Search Results (67)

Background: Vitiligo, a chronic depigmentation disorder driven by oxidative stress and immune dysregulation, remains poorly understood mechanistically. The Keap1/NRF2/ARE pathway is critical for melanocyte protection against oxidative damage; however, the role of Cullin-3 (CUL3), a scaffold for E3 ubiquitin ligases that regulate NRF2 degradation, and its interplay with inflammatory mediators in vitiligo pathogenesis are underexplored. This study investigates CUL3, NRF2, and the associated regulatory networks in vitiligo, integrating clinical profiling and computational docking to identify therapeutic targets. Methods: A case-control study compared non-segmental vitiligo patients with age-/sex-matched controls. Lesional skin biopsies were analyzed by qRT-PCR for the expression of CUL3, NRF2, miRNA-146a, FOXP3, NF-κB, IL-6, TNF-α, and P53. Molecular docking was used to evaluate vitexin’s binding affinity to Keap1, validated by root mean square deviation (RMSD) calculations. Results: Patients with vitiligo exhibited significant downregulation of CUL3 (0.27 ± 0.03 vs. 1 ± 0.58; p = 0.013), NRF2 (0.37 ± 0.26 vs. 1 ± 0.8; p = 0.001), and FOXP3 (0.09 ± 0.2 vs. 1 ± 0.3; p = 0.001), alongside the upregulation of miRNA-146a (4.7 ± 1.9 vs. 1 ± 0.8; p = 0.001), NF-κB (4.7 ± 1.9 vs. 1 ± 0.5; p = 0.001), IL-6 (2.8 ± 1.5 vs. 1 ± 0.4; p = 0.001), and TNF-α (2.2 ± 1.1 vs. 1 ± 0.3; p = 0.001). P53 showed no differential expression (p > 0.05). Docking revealed a strong binding of vitexin to Keap1 (RMSD: 0.23 Å), mirroring the binding of the control ligand CDDO-Im. Conclusions: Dysregulation of the CUL3/Keap1/NRF2 axis and elevated miRNA-146a levels correlate with vitiligo progression, suggesting a role for oxidative stress and immune imbalance. Vitexin’s high-affinity docking to Keap1 positions it as a potential modulator of the NRF2 pathway, offering novel therapeutic avenues. This study highlights the translational potential of targeting the ubiquitin–proteasome and antioxidant pathways in the management of vitiligo. Full article

Using a multidisciplinary approach, this paper was designed to prepare, identify, and characterize novel maize antioxidant cyclic peptides from protein hydrolysate of corn gluten meal (CGM). A bioinformatics approach was used to identify the best protease, and the results showed that papain+subtilisin was most likely to produce antioxidant cyclic peptides. The result of the enzymatic hydrolysis validation experiment showed that hydrolysate by papain+subtilisin yielded the highest concentration of cyclic peptide (67.14 ± 1.88%) and remarkable DPPH, ABTS, and hydroxyl radical scavenging rates (81.06 ± 2.23%, 82.82 ± 1.83%, and 47.44 ± 2.43%, respectively) compared to other hydrolysates. Eleven antioxidant cyclic peptides were identified in the protein hydrolysate of CGM through sequential purification and mass spectrometry analysis. The results of molecular docking analysis indicated that the cyclic peptides can form stable hydrogen bonds and hydrophobic interactions with the key amino acid residues of Kelch-like ECH-associated protein 1 (Keap1). Cyclic peptides may regulate the Keap1-Nrf2 pathway by occupying the Kelch domain of Keap1, inhibiting the ubiquitination degradation of Nrf2 (nuclear factor erythroid 2-related factor 2), thereby stabilizing the Nrf2 protein and activating the antioxidant gene network. This study underlined the bioinformatics approach for antioxidant cyclic peptide discovery, which is time- and cost-effective and promotes new cyclic peptide drugs or functional food development. Full article

