Nrf2 Transcriptional Activity Research Articles

M6A demethylation of FOSL1 mRNA protects hepatoma cells against necrosis under glucose deprivation.

Stress-adaptive mechanisms enabling cancer cells to survive under glucose deprivation remain elusive. N6-methyladenosine (m6A) modification plays important roles in determining cancer cell fate and cellular stress response to nutrient deficiency. However, whether m6A modification functions in the regulation of cancer cell survival under glucose deprivation is unknown. Here, we found that glucose deprivation reduced m6A modification levels. Increasing m6A modification resulted in increased hepatoma cell necrosis under glucose deprivation, whereas decreasing m6A modification had an opposite effect. Integrated m6A-seq and RNA-seq revealed potential targets of m6A modification under glucose deprivation, including the transcription factor FOSL1; further, glucose deprivation upregulated FOSL1 by inhibiting FOSL1 mRNA decay in an m6A-YTHDF2-dependent manner through reducing m6A modification in its exon1 and 5'-UTR regions. Functionally, FOSL1 protected hepatoma cells against glucose deprivation-induced necrosis in vitro and in vivo. Mechanistically, FOSL1 transcriptionally repressed ATF3 by binding to its promoter. Meanwhile, ATF3 and MAFF interacted via their leucine zipper domains to form a heterodimer, which competed with NRF2 for binding to antioxidant response elements in the promoters of NRF2 target genes, thereby inhibiting their transcription. Consequently, FOSL1 reduced the formation of the ATF3-MAFF heterodimer, thereby enhancing NRF2 transcriptional activity and the antioxidant capacity of glucose-deprived-hepatoma cells. Thus, FOSL1 alleviated the necrosis-inducing effect of glucose deprivation-induced reactive oxygen species accumulation. Collectively, our study uncovers the protective role of m6A-FOSL1-ATF3 axis in hepatoma cell necrosis under glucose deprivation, and may provide new targets for cancer therapy.

Abstract LB169: Using a global reporter on transcript abundance to identify a selective inhibitor of Nrf2 signaling in KEAP1-mutant non small cell lung cancer

Abstract Loss-of-function mutations in Kelch-like ECH-associated protein 1 (KEAP1) are common in non-small cell lung cancer (NSLC) and result in particularly poor prognosis. Such mutations activate the transcription factor Nrf2, leading to aberrant expression of chemoprotective genes and rendering cells resistant to chemotherapy. We used our chemo-transcriptomic platform, termed GRETA, for Global Reporter on Transcript Abundance, to identify compounds that inhibit Nrf2 transcription factor activity in KEAP1-mutant NSCLC cell lines. After defining the transcriptomic signature imparted by constitutive activation of Nrf2 in a KEAP1 KO NCI-H1975 cell line, we generated over 10,000 transcriptomes to identify compounds that dampen the Nrf2-overactive transcriptional signature. One such compound, ARP-4316, displays selectivity for KEAP1-mutant cell lines, reduces nuclear Nrf2 protein levels, and reduces Nrf2 transcriptional activity in an ARE reporter assay. Furthermore, ARP-4316 synergizes with 5-fluorouracil in two KEAP1-mutant cell models, A549 and NCI-H2122, suggesting that ARP-4316 may sensitize KEAP1 mutant cell lines to chemotherapy by inhibiting Nrf2 transcriptional activity. Efforts to identify the cellular target and mechanism of action of ARP-4316 are underway. Citation Format: Timothy J. Read, Leah Damon, Reo Yoo, Carter Grochala, Cole Liechty, Christy Rhine, Jenna Rimel, David Simpson, Riley Lundblad, Casey Pham, Richard Steel, Joey Azofeifa. Using a global reporter on transcript abundance to identify a selective inhibitor of Nrf2 signaling in KEAP1-mutant non small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB169.

Mechanisms of PM10 Disruption of the Nrf2 Pathway in Cornea.

