs can play opposite or similar roles depending on cell context; they can cross-regulate each other's expression. In a previous study, we have shown that FOXP1 contributes to normal human HSP and acute myeloid leukemia (AML) cell growth. Here we investigated the role of FOXP1 in HSPCs and AML cell oxidative stress defense in human context. FOXP1 expression level was associated with inferior survival outcome of cytogenetically normal (CN) AML patients. FOXP1 knockdown enhanced superoxide anion levels of human committed CD34+CD38+ but not stem cell-enriched CD34+CD38- HSPCs, and AML cells in vitro. It triggered enhanced NRF2 activity and increased cell oxidative stress. FOXP1 had no impact on FOXO1/3/4 expression in these cells; genetic and pharmacological inhibition of FOXOs did not change superoxide anion levels of human HSPCs and AML cells. Also, FOXP1 antioxidant activity was independent of superoxide dismutase (SOD)1-2 or catalase expression changes. Instead, FOXP1 upregulated expression of the stress sensor SIRT1 by stabilizing SIRT1 protein. FOXP1 loss sensitized AML cells to chemotherapy. Altogether, this study identified FOXP1 as a new safeguard against myeloid progenitor oxidative stress, which works independently of FOXOs but through SIRT1, and contributes to AML chemoresistance. It proposes FOXP1 expression/activity as a promising target to overcome drug-resistance of AML HSPCs.

CRISPR/Cas9-Based Screening of FDA-Approved Drugs for NRF2 Activation: A Novel Approach to Discover Therapeutics for Non-Alcoholic Fatty Liver Disease.

With the rising prevalence of obesity, non-alcoholic fatty liver disease (NAFLD) now affects 20-25% of the global population. NAFLD, a progressive condition associated with oxidative stress, can result in cirrhosis and liver cancer in 10% and 3% of patients suffering NAFLD, respectively. Therapeutic options are currently limited, emphasizing the need for novel treatments. In this study, we examined the potential of activating the transcription factor NRF2, a crucial player in combating oxidative stress, as an innovative approach to treating NAFLD. Utilizing a CRISPR/Cas9-engineered human HEK293T cell line, we were able to monitor the expression of heme oxygenase-1 (HMOX1), an NRF2 target, using a Nanoluc luciferase tag. Our model was validated using a known NRF2 activator, after which we screened 1200 FDA-approved drugs, unearthing six compounds (Disulfiram, Thiostrepton, Auranofin, Thimerosal, Halofantrine, and Vorinostat) that enhanced NRF2 activity and antioxidant response. These compounds demonstrated protective effects against oxidative stress induced by hydrogen peroxide and lipid droplets accumulation in vitro with hepatoma HUH-7 cells. Our study underscores the utility of CRISPR/Cas9 tagging with Nanoluc luciferase in identifying potential NRF2 activators, paving the way for potential NAFLD therapeutics.

Enhanced liquidity of p62 droplets mediated by Smurf1 links Nrf2 activation and autophagy

BackgroundMacro-autophagy/Autophagy is an evolutionarily well-conserved recycling process to maintain the balance through precise spatiotemporal regulation. However, the regulatory mechanisms of biomolecular condensates by the key adaptor protein p62 via liquid-liquid phase separation (LLPS) remain obscure.ResultsIn this study, we showed that E3 ligase Smurf1 enhanced Nrf2 activation and promoted autophagy by increasing p62 phase separation capability. Specifically, the Smurf1/p62 interaction improved the formation and material exchange of liquid droplets compared with p62 single puncta. Additionally, Smurf1 promoted the competitive binding of p62 with Keap1 to increase Nrf2 nuclear translocation in p62 Ser349 phosphorylation-dependent manner. Mechanistically, overexpressed Smurf1 increased the activation of mTORC1 (mechanistic target of rapamycin complex 1), in turn leading to p62 Ser349 phosphorylation. Nrf2 activation increased the mRNA levels of Smurf1, p62, and NBR1, further promoting the droplet liquidity to enhance oxidative stress response. Importantly, we showed that Smurf1 maintained cellular homeostasis by promoting cargo degradation through the p62/LC3 autophagic pathway.ConclusionsThese findings revealed the complex interconnected role among Smurf1, p62/Nrf2/NBR1, and p62/LC3 axis in determining Nrf2 activation and subsequent clearance of condensates through LLPS mechanism.

Is Nrf2 Behind Endogenous Neuroprotection of the Hippocampal CA2-4,DG Region?

The transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2) is the master regulator of genes known to be involved in antioxidant, and anti-inflammatory processes, metabolic regulation, and other cellular functions. Here, we also hypothesize a core role for it in endogenous neuroprotection, i.e., the natural adaptive mechanisms protecting the brain from ischemia–reperfusion (I/R) episode. An example of endogenous neuroprotection is ischemia-resistance of the hippocampal regions comprising the CA2, CA3, CA4 and dentate gyrus subfields (here abbreviated to CA2-4,DG) which can be contrasted with the ischemia-vulnerable CA1 region. In the work detailed here, we used a gerbil model of transient cerebral ischemia to examined Nrf2 activation in CA1 and CA2-4,DG, in a control group, and post I/R episode. Data obtained indicate enhanced Nrf2 activity in CA2-4,DG as compared with CA1 in the control, with this difference seen to persist even after I/R. While I/R does indeed cause further activation of Nrf2 in CA2-4,DG, it is associated with slight and transient activation in CA1. Sub-regional differences in Nrf2 activity correlate with immunoreactivity of Keap1 (an Nrf2 suppressor) and Nrf2 target proteins, including heme oxygenase 1, the catalytic and modulatory sub-units of glutamate-cysteine ligase, and glutathione peroxidase 1. Pharmacological Nrf2 activation by sulforaphane results in protection of CA1 after I/R episode. Our results therefore suggest that high Nrf2 activity in CA2-4,DG may guarantee resistance of this region to I/R, potentially explaining the differential sensitivities of the hippocampal regions.

BP5 alleviates endotoxemia-induced acute lung injury by activating Nrf2 via dual regulation of the Keap1-Nrf2 interaction and the Akt (Ser473)/GSK3β (Ser9)/Fyn pathway.

Oxidative stress and inflammation play a crucial role in the pathogenesis of acute lung injury (ALI). Previously, pentapeptide bursopentin (BP5, Cys-Lys-Arg-Val-Tyr) was reported to possess significant antioxidant activity and inhibit lipopolysaccharides (LPS)-induced NF-κB activation in vitro, whereas little is known about its effects in vivo. In this study, we explored the effects of BP5 on endotoxemia-induced ALI in mice and the underlying molecular mechanisms. Our studies revealed that BP5 markedly improved survival and effectively alleviated lung injury by reducing overoxidation and excessive inflammatory response in endotoxemia mice. In LPS-stimulated mouse primary macrophages and RAW 264.7cells, BP5 also exhibited antioxidant and anti-inflammatory properties by enhancing Nrf2 activation. Importantly, these beneficial effects were abolished by Nrf2 knockdown. To further elucidate the underlying mechanisms, we performed localized surface plasmon resonance (LSPR) assays, molecular docking, together with cell-based studies, and found that BP5 inhibited the Keap1-Nrf2 interaction to promote Nrf2 nuclear translocation and activation. Moreover, BP5-induced Nrf2 activation was shown to be accompanied by an increase in the phosphorylation of Akt (at Ser473) and GSK3β (at Ser9), and a decrease in Fyn nuclear accumulation both in vitro and in vivo. Pharmacologically inhibiting phosphorylation of Akt and GSK3β obviously enhanced Fyn nuclear accumulation in RAW 264.7cells, which partially attenuated the promoting effect of BP5 on Nrf2 nuclear accumulation and activation. Furthermore, In Nrf2-/- mice, the protective effects of BP5 on the endotoxemia-induced ALI in WT mice were largely vanished. Our findings indicated that BP5 effectively protected endotoxemia-induced ALI against oxidative stress and inflammatory response, which are largely dependent on activation of the Nrf2 pathway. Underlying mechanisms include dual regulation of the Keap-Nrf2 interaction and the Akt (Ser473)/GSK3β (Ser9)/Fyn pathway.

Cycloastragenol suppresses M1 and promotes M2 polarization in LPS-stimulated BV-2 cells and ischemic stroke mice