The transcription factor erythroid 2-related factor 2 (Nrf2) is a central regulator of cellular defense mechanisms against oxidative stress and inflammation. Keap1 (Kelch-like ECH-associated protein 1) regulates Nrf2 activity by ubiquitination-mediated cytoplasmic retention, thereby suppressing its nuclear translocation and subsequent transcriptional activation of genes encoding phase II detoxifying enzymes. Using a structure-based virtual screening approach, we screened ~16,000 natural compounds to identify Keap1-Nrf2 PPI inhibitors. Nine compounds were identified based on their high binding affinities and favorable interactions with Keap1, primarily through non-covalent interactions. To validate the binding stability of these inhibitors, molecular dynamics (MD) simulations were performed, confirming the robustness of the Keap1–inhibitor complexes over time. Subsequent in vitro assays on human epithelial keratinocyte cells (HaCaT) revealed that six of these compounds notably upregulated Nrf2 mRNA expression, regis tering increases from 23% to 50% in comparison to the control. Notably, chebulinic acid emerged as the most potent compound, demonstrating the greatest elevation in Nrf2 expression. Penetration studies further showed that chebulinic acid, when encapsulated in silk fibroin, achieved a 0.14% penetration rate after 24 h though it could not penetrate into the stratum corneum alone. This result highlighted the potential of chebulinic acid in the use of anti-aging skincare formulations. Collectively, our findings affirmed that molecular docking is a reliable and effective approach for the identification of novel anti-aging agents targeting the Keap1-Nrf2 pathway. Full article

Ubiquitin-specific protease 32 (USP32), a deubiquitylating enzyme that controls the ubiquitin process, is overexpressed in multiple cancers and serves as a promising therapeutic target for cancer therapy. Drugs targeting ferroptosis have exhibited promising anticancer activity. Lycobetaine (LBT), a natural alkaloid, holds promise against various cancers, yet its specific targets and anticancer mechanisms remain unclear. In this study, we show that LBT induced ferroptosis in lung squamous cell carcinoma (LUSC) cells, accompanied by glutathione depletion and the accumulation of lipid peroxidation, malondialdehyde, and ferrous iron. Mechanistically, drug affinity responsive target stability-based mass spectrometry analysis, molecular dynamics simulations, and a cellular thermal shift assay confirmed that USP32 is a potential target of LBT in LUSC cells. Moreover, a strong interaction between USP32 and nuclear factor erythroid 2-related factor 2 (NRF2) was found via immunoprecipitation–mass spectrometry and co-immunoprecipitation. In addition, the ubiquitination assay results demonstrated that LBT treatment significantly increased NRF2 ubiquitination and degradation by targeting USP32. Importantly, USP32 overexpression effectively attenuated the effects of LBT on proliferation and ferroptosis in LUSC cells. In orthotopic LUSC xenografts, the administration of LBT significantly inhibited tumor growth and metastasis and induced ferroptosis by targeting the USP32–NRF2 signaling axis. Taken together, these data suggest that LBT exerts its anticancer effects by inhibiting USP32-mediated NRF2 deubiquitination to induce ferroptosis and that LBT may serve as a prospective USP32-targeting agent for LUSC treatment. Full article

Cannabinoids include both endogenous endocannabinoids and exogenous phytocannabinoids, such as cannabidiol (CBD), and have potential as therapeutic agents in cancer treatment due to their selective anticancer activities. CBD exhibits both antioxidant and pro-oxidant effects depending on its concentration and cell types. These properties allow CBD to influence oxidative stress responses and potentially enhance the efficacy of antitumor therapies. In this study, we treated U87MG glioma cells with low dose (1 μM) CBD and evaluated its molecular effects. Our findings indicate that CBD reduced cell viability by 20% (p < 0.05) through the alteration of mitochondrial membrane potential. The alteration of redox status by CBD caused an attempt to rescue mitochondrial functionality through nuclear localization of the GABP transcription factor involved in mitochondria biogenesis. Moreover, CBD treatment caused an increase in autophagic flux, as supported by the increase in Beclin-1 and the ratio of LC3-II/LC3-I. Due to mitochondria functionality alteration, pro-apoptotic proteins were induced without activating apoptotic effectors Caspase-3 or Caspase-7. The study of the transcription factor NRF2 and the ubiquitin-binding protein p62 expression revealed an increase in their levels in CBD-treated cells. In conclusion, low-dose CBD makes U87MG cells more vulnerable to cytotoxic effects, reducing cell viability and mitochondrial dynamics while increasing autophagic flux and redox systems. This explains the mechanisms by which glioma cells respond to CBD treatment. These findings highlight the therapeutic potential of CBD, suggesting that modulating NRF2 and autophagy pathways could represent a promising strategy for glioblastoma treatment. Full article