We have previously shown that PM10 exposure causes oxidative stress and reduces Nrf2 protein levels, and SKQ1 pre-treatment protects against this damage in human corneal epithelial cells (HCE-2). The current study focuses on uncovering the mechanisms underlying acute PM10 toxicity and SKQ1-mediated protection. HCE-2 were pre-treated with SKQ1 and then exposed to 100 μg/mL PM10. Cell viability, oxidative stress markers, programmed cell death, DNA damage, senescence markers, and pro-inflammatory cytokines were analyzed. Nrf2 cellular location and its transcriptional activity were determined. Effects of the Nrf2 inhibitor ML385 were similarly evaluated. Data showed that PM10 decreased cell viability, Nrf2 transcriptional activity, and mRNA levels of antioxidant enzymes, but increased p-PI3K, p-NFκB, COX-2, and iNOS proteins levels. Additionally, PM10 exposure significantly increased DNA damage, phosphor-p53, p16 and p21 protein levels, and β-galactosidase (β-gal) staining, which confirmed the senescence. SKQ1 pre-treatment reversed these effects. ML385 lowered the Nrf2 protein levels and mRNA levels of its downstream targets. ML385 also abrogated the protective effects of SKQ1 against PM10 toxicity by preventing the restoration of cell viability and reduced oxidative stress. In conclusion, PM10 induces inflammation, reduces Nrf2 transcriptional activity, and causes DNA damage, leading to a senescence-like phenotype, which is prevented by SKQ1.

Abstract 638: FGFR4 mediates nuclear accumulation of NRF2 by binding to P62 in gastric cancer

Abstract Background: Helicobacter pylori (H. pylori) is the major cause of gastric cancer. Fibroblast growth factor receptor 4 (FGFR4) is a member of the highly conserved tyrosine kinase family known to be activated in cancer. Nuclear Factor Erythroid 2-Related Factor 2 (NRF2), a transcription factor, is essential for the antioxidant response and exhibits cytoprotective properties against cellular stress and oxidative damage. This study investigates the novel functions of FGFR4 and its role in regulating the antioxidant response in gastric tumorigenesis. Methods: We used public datasets, gastric tissues from mice and human, and gastric cancer cell lines. In vitro and in vivo H. pylori infections were performed. Western blot, RT-qPCR, Immunofluorescence, flow cytometry, Immunohistochemistry, Immunoprecipitation, Proximity ligation assay and Luciferase reporter assays were performed. Results: The analysis of a public dataset revealed that overexpression of FGFR4 is associated with an oxidative stress signature and poor survival. Gene set enrichment analysis (GSEA) showed that tissue samples with high FGFR4 levels had an enrichment of NRF2 signature genes. In vitro cell models infected with H. pylori showed an increase in reactive oxygen species (ROS). Knockdown of FGFR4 resulted in significant reductions in NRF2 protein and transcriptional activity, leading to increased ROS levels and DNA damage. Immunofluorescence analysis using various models confirmed that FGFR4 knockdown reversed the accumulation of NRF2 in the nucleus. Dysplastic and neoplastic gastric lesions in the TFF1-KO mouse model exhibited high levels of both FGFR4 and NRF2. Pharmacologic inhibition or knockdown of FGFR4 significantly decreased NRF2 levels, as well as the size and number of gastric cancer spheroids. Mechanistically, elevated levels of P62 protein were associated with FGFR4 expression. Binding between FGFR4 and P62 proteins, which competes with NRF2-KEAP1 interaction, was detected using immunoprecipitation and proximity ligation assay. Immunohistochemistry analysis on a tissue microarray showed increased FGFR4 immunostaining in HGD/Cancer samples compared to adjacent normal samples, and this increase correlated with an increase in NRF2 and P62 levels. Conclusion: These findings revealed that FGFR4 has a distinct functional role in promoting gastric carcinogenesis. FGFR4 binds to P62 to inhibit the interaction between NRF2 and KEAP1, allowing NRF2 to avoid degradation facilitating its translocation and accumulation in the nucleus. The use of FGFR4 inhibitors is a viable treatment option that warrants further research in patients with gastric cancer. Citation Format: Nadeem S. Bhat, Mohammed Soutto, Xing Zhang, Zheng Chen, Shoumin Zhu, Selma Maacha, Melanie Genoula, Dunfa Peng, Heng Lu, Oliver G McDonald, Xi Steven Chen, Longlong Cao, Zekuan Xu, Wael El-Rifai. FGFR4 mediates nuclear accumulation of NRF2 by binding to P62 in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 638.

ARID1A loss is associated with increased NRF2 signaling in human head and neck squamous cell carcinomas.