There are two distinct phenotypes of activated microglia, pro-inflammatory M1 and anti-inflammatory M2. Accumulating evidence indicates that shifting the microglial polarization from M1 to M2 is a potential strategy for the treatment of neuroinflammation-associated brain diseases, including ischemic stroke. Cycloastragenol (CAG) is a hydrolysis product of astragaloside IV, the major active component of Astragalus radix. We have previously demonstrated that CAG has anti-inflammatory effect in a mouse model of ischemic stroke. This study investigated the effect of CAG on the phenotype polarization of microglia in lipopolysaccharide (LPS)-stimulated BV-2 mouse microglial cells and ischemic stroke mice. In LPS-treated BV-2 cells, we found that CAG significantly reduced the expression of M1 markers, including pro-inflammatory cytokines and enzymes. In contrast, CAG promoted the expression of M2 markers, including anti-inflammatory cytokines and growth factor. In addition, CAG inhibited the activation of nuclear factor-κB (NF-κB) and enhanced the activation of nuclear factor E2-related factor 2 (Nrf2) and the expression of its downstream heme oxygenase-1 (HO-1). Furthermore, CAG also inhibited levels of M1 markers, promoted those of M2 markers, and enhanced Nrf2 activation and HO-1 expression in ischemic mouse brain. Importantly, the effect of CAG on M2 markers, but not M1 markers, was reversed by Nrf2 siRNA in LPS-stimulated BV-2 cells. Together, our results suggested that CAG promoted microglial M2 and suppressed M1 polarization through activating Nrf2 and inhibiting NF-κB, respectively, in LPS-stimulated BV-2 cells and ischemic mouse brain. CAG is a promising candidate for the treatment of neuroinflammation-related diseases, including ischemic stroke.

Sequestered SQSTM1/p62 crosstalk with Keap1/NRF2 axis in hPDLCs promotes oxidative stress injury induced by periodontitis

Periodontitis is a recognized multifactorial inflammatory chronic disease, however, the exact role of oxidative stress in the pathogenesis of periodontitis is undefined. This study aims to imply the mechanism of NRF2-regulated oxidative stress and inflammatory responses under periodontitis and explored the novelty therapeutic targets. We first demonstrate that redox imbalance caused by inhibited NRF2 signaling pathway is induced in periodontium during hypoxia and bacterial events. Then we propose that LPS from P. gingivalis and hypoxia stimuli could inhibit hPDLCs proliferation and GSH level, promote ROS production, lipid peroxidation level, and pro-inflammatory cytokines such as IL-6, TNF-α, and IL-17 level caused by the inhibited PI3K/AKT/mTOR pathway and sequential sequestered crosstalk between selective autophagy SQSTM1/p62 and Keap1/NRF2 axis accompanied by the reinforced NRF2 ubiquitination degradation and inactivated NRF2 nuclear translocation. Overexpression of NRF2 and SQSTM1 can protect hPDLCs from oxidative stress and inflammation exacerbation because of enhanced NRF2 activity. Further, the antioxidant and anti-inflammation potential of puerarin is verified in vitro and in experimental periodontitis in mice through diminishing above negative feedback loop mechanically. Altogether, we speculate that NRF2-mediated redox homeostasis is a profound candidate for one of the prominent roles in periodontitis pathogenesis and suggest puerarin as a promising therapeutic target.

Methyl eugenol protects the kidney from oxidative damage in mice by blocking the Nrf2 nuclear export signal through activation of the AMPK/GSK3β axis.

Disrupted redox homeostasis contributes to renal ischemia-reperfusion (IR) injury. Abundant natural products can activate nuclear factor erythroid-2-related factor 2 (Nrf2), thereby providing therapeutic benefits. Methyl eugenol (ME), an analog of the phenolic compound eugenol, has the ability to induce Nrf2 activity. In this study, we investigated the protective effects of ME against renal oxidative damage in vivo and in vitro. An IR-induced acute kidney injury (AKI) model was established in mice. ME (20 mg·kg-1·d-1, i.p.) was administered to mice on 5 consecutive days before IR surgery. We showed that ME administration significantly attenuated renal destruction, improved the survival rate, reduced excessive oxidative stress and inhibited mitochondrial lesions in AKI mice. We further demonstrated that ME administration significantly enhanced Nrf2 activity and increased the expression of downstream antioxidative molecules. Similar results were observed in vitro in hypoxia/reoxygenation (HR)-exposed proximal tubule epithelial cells following pretreatment with ME (40 μmol·L-1). In both renal oxidative damage models, ME induced Nrf2 nuclear retention in tubular cells. Using specific inhibitors (CC and DIF-3) and molecular docking, we demonstrated that ME bound to the binding pocket of AMPK with high affinity and activated the AMPK/GSK3β axis, which in turn blocked the Nrf2 nuclear export signal. In addition, ME alleviated the development of renal fibrosis induced by nonfatal IR, which is frequently encountered in the clinic. In conclusion, we demonstrate that ME modulates the AMPK/GSK3β axis to regulate the cytoplasmic-nuclear translocation of Nrf2, resulting in Nrf2 nuclear retention and thereby enhancing antioxidant target gene transcription that protects the kidney from oxidative damage.