Harsh acid oxidation of activated charcoal transforms an insoluble carbon-rich source into water-soluble, disc structures of graphene decorated with multiple oxygen-containing functionalities. We term these pleiotropic nano-enzymes as “pleozymes”. A broad redox potential spans many crucial redox reactions including the oxidation of hydrogen sulfide (H₂S) to polysulfides and thiosulfate, dismutation of the superoxide radical (O₂⁻*), and oxidation of NADH to NAD⁺. The oxidation of H₂S is predicted to enhance protein persulfidation—the attachment of sulfur to cysteine residues. Persulfidated proteins act as redox intermediates, and persulfidation protects proteins from irreversible oxidation and ubiquitination, providing an important means of signaling. Protein persulfidation is believed to decline in several neurological disorders and aging. Importantly, and consistent with the role of persulfidation in signaling, the master antioxidant transcription factor Nrf2 is regulated by Keap1’s persulfidation. Here, we demonstrate that pleozymes increased overall protein persulfidation in cells from apparently healthy individuals and from individuals with the mitochondrial protein mutation responsible for Friedreich’s ataxia. We further find that pleozymes specifically enhanced Keap1 persulfidation, with subsequent increased accumulation of Nrf2 and Nrf2’s antioxidant targets. Full article

Aspiration pneumonia exacerbates swallowing and respiratory muscle atrophy. It induces respiratory muscle atrophy through three steps: proinflammatory cytokine production, caspase-3 and calpain, and then ubiquitin–proteasome activations. In addition, autophagy induces swallowing muscle atrophy. Nrf2 is the central detoxifying and antioxidant gene whose function in aspiration pneumonia is unclear. We explored the role of Nrf2 in aspiration pneumonia by examining swallowing and respiratory muscle mass and function using wild-type and Nrf2-knockout mice. Pepsin and lipopolysaccharide aspiration challenges caused aspiration pneumonia. The swallowing (digastric muscles) and respiratory (diaphragm) muscles were isolated. Quantitative RT-PCR and Western blotting were used to assess their proteolysis cascade. Pathological and videofluoroscopic examinations evaluated atrophy and swallowing function, respectively. Nrf2-knockouts showed exacerbated aspiration pneumonia compared with wild-types. Nrf2-knockouts exhibited more persistent and intense proinflammatory cytokine elevation than wild-types. In both mice, the challenge activated calpains and caspase-3 in the diaphragm but not in the digastric muscles. The digastric muscles showed extended autophagy activation in Nrf2-knockouts compared to wild-types. The diaphragms exhibited autophagy activation only in Nrf2-knockouts. Nrf2-knockouts showed worsened muscle atrophies and swallowing function compared with wild-types. Thus, activation of Nrf2 may alleviate inflammation, muscle atrophy, and function in aspiration pneumonia, a major health problem for the aging population, and may become a therapeutic target. Full article