Prior to the next generation sequencing and characterization of the tumor genome landscape, mutations in the SWI/SNF chromatin remodeling complex and the KEAP1-NRF2 signaling pathway were underappreciated. While these two classes of mutations appeared to independently contribute to tumor development, recent reports have demonstrated a mechanistic link between these two regulatory mechanisms in specific cancer types and cell models. In this work, we expand upon these data by exploring the relationship between mutations in BAF and PBAF subunits of the SWI/SNF complex and activation of NRF2 signal transduction across many cancer types. ARID1A/B mutations were strongly associated with NRF2 transcriptional activity in head and neck squamous carcinomas (HNSC). Many additional tumor types showed significant association between NRF2 signaling and mutation of specific components of the SWI/SNF complex. Different effects of BAF and PBAF mutations on the polarity of NRF2 signaling were observed. Overall, our results support a context-dependent functional link between SWI/SNF and NRF2 mutations across human cancers and implicate ARID1A inactivation in HPV-negative HNSC in promoting tumor progression and survival through activation of the KEAP1-NRF2 signaling pathway. The tumor-specific effects of these mutations open a new area of study for how mutations in the KEAP1-NRF2 pathway and the SWI/SNF complex contribute to cancer.

Enhanced ·OH-Scavenging Activity of Cu-CeOx Nanozyme via Resurrecting Macrophage Nrf2 Transcriptional Activity Facilitates Diabetic Wound Healing.

Diabetic wounds are a prevalent and devastating complication of diabetes, which may impede their healing and regeneration. In diabetic wounds, excess reactive oxygen species (ROS) activate the nuclear factor kappa-B pathway, leading to transcriptional silencing of nuclear factor erythroid 2-related factor 2 (Nrf2), resulting in a vicious cycle of oxidative stress and inflammation. Conventional nanozymes have limitations in preventing the continuous production of ROS, including the most oxidizing reactive hydroxyl radical (·OH), although they can remove pre-existing ROS. Herein, a novel antioxidant nanoplatform addresses this challenge by incorporating JSH-23 into the mesoporous of cupric-doped cerium oxide nanozymes. Additionally, for rapid wound adaptability and durable tissue adhesion, a nanozyme hydrogel spray consisting of oxidized sodium alginate and methacrylate gelatin is constructed, named OG@CCJs. This platform resurrects Nrf2 transcriptional activity of macrophages in vitro, curbing the production of ROS at its source, particularly ·OH, while enabling the nanozymes to scavenge previously generated ROS. OG@CCJs significantly alleviate oxidative stress in diabetic wounds in vivo, promoting wound healing. Overall, the proposed nanozyme-hydrogel spray with enhanced ·OH-scavenging activity uses a "two-track" antioxidant strategy to rebuild the antioxidant defense barrier of macrophages. This pioneering approach highlights the tremendous potential of OG@CCJs for facilitating diabetic wound healing.

Sequestosome 1 (p62) mitigates hypoxia-induced cardiac dysfunction by stabilizing hypoxia-inducible factor 1α and nuclear factor erythroid 2-related factor 2.

Heart failure due to ischaemic heart disease (IHD) is a leading cause of mortality worldwide. A major contributing factor to IHD-induced cardiac damage is hypoxia. Sequestosome 1 (p62) is a multi-functional adaptor protein with pleiotropic roles in autophagy, proteostasis, inflammation, and cancer. Despite abundant expression in cardiomyocytes, the role of p62 in cardiac physiology is not well understood. We hypothesized that cardiomyocyte-specific p62 deletion evokes hypoxia-induced cardiac pathology by impairing hypoxia-inducible factor 1α (Hif-1α) and nuclear factor erythroid 2-related factor 2 (Nrf2) signalling. Adult mice with germline deletion of cardiomyocyte p62 exhibited mild cardiac dysfunction under normoxic conditions. Transcriptomic analyses revealed a selective impairment in Nrf2 target genes in the hearts from these mice. Demonstrating the functional importance of this adaptor protein, adult mice with inducible depletion of cardiomyocyte p62 displayed hypoxia-induced contractile dysfunction, oxidative stress, and cell death. Mechanistically, p62-depleted hearts exhibit impaired Hif-1α and Nrf2 transcriptional activity. Because findings from these two murine models suggested a cardioprotective role for p62, mechanisms were evaluated using H9c2 cardiomyoblasts. Loss of p62 in H9c2 cells exposed to hypoxia reduced Hif-1α and Nrf2 protein levels. Further, the lack of p62 decreased Nrf2 protein expression, nuclear translocation, and transcriptional activity. Repressed Nrf2 activity associated with heightened Nrf2-Keap1 co-localization in p62-deficient cells, which was concurrent with increased Nrf2 ubiquitination facilitated by the E3 ligase Cullin 3, followed by proteasomal-mediated degradation. Substantiating our results, a gain of p62 in H9c2 cells stabilized Nrf2 and increased the transcriptional activity of Nrf2 downstream targets. Cardiac p62 mitigates hypoxia-induced cardiac dysfunction by stabilizing Hif-1α and Nrf2.