Galangin Reverses H2O2-Induced Dermal Fibroblast Senescence via SIRT1-PGC-1α/Nrf2 Signaling.

UV radiation and H2O2 are the primary factors that cause skin aging. Both trigger oxidative stress and cellular aging. It has been reported that deacetylase silent information regulator 1 (SIRT1), a longevity gene, enhances activation of NF-E2-related factor-2 (Nrf2), as well as its downstream key antioxidant gene hemeoxygenase-1 (HO-1), to protect cells against oxidative damage by deacetylating the transcription coactivator PPARγ coactivator-1α (PGC-1α). Galangin, a flavonoid, possesses anti-oxidative and anti-inflammatory potential. In the present study, we applied Ultraviolet B/H2O2-induced human dermal fibroblast damage as an in vitro model and UVB-induced photoaging of C57BL/6J nude mice as an in vivo model to investigate the underlying dermo-protective mechanisms of galangin. Our results indicated that galangin treatment attenuates H2O2/UVB-induced cell viability reduction, dermal aging, and SIRT1/PGC-1α/Nrf2 signaling activation. Furthermore, galangin treatment enhanced Nrf2 activation and nuclear accumulation, in addition to inhibiting Nrf2 degradation. Interestingly, upregulation of antioxidant response element luciferase activity following galangin treatment indicated the transcriptional activation of Nrf2. However, knockdown of SIRT1, PGC-1α, or Nrf2 by siRNA reversed the antioxidant and anti-aging effects of galangin. In vivo evidence further showed that galangin treatment, at doses of 12 and 24 mg/kg on the dorsal skin cells of nude mice resulted in considerably reduced UVB-induced epidermal hyperplasia and skin senescence, and promoted SIRT1/PGC-1α/Nrf2 signaling. Furthermore, enhanced nuclear localization of Nrf2 was observed in galangin-treated mice following UVB irradiation. In conclusion, our data indicated that galangin exerts anti-photoaging and antioxidant effects by promoting SIRT1/PGC-1α/Nrf2 signaling. Therefore, galangin is a potentially promising agent for cosmetic skin care products against UV-induced skin aging.

Inhibition ofUSP15ameliorates high‐glucose‐induced oxidative stress and inflammatory injury in podocytes through regulation of theKeap1/Nrf2signaling

Ubiquitin-specific peptidase 15 (USP15) is implicated in the pathogenesis of numerous diseases. However, whether USP15 plays a role in diabetic nephropathy remains undetermined. This project was designed to determine the potential role of USP15 in mediating high glucose (HG)-induced podocyte injury, a key event in the pathogenesis of diabetic nephropathy. We found that USP15 levels were elevated in podocytes after HG stimulation. Inhibition of USP15 led to decreases in HG-evoked apoptosis, oxidative stress, and inflammation in podocytes. Further investigation showed that inhibition of USP15 enhanced the activation of NF-E2-related factor 2 (Nrf2) and expression of Nrf2 target genes in HG-simulated podocytes. Moreover, depletion of Kelch-like ECH-associated protein 1 (Keap1) diminished the regulatory effect of USP15 inhibition on Nrf2 activation. In addition, Nrf2 suppression reversed USP15-inhibition-induced protective effects in HG-injured podocytes. Taken together, these data indicate that USP15 inhibition protects podocytes from HG-induced injury by enhancing Nrf2 activation via Keap1.

Senescence marker protein 30 (SMP30) protects against high glucose-induced apoptosis, oxidative stress and inflammatory response in retinal ganglion cells by enhancing Nrf2 activation via regulation of Akt/GSK-3β pathway

Senescence marker protein 30 (SMP30) is an aging-related protein that participates in the regulation of tissue damage under various pathological conditions. However, the role of SMP30 in mediating high glucose (HG)-induced injury of retinal ganglion cells (RGCs) has not been fully determined. We found that SMP30 expression declined during HG stimulation in RGCs. Cellular functional studies showed that the up-regulation of SMP30 dramatically prohibited HG-evoked apoptosis, oxidative stress and inflammatory response in RGCs. Mechanism research reported that SMP30 overexpression led to the enhancement of nuclear factor erythroid 2-related factor (Nrf2) activation in HG-stimulated RGCs. Moreover, SMP30 overexpression enhanced the phosphorylation of Akt and glucogen synthase kinase-3β (GSK-3β), and the suppression of Akt markedly abolished SMP30-mediated Nrf2 activation in HG-stimulated RGCs. Additionally, the suppression of Nrf2 substantially reversed SMP30-overexpression-induced anti-HG injury effects in RGCs. Overall, these findings suggest that SMP30 protects against HG injury of RGCs by potentiating Nrf2 through regulation of the Akt/GSK-3β pathway. Our work underscores that SMP30/Akt/GSK-3β/Nrf2 may exert a vital role in mediating the injury of RGCs during diabetic retinopathy.