Ferroptosis is a recently identified iron-dependent programmed cell death with lipid peroxide accumulation and condensation and compaction of mitochondria. A recent study indicated that ferroptosis plays a pivotal role in ischemic cardiac injury with the mechanisms remain largely unknown. This study demonstrates that when an iron overload occurs in the ischemia/reperfusion cardiac tissues, which initiates myocardial ferroptosis, the expression levels of mitochondrial inner membrane protein MPV17 are reduced. Overexpression of MPV17 delivered via adenovirus significantly reduced ferroptosis in both cardiomyocytes with high levels of iron and cardiac I/R tissues. Mitochondrial glutathione (mtGSH), crucial for reactive oxygen species scavenging and mitochondrial homeostasis maintenance, is depleted in myocardial ferroptosis caused by iron overload. This mechanistic study shows that MPV17 can increase mitochondrial glutathione levels through maintaining the protein homeostasis of SLC25A10, which is a mitochondrial inner-membrane glutathione transporter. The absence of MPV17 in iron overload resulted in the ubiquitination-dependent degradation of SLC25A10, leading to impaired mitochondrial glutathione import. Moreover, we found that MPV17 was the targeted gene of Nrf2, which plays a pivotal role in preventing lipid peroxide accumulation and ferroptosis. The decreased expression levels of Nrf2 led to the inactivation of MPV17 in iron overload-induced myocardial ferroptosis. In summary, this study demonstrates the critical role of MPV17 in protecting cardiomyocytes from ferroptosis and elucidates the Nrf2-MPV17-SLC25A10/mitochondrial glutathione signaling pathway in the regulation of myocardial ferroptosis. Full article

Mangifera indica L., a member of the Anacardiaceae family, is widely cultivated across the globe. The leaves of M. indica are renowned for their medicinal properties, attributed to the abundance of bioactive compounds. This study investigated the effects of mango leaf extract on oxidative stress in HeLa cells. Notably, the n-hexane fraction (MLHx) significantly enhanced antioxidant response element (ARE)-luciferase activity at a concentration of 100 µg/mL, surpassing other fractions. MLHx also promoted the expression of HO-1 mRNA by increasing nuclear NRF2 levels. The molecular mechanism of MLHx involves increased phosphorylation of ERK1/2 and stabilization of NRF2. Bioactivity-guided isolation resulted in the identification of six oxylipins: 13(R)-hydroxy-octadeca-(9Z,11E,15Z)-trienoic acid (C-1), 9(R)-hydroxy-octadeca-(10E,12Z,15Z)-trienoic acid (C-2), 13(R)-hydroxy-(9Z,11E)-octadecadienoic acid (C-3), 9(R)-hydroxy-(10E,12Z)-octadecadienoic acid (C-4), 9-oxo-(10E,12E)-octadecadienoic acid (C-5), and 9-oxo-(10E,12Z)-octadecadienoic acid (C-6). These structures were elucidated using comprehensive spectroscopic techniques, including MS and 1H NMR. Additionally, compounds C-7 (9-oxo-(10E,12Z,15Z)-octadecatrienoic acid) and 8 (13-oxo-(9E,11E)-octadecadienoic acid) were characterized by LC-MS/MS mass fragmentation. This study reports the isolation of compounds 1–6 from M. indica for the first time. When tested for their effect on NRF2 activity in HeLa cells, compounds 3, 5, and 6 showed strong stimulation of ARE-luciferase activity in a dose-dependent manner. Full article