SN-38, an active metabolite of irinotecan, inhibits transcription of nuclear factor erythroid 2-related factor 2 and enhances drug sensitivity of colorectal cancer cells.

Nuclear factor erythroid 2-related factor 2 (Nrf2) significantly contributes to drug resistance of cancer cells, and Nrf2 inhibitors have been vigorously pursued. Repurposing of existing drugs, especially anticancer drugs, is a straightforward and promising strategy to find clinically available Nrf2 inhibitors and effective drug combinations. Topoisomerase inhibitors SN-38 (an active metabolite of irinotecan), topotecan, mitoxantrone, and epirubicin were found to significantly suppress Nrf2 transcriptional activity in cancer cells. SN-38, the most potent one among them, significantly inhibited the transcription of Nrf2, as indicated by decreased mRNA level and binding of RNA polymerase II to NFE2L2 gene, while no impact on Nrf2 protein or mRNA degradation was observed. SN-38 synergized with Nrf2-sensitive anticancer drugs such as mitomycin C in killing colorectal cancer cells, and irinotecan and mitomycin C synergistically inhibited the growth of SW480 xenografts in nude mice. Our study identified SN-38 and three other topoisomerase inhibitors as Nrf2 inhibitors, revealed the Nrf2-inhibitory mechanism of SN-38, and indicate that clinically feasible drug combinations could be designed based on their interactions with Nrf2 signaling.

The Multifaceted Roles of NRF2 in Cancer: Friend or Foe?

Physiological concentrations of reactive oxygen species (ROS) play vital roles in various normal cellular processes, whereas excessive ROS generation is central to disease pathogenesis. The nuclear factor erythroid 2-related factor 2 (NRF2) is a critical transcription factor that regulates the cellular antioxidant systems in response to oxidative stress by governing the expression of genes encoding antioxidant enzymes that shield cells from diverse oxidative alterations. NRF2 and its negative regulator Kelch-like ECH-associated protein 1 (KEAP1) have been the focus of numerous investigations in elucidating whether NRF2 suppresses tumor promotion or conversely exerts pro-oncogenic effects. NRF2 has been found to participate in various pathological processes, including dysregulated cell proliferation, metabolic remodeling, and resistance to apoptosis. Herein, this review article will examine the intriguing role of phase separation in activating the NRF2 transcriptional activity and explore the NRF2 dual impacts on tumor immunology, cancer stem cells, metastasis, and long non-coding RNAs (LncRNAs). Taken together, this review aims to discuss the NRF2 multifaceted roles in both cancer prevention and promotion while also addressing the advantages, disadvantages, and limitations associated with modulating NRF2 therapeutically in cancer treatment.

The Role of the NRF2 Pathway in the Pathogenesis of Viral Respiratory Infections.

In humans, acute and chronic respiratory infections caused by viruses are associated with considerable morbidity and mortality. Respiratory viruses infect airway epithelial cells and induce oxidative stress, yet the exact pathogenesis remains unclear. Oxidative stress activates the transcription factor NRF2, which plays a key role in alleviating redox-induced cellular injury. The transcriptional activation of NRF2 has been reported to affect both viral replication and associated inflammation pathways. There is complex bidirectional crosstalk between virus replication and the NRF2 pathway because virus replication directly or indirectly regulates NRF2 expression, and NRF2 activation can reversely hamper viral replication and viral spread across cells and tissues. In this review, we discuss the complex role of the NRF2 pathway in the regulation of the pathogenesis of the main respiratory viruses, including coronaviruses, influenza viruses, respiratory syncytial virus (RSV), and rhinoviruses. We also summarize the scientific evidence regarding the effects of the known NRF2 agonists that can be utilized to alter the NRF2 pathway.

Fibroblast growth factor receptor-4 mediates activation of Nuclear Factor Erythroid 2-Related Factor-2 in gastric tumorigenesis