Tripartite motif-containing protein 16 protects against myocardial ischemia/reperfusion injury by affecting the Keap1/Nrf2 axis.

Tripartite motif-containing protein (TRIM16) is a newly identified oxidative-stress-responsive protein. Oxidative stress is a hallmark of myocardial ischemia/reperfusion (I/R) injury and contributes to the cardiac injury. To date, whether TRIM16 plays a role in mediating oxidative stress during myocardial I/R injury is undetermined. The work is devoted to evaluate the possible relevance of TRIM16 in myocardial I/R injury. TRIM16 induction by myocardial hypoxia/reoxygenation (H/R) injury in vitro or myocardial I/R injury in vivo was observed. TRIM16 overexpression alleviated H/R-induced injury of rat cardiomyocytes. TRIM16 overexpression markedly attenuated cardiac injury, infarct size, and myocardial apoptosis induced by myocardial I/R injury. Further research revealed that TRIM16 was capable of enhancing Nrf2 activation via the regulation of Keap1. The inhibition of Nrf2 diminished TRIM16-overexpression-mediated cardioprotective effects. Overall, this work demonstrates that TRIM16 protects against myocardial I/R injury via affecting the Keap1/Nrf2 axis. This work offers new insights into the molecular mechanism underlying myocardial I/R injury and proposes TRIM16 as an attractive candidate target for cardioprotection.

Ferulic acid inhibits LPS-induced apoptosis in bovine mammary epithelial cells by regulating the NF-\u03baB and Nrf2 signalling pathways to restore mitochondrial dynamics and ROS generation

In bovine mammary epithelial cells (BMECs), a cascade of inflammatory reactions induced by lipopolysaccharide (LPS) has been shown to result in cell injury and apoptosis. The present study aims to reveal the protective effect of ferulic acid (FA) on LPS-induced BMEC apoptosis and explore its potential molecular mechanisms. First, we showed that FA had low cytotoxicity to BMECs and significantly decreased cell apoptosis and the proinflammatory response induced by LPS. Next, FA blocked LPS-induced oxidative stress by restoring the balance of the redox state and inhibiting mitochondrial dysfunction, the main contributor to LPS-induced apoptosis and ROS generation. Furthermore, the relief of inflammation and redox disturbance in the FA preconditioning group were accompanied by weaker NF-κB activation, enhanced Nrf2 activation and maintained cell viability compared to the LPS group. When BMECs were treated with FA alone, we observed that Nrf2 activation was induced before the inhibition of NF-κB activation and that the Keap1–Nrf2 relationship was disturbed. We concluded that FA prevented LPS-induced BMEC apoptosis by reversing the dominant relationship between NF-κB and Nrf2.

Combinations of Phytochemicals More Efficiently than Single Components Activate Nrf2 and Induce the Expression of Antioxidant Enzymes in Pancreatic Cancer Cells

Cancer prevention particularly related to aging can be improved by the use of phytochemicals combinations. In this study, we evaluated the effect of phenethyl isothiocyanate (PEITC), xanthohumol (XAN), indole-3-carbinol (I3C), and resveratrol (RES) and their combinations on the Nrf2 signaling pathway. Human pancreatic cancer cells MIA-Pa-Ca-2 were treated with the phytochemicals alone or their equimolar mixture for 24 h and activation of Nrf2 and expression of its target genes were evaluated. Phytochemicals alone enhanced Nrf2 activation and expression, but their combinations were more efficient. The mixture of XAN and PEITC was found to be the most potent modulator of the Nrf2 pathway. Moreover, increased levels of P-Nrf2 and P-JNK and decreased level of P-GSK-3β suggested possible activation of Nrf2 through modulation of these kinases. The combinations of XAN with PEITC and RES with PEITC increased mostly the expression of SOD, GSTP, CAT, and GPx. XAN and PEITC mixture induced the cell cycle arrest in G0/G1 phase and increased apoptotic and autophagy markers. These results indicate that combinations of phytochemicals resembling that occurring in natural diets may efficiently modulate the signaling pathways, which proper function is important for pancreatic cancer prophylaxis or improving the results of conventional therapy.