Helicobacter pylori (H. pylori) is the leading risk factor for gastric carcinogenesis. Fibroblast growth factor receptor 4 (FGFR4) is a member of transmembrane tyrosine kinase receptors that are activated in cancer. We investigated the role of FGFR4 in regulating the cellular response to H. pylori infection in gastric cancer. High levels of oxidative stress signature and FGFR4 expression were detected in gastric cancer samples. Gene set enrichment analysis (GSEA) demonstrated enrichment of NRF2 signature in samples with high FGFR4 levels. H. pylori infection induced reactive oxygen species (ROS) with a cellular response manifested by an increase in FGFR4 with accumulation and nuclear localization NRF2. Knocking down FGFR4 significantly reduced NRF2 protein and transcription activity levels, leading to higher levels of ROS and DNA damage following H. pylori infection. We confirmed the induction of FGFR4 and NRF2 levels using mouse models following infection with a mouse-adapted H. pyloristrain. Pharmacologic inhibition of FGFR4 using H3B-6527, or its knockdown, remarkably reduced the level of NRF2 with a reduction in the size and number of gastric cancer spheroids. Mechanistically, we detected binding between FGFR4 and P62 proteins, competing with NRF2-KEAP1 interaction, allowing NRF2 to escape KEAP1-dependent degradation with subsequent accumulation and translocation to the nucleus. These findings demonstrate a novel functional role of FGFR4 in cellular homeostasis via regulating the NRF2 levels in response to H. pylori infection in gastric carcinogenesis, calling for testing the therapeutic efficacy of FGFR4 inhibitors in gastric cancer models.

MiR-630 Promotes Radioresistance by Induction of Anti-Apoptotic Effect via Nrf2-GPX2 Molecular Axis in Head-Neck Cancer.

Head and neck cancer (HNC) ranks among the top ten prevalent cancers worldwide. Radiotherapy stands as a pivotal treatment component for HNC; however, radioresistance in cancerous cells often leads to local recurrence, becoming a substantial factor in treatment failure. MicroRNAs (miRNAs) are compact, non-coding RNAs that regulate gene expression by targeting mRNAs to inhibit protein translation. Although several studies have indicated that the dysregulation of miRNAs is intricately linked with malignant transformation, understanding this molecular family's role in radioresistance remains limited. This study determined the role of miR-630 in regulating radiosensitivity in HNC. We discovered that miR-630 functions as an oncomiR, marked by its overexpression in HNC patients, correlating with a poorer prognosis. We further delineated the malignant function of miR-630 in HNC cells. While it had a minimal impact on cell growth, the miR-630 contributed to radioresistance in HNC cells. This result was supported by decreased cellular apoptosis and caspase enzyme activities. Moreover, miR-630 overexpression mitigated irradiation-induced DNA damage, evidenced by the reduced levels of the γ-H2AX histone protein, a marker for double-strand DNA breaks. Mechanistically, the overexpression of miR-630 decreased the cellular ROS levels and initiated Nrf2 transcriptional activity, resulting in the upregulation of the antioxidant enzyme GPX2. Thus, this study elucidates that miR-630 augments radioresistance by inducing an anti-apoptotic effect via the Nrf2-GPX2 molecular axis in HNC. The modulation of miR-630 may serve as a novel radiosensitizing target for HNC.

Proximity proteomic analysis of the NRF family reveals the Parkinson's disease protein ZNF746/PARIS as a co-complexed repressor of NRF2.

The nuclear factor erythroid 2-related factor 2 (NRF2) transcription factor activates cytoprotective and metabolic gene expression in response to various electrophilic stressors. Constitutive NRF2 activity promotes cancer progression, whereas decreased NRF2 function contributes to neurodegenerative diseases. We used proximity proteomic analysis to define protein networks for NRF2 and its family members NRF1, NRF3, and the NRF2 heterodimer MAFG. A functional screen of co-complexed proteins revealed previously uncharacterized regulators of NRF2 transcriptional activity. We found that ZNF746 (also known as PARIS), a zinc finger transcription factor implicated in Parkinson's disease, physically associated with NRF2 and MAFG, resulting in suppression of NRF2-driven transcription. ZNF746 overexpression increased oxidative stress and apoptosis in a neuronal cell model of Parkinson's disease, phenotypes that were reversed by chemical and genetic hyperactivation of NRF2. This study presents a functionally annotated proximity network for NRF2 and suggests a link between ZNF746 overexpression in Parkinson's disease and inhibition of NRF2-driven neuroprotection.

Selenium bioavailability modulates the sensitivity of thyroid cells to iodide excess

IntroductionIodide is an essential micronutrient for the synthesis of thyroid hormones and its imbalance is involved in the origin of different thyroid pathological processes. Selenium (Se) is another essential trace element that contributes to thyroid preservation through the control of the redox homeostasis. Different studies have demonstrated that sodium-iodide-symporter (NIS) is downregulated in the presence of iodide excess and Se supplementation reverses this effect. We also demonstrated that NOX4-derived ROS are involved in NIS repression induced by iodide excess. The aim of this study was to investigate how Se bioavailability is decisive in the sensitivity to iodide excess on a differentiated rat thyroid cell line (FRTL-5). ResultsWe demonstrated that siRNA-mediated silencing of Nox4 suppressed AKT phosphorylation induced by iodide excess. Iodide increases TGF-β1 mRNA expression, AKT phosphorylation, ROS levels and decreases GPX1 and TXRND1 mRNAs expression while Se reversed these effects. Furthermore, iodide induced Nrf2 transcriptional activity only in Se-supplemented cultures, suggesting that Se positively influences Nrf2 activation and selenoenzyme response in FRTL-5. Se, also inhibited NF-κB phosphorylation induced by iodide excess. In addition, we found that iodide excess decreased total phosphatase activity and PTP1B and PTEN mRNA expression. Se supply restored only PTEN mRNA expression. Finally, we studied the 2-α-iodohexadecanal (2-IHD) effects since it has been proposed as intermediary of iodide action on thyroid autoregulation. 2-IHD stimulated PI3K/AKT activity and reduced NIS expression by a ROS-independent mechanism. Also, we found that 2-IHD increased TGF-β1 mRNA and TGF-β inhibitor (SB431542) reverses the 2-IHD inhibitory effect on NIS mRNA expression, suggesting that TGF-β1 signaling pathway could be involved. Although Se reduced 2-IHD-induced TGFB1 levels, it could not reverse its inhibitory effect on NIS expression. ConclusionOur study suggests that Se bioavailability may improve the expression of antioxidant genes through the activation of Nrf2, interfere in PI3K/AKT signaling and NIS expression by redox modulation.

Histone Deacetylase 3 Inhibition Ameliorates Microglia-Mediated Neuro-Inflammation Via the SIRT1/Nrf2 Pathway After Traumatic Spinal Cord Injury

Background Microglial-induced inflammation plays a crucial role in the pathophysiological process of nervous system injury, however, still lacks effective therapeutic agents. Previously, we discovered that the inhibition of histone deacetylase 3 (HDAC3) exerts anti-inflammatory effects after traumatic spinal cord injury (SCI), whereas little is known about its underlying mechanism. Therefore, the present study aimed to explore the effects and potential mechanisms of HDAC3 on neuroinflammation and microglial function. Methods Rats were randomized into 4 groups: sham group, SCI group, SCI + vehicle group, and SCI + RGF966 group. To examine the effect of HDAC3 on neurological deficit after SCI, we gathered data using the Basso Beattie Bresnahan locomotion scale, the inclined plane test, the blood-spinal cord barrier, junction protein expression, and Nissl staining. We also evaluated microglial activation and inflammatory factor levels. Immunofluorescence analysis, immunohistochemical analysis, western blotting, and quantitative real-time polymerase chain reaction were performed to examine the regulation of the Sirtuin 1 (SIRT1)/nuclear factor-erythroid 2-related factor 2 (Nrf2) pathway. Results The results showed that HDAC3 inhibition significantly ameliorated Basso–Beattie–Bresnahan (BBB) permeability, brain edema, and improved neurological functions and junction protein levels. Additionally, HDAC3 inhibition significantly inhibited microglial activation, thereby reducing the levels of SCI-induced pro-inflammatory factors. Moreover, HDAC3 inhibition dramatically enhanced the expression of SIRT1 and increased both Nrf2 nuclear accumulation and transcriptional activity, thereby increasing downstream heme oxygenase-1 and NAD(P)H quinone oxidoreductase 1 expression. Conclusions The results of this study suggest that HDAC3 inhibition protects the spinal cord from injury following SCI by inhibiting SCI-induced microglial activation and the subsequent inflammatory response via SIRT1/Nrf2 signaling pathway, highlighting HDAC3 as a potential therapeutic target for the treatment of SCI.

In vivo measurement of mitochondrial ROS production in mouse models of photoreceptor degeneration

Retinitis pigmentosa (RP) is a disease characterised by photoreceptor cell death. It can be initiated by mutations in a number of different genes, primarily affecting rods, which will die first, resulting in loss of night vision. The secondary death of cones then leads to loss of visual acuity and blindness. We set out to investigate whether increased mitochondrial reactive oxygen species (ROS) formation, plays a role in this sequential photoreceptor degeneration. To do this we measured mitochondrial H2O2 production within mouse eyes in vivo using the mass spectrometric probe MitoB. We found higher levels of mitochondrial ROS that preceded photoreceptor loss in four mouse models of RP: Pde6brd1/rd1; Prhp2rds/rds; RPGR−/−; Cln6nclf. In contrast, there was no increase in mitochondrial ROS in loss of function models of vision loss (GNAT−/−, OGC), or where vision loss was not due to photoreceptor death (Cln3). Upregulation of Nrf2 transcriptional activity with dimethylfumarate (DMF) lowered mitochondrial ROS in RPGR−/− mice. These findings have important implications for the mechanism and treatment of RP.