Glutaredoxin 1 protects neurons from oxygen-glucose deprivation/reoxygenation (OGD/R)-induced apoptosis and oxidative stress via the modulation of GSK-3β/Nrf2 signaling.

Increasing evidence has indicated that glutaredoxin 1 (GRX1) is a potent antioxidant protein that promotes cell survival under conditions of oxidative stress. Oxidative stress-induced neuronal injury contributes to cerebral ischemia/reperfusion injury. However, the role of GRX1-mediated antioxidant defense against neuronal damage during cerebral ischemia/reperfusion injury has not been thoroughly investigated. Thus, the objective of this study was to evaluate whether GRX1 protects neurons against oxygen-glucose deprivation/reoxygenation (OGD/R)-evoked oxidative stress injury in an in vitro model of cerebral ischemia/reperfusion injury. Our data revealed that GRX1 was induced by OGD/R treatment in neurons. Functional assays indicated that loss of GRX1 exacerbated OGD/R-induced apoptosis and the generation of reactive oxygen species (ROS), while GRX1 up-regulation protected against OGD/R-evoked neuronal injury. Further investigation revealed that GRX1 promoted the nuclear expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and enhanced transcription of the Nrf2/antioxidant response element (ARE) in GOD/R-exposed neurons. Furthermore, GRX1 promoted the activation of Nrf2/ARE associated with the modulation of glycogen synthase kinase-3β (GSK-3β). GSK-3β inhibition blocked GRX1 knockdown-mediated suppression of Nrf2 activation. Notably, the suppression of Nrf2 partially reversed GRX1-mediated anti-oxidative stress injury in OGD/R-exposed neurons. In summary, these findings indicate that GRX1 protects neurons against OGD/R-induced oxidative stress injury by enhancing Nrf2 activation via the modulation of GSK-3β. Our study suggests that GRX1 is a potential neuroprotective protein that protects against cerebral ischemia/reperfusion injury.

The Vitamin D Receptor As A Therapeutic Target In Oxidative Stress And Nrf‐2 Signaling: Role Of Vitamin D And Urolithin A

Vitamin D is an essential nutrient, commonly acquired via dietary intake and/or from endogenous cutaneous synthesis in response to ultraviolet radiation. The biologically active form of vitamin D, 1,25-dihydroxyvitamin D (1,25D), binds to the vitamin D receptor (VDR) and stimulates formation of an active hetero-complex with the retinoid X receptor (RXR). This VDR-RXR heterodimer controls vitamin D-regulated genes in such target tissues as kidney and colon, modulates immune defenses, and controls cellular proliferation. VDR may also play a significant role in preventing oxidative damage, potentially delaying the aging process, and serving as an anti-carcinogenic mediator. We hypothesize that VDR may target genes encoding antioxidant enzymes which contain antioxidant-responsive elements (AREs) that act as binding sites for transcriptional regulators such as nuclear factor (erythroid-derived 2)-like 2 (Nrf-2). This study aims to investigate the influence of vitamin D-VDR signaling on Nrf-2 activity. In order to probe a possible molecular mechanism, an ARE-luciferase reporter plasmid was employed to measure Nrf-2 activity in human embryonic kidney cells (HEK-293) in the presence of 1,25D/VDR. Results indicate cells transfected with both Nrf-2 and VDR displayed Nrf-2 activity that was modulated in a 1,25D- and VDR-dependent manner; with low 1,25D enhancing Nrf-2 activity while higher concentrations inhibited Nrf-2. When treating cells with 1,25D and/or urolithin-A (UA), a nutraceutical hypothesized to cooperate with vitamin D, Nrf-2 activity was instead consistently upregulated. Moreover, in qPCR studies with Nrf-2 target genes GCLC and HMOX1, UA and 1,25D treatment resulted in similar enhancement and/or suppression of Nrf-2, consistent with the luciferase-based assays. Collectively, these results imply that VDR likely targets Nrf-2 genes indirectly perhaps by influencing the activity of Nrf-2 transfactors and/or by post-translational modification of Nrf-2 to either activate or suppress Nrf-2-directed gene regulation. The modulation of Nrf-2 activity by a VDR-mediated pathway identifies a possible regulatory role for vitamin D in anti-oxidation and establishes the significance of vitamin D to human senescence and aging.