Zhilong Huoxue Tongyu capsule attenuates intracerebral hemorrhage induced redox imbalance by modulation of Nrf2 signaling pathway.

Background: One of the severely debilitating and fatal subtypes of hemorrhagic stroke is intracerebral hemorrhage (ICH), which lacks an adequate cure at present. The Zhilong Huoxue Tongyu (ZLHXTY) capsule has been utilized effectively since last decade to treat ICH, in some provinces of China but the scientific basis for its mechanism is lacking. Purpose: To investigate the neuroprotective role of ZLHXTY capsules for ICH-induced oxidative injury through the regulation of redox imbalance with the Nrf2 signaling pathway. Methods: Autologous blood injection model of ICH in C57BL/6J mice was employed. Three treatment groups received ZLHXTY once daily through oral gavage at doses 0.35g/kg, 0.7g/kg, and 1.4g/kg, started after 2h and continued for 72h of ICH induction. The neurological outcome was measured using a balance beam test. Serum was tested for inflammatory markers IL-1β, IL-6, and TNF-α through ELISA, oxidative stress through hydrogen peroxide content assay, and antioxidant status by total antioxidant capacity (T-AOC) assay. Nuclear extract from brain tissue was assayed for Nrf2 transcriptional factor activity. RT-qPCR was performed for Nfe2l2, Sod1, Hmox1, Nqo1, and Mgst1; and Western blotting for determination of protein expression of Nrf2, p62, Pp62, Keap, HO1, and NQO1. Fluoro-jade C staining was also used to examine neuronal damage. Results: ZLHXTY capsule treatment following ICH demonstrated a protective effect against oxidative brain injury. Neurological scoring showed improvement in behavioral outcomes. ELISA-based identification demonstrated a significant decline in the expression of serum inflammatory markers. Hydrogen peroxide content in serum was found to be reduced. The total antioxidant capacity was also reduced in serum, but the ZLHXTY extract showed a concentration-dependent increase in T-AOC speculating at its intrinsic antioxidant potential. Nrf2 transcriptional factor activity, mRNA and protein expression analyses revealed normalization of Nrf2 and its downstream targets, which were previously elevated as a result of oxidative stress induced by ICH. Neuronal damage was also reduced markedly after ZLHXTY treatment as revealed by Fluoro-jade C staining. Conclusion: ZLHXTY capsules possess an intrinsic antioxidant potential that can modulate the ICH-induced redox imbalance in the brain as revealed by the normalization of Nrf2 and its downstream antioxidant targets.

Geniposide ameliorates dextran sulfate sodium-induced ulcerative colitis via KEAP1-Nrf2 signaling pathway

Ethnopharmacological relevanceThe dried fruit of Gardenia jasminoides Ellis (Zhizi in Chinese) is a traditional medicine used for thousands of years in China, Japan and Korea. Zhizi was recorded in Shennong Herbal, as a folk medicine, it reduces fever and treats gastrointestinal disturbance with antiphlogistic effects. Geniposide, an iridoid glycoside, is an important bioactive compound derived from Zhizi and possesses remarkable antioxidant and anti-inflammatory capacities. The pharmacological efficacy of Zhizi is highly related to the antioxidant and anti-inflammatory effects of geniposide. Aim of the studyUlcerative colitis (UC) is a common chronic gastrointestinal disease as a global public health threat. Redox imbalance is an essential factor in the progression and recurrence of UC. This study aimed to explore the therapeutic effect of geniposide on colitis and uncover the underlying mechanisms of geniposide-mediated antioxidant and anti-inflammatory activities. Experimental designThe study design involved investigating the novel mechanism by which geniposide ameliorates dextran sulfate sodium (DSS)-induced colitis in vivo and lipopolysaccharide (LPS)-challenged colonic epithelial cells in vitro. Materials and methodsThe protective effect of geniposide against colitis was evaluated by histopathologic observation and biochemical analysis of colonic tissues in DSS-induced colitis mice. The antioxidant and anti-inflammatory effects of geniposide were evaluated in both DSS-induced colitis mice and LPS-challenged colonic epithelial cells. Immunoprecipitation, drug affinity responsive target stability (DARTS), and molecular docking were performed to identify the potential therapeutic target of geniposide and the potential binding sites and patterns. ResultsGeniposide ameliorated the symptoms of DSS-induced colitis and colonic barrier injury, inhibited pro-inflammatory cytokine expression, and suppressed activation of the NF-κB signaling in colonic tissues of DSS-challenged mice. Geniposide also ameliorated lipid peroxidation and restored redox homeostasis in DSS-treated colonic tissues. In addition, in vitro experiments also showed that geniposide exhibited significant anti-inflammatory and antioxidant activity, as evidenced by suppressed IκB-α and p65 phosphorylation and IκB-α degradation, and enhanced the phosphorylation and transcriptional activity of Nrf2 in LPS-treated Caco2 cells. ML385, a specific Nrf2 inhibitor, abolished the protective effect of geniposide against LPS-induced inflammation. Mechanistically, geniposide could bind to KEAP1, thereby disrupting the interaction between KEAP1 and Nrf2, preventing Nrf2 from degradation and activating the Nrf2/ARE signaling pathway, ultimately suppressing the onset of inflammation caused by redox imbalance. ConclusionsGeniposide ameliorates colitis by activation of Nrf2/ARE signaling, while preventing colonic redox imbalance and inflammatory damage, indicating that geniposide can be considered as a promising lead compound for the treatment of colitis.

S-lactoyl modification of KEAP1 by a reactive glycolytic metabolite activates NRF2 signaling.

KEAP1 (Kelch-like ECH-associated protein), a cytoplasmic repressor of the oxidative stress responsive transcription factor Nuclear factor erythroid 2-related factor 2 (NRF2), senses the presence of electrophilic agents by modification of its sensor cysteine residues. In addition to xenobiotics, several reactive metabolites have been shown to covalently modify key cysteines on KEAP1, although the full repertoire of these molecules and their respective modifications remain undefined. Here, we report the discovery of sAKZ692, a small molecule identified by high-throughput screening that stimulates NRF2 transcriptional activity in cells by inhibiting the glycolytic enzyme pyruvate kinase. sAKZ692 treatment promotes the buildup of glyceraldehyde 3-phosphate, a metabolite which leads to S-lactate modification of cysteine sensor residues of KEAP1, resulting in NRF2-dependent transcription. This work identifies a posttranslational modification of cysteine derived from a reactive central carbon metabolite and helps further define the complex relationship between metabolism and the oxidative stress-sensing machinery of the cell.

Panax quinquefolius saponin inhibits vascular smooth muscle cell calcification via activation of nuclear factor-erythroid 2-related factor 2

BackgroundPanax quinquefolius saponin (PQS) is the main active component of Panax quinquefolius. Emerging evidence suggests that PQS exerts beneficial effects against cardiovascular diseases. However, the role and mechanism of PQS in vascular calcification are not unclear. The present study investigated the effects of PQS on the calcification of vascular smooth muscle cell (VSMCs).MethodsThe present study used calcification medium containing 3 mM inorganic phosphate (Pi) to induce rat VSMCs calcification. We investigated the effects of PQS on VSMCs calcification using alizarin red staining and alkaline phosphatase (ALP) activity assays. The intracellular reactive oxygen species (ROS) levels and the transcriptional activity of nuclear factor-erythroid 2-related factor 2 (Nrf2) were determined. The mRNA and protein expression levels of Nrf2, the antioxidant gene heme oxygenase-1 (HO-1), osteogenic markers, including runt-related transcription factor 2 (Runx2) and bone morphogenetic protein 2 (BMP2), and Kelch-like ECH-associated protein 1 (Keap1) were also measured.ResultsTreatment with Pi significantly increased intracellular calcium deposition and ALP activity, which were suppressed by PQS in a concentration-dependent manner. During VSMCs calcification, PQS inhibited the mRNA and protein expression of Runx2 and BMP2. PQS treatment reduced intracellular ROS production and significantly upregulated Nrf2 transcriptional activity and the expression of Nrf2 and its target antioxidant gene HO-1. PQS suppressed the Pi-induced protein expression of Keap1, which is an endogenous inhibitor of Nrf2. Keap1 siRNA treatment induced Nrf2 expression and downregulated Runx2 expression in the presence of Pi and PQS.ConclusionTaken together, these findings suggest that PQS could effectively inhibit VSMCs calcification by ameliorating oxidative stress and regulating osteogenic genes via the promotion of Nrf2 expression.