TRIM44, a tripartite motif (TRIM) family member, is pivotal in linking the ubiquitin-proteasome system (UPS) to autophagy in multiple myeloma (MM). However, its prognostic impact and therapeutic potential remain underexplored. Here, we report that TRIM44 overexpression is associated with poor prognosis in a Multiple Myeloma Research Foundation (MMRF) cohort of 858 patients, persisting across primary and recurrent MM cases. TRIM44 expression notably increases in advanced MM stages, indicating its potential role in disease progression. Single-cell RNA sequencing across MM stages showed significant TRIM44 upregulation in smoldering MM (SMM) and MM compared to normal bone marrow, especially in patients with t(4;14) cytogenetic abnormalities. This analysis further identified high TRIM44 expression as predictive of lower responsiveness to proteasome inhibitor (PI) treatments, underscoring its critical function in the unfolded protein response (UPR) in TRIM44-high MM cells. Our findings also demonstrate that TRIM44 facilitates SQSTM1 oligomerization under oxidative stress, essential for its phosphorylation and subsequent autophagic degradation. This process supports the survival of PI-resistant MM cells by activating the NRF2 pathway, which is crucial for oxidative stress response and, potentially, other chemotherapy-induced stressors. Additionally, TRIM44 counters the TRIM21-mediated suppression of the antioxidant response, enhancing MM cell survival under oxidative stress. Collectively, our discoveries highlight TRIM44’s significant role in MM progression and resistance to therapy, suggesting its potential value as a therapeutic target. Full article

Chikungunya (CHIKV) and Mayaro (MAYV) viruses are arthritogenic alphaviruses that promote an incapacitating and long-lasting inflammatory muscle–articular disease. Despite studies pointing out the importance of skeletal muscle (SkM) in viral pathogenesis, the long-term consequences on its physiology and the mechanism of persistence of symptoms are still poorly understood. Combining molecular, morphological, nuclear magnetic resonance imaging, and histological analysis, we conduct a temporal investigation of CHIKV and MAYV replication in a wild-type mice model, focusing on the impact on SkM composition, structure, and repair in the acute and late phases of infection. We found that viral replication and induced inflammation promote a rapid loss of muscle mass and reduction in fiber cross-sectional area by upregulation of muscle-specific E3 ubiquitin ligases MuRF1 and Atrogin-1 expression, both key regulators of SkM fibers atrophy. Despite a reduction in inflammation and clearance of infectious viral particles, SkM atrophy persists until 30 days post-infection. The genomic CHIKV and MAYV RNAs were still detected in SkM in the late phase, along with the upregulation of chemokines and anti-inflammatory cytokine expression. In agreement with the involvement of inflammatory mediators on induced atrophy, the neutralization of TNF and a reduction in oxidative stress using monomethyl fumarate, an agonist of Nrf2, decreases atrogen expression and atrophic fibers while increasing weight gain in treated mice. These data indicate that arthritogenic alphavirus infection could chronically impact body SkM composition and also harm repair machinery, contributing to a better understanding of mechanisms of arthritogenic alphavirus pathogenesis and with a description of potentially new targets of therapeutic intervention. Full article

Keap1 interacts with Nrf2 by assisting in its ubiquitination and subsequent proteolysis. By preventing ROS accumulation during RANKL-induced osteoclastogenesis, Nrf2 activation can prevent the differentiation of osteoclasts. Additionally, inhibiting the Keap1-Nrf2 PPI can be an effective strategy for triggering Nrf2 to regulate oxidative stress. Structure-based virtual screening was performed to discover a potentially novel Keap1-Nrf2 PPI inhibitor wherein KCB-F06 was identified. The inhibitory effects of KCB-F06 on osteoclastogenesis were investigated in vitro through TRAP staining and bone resorption assays. An ovariectomy-induced osteoporosis mouse model was applied to evaluate KCB-F06’s therapeutic effects in vivo. Lastly, the underlying mechanisms were explored using real-time PCR, Western blotting, and co-IP assays. KCB-F06 was discovered as a novel Keap1-Nrf2 PPI inhibitor. As a result, the expression of antioxidants (HO-1 and NQO1) was suppressed, hence reducing ROS accumulation during osteoclastogenesis. Subsequently, this caused the inactivation of RANKL-induced IKB/NF-kB signaling. This eventually led to the downregulation of osteoclast-specific proteins including NFATc1, which is an essential transcription factor for osteoclastogenesis. These results demonstrated that Nrf2 activation in osteoclasts is a valuable tool for osteoclastic bone loss management. In addition, KCB-F06 presents as an alternative candidate for treating osteoclast-related bone diseases and as a novel small molecule that can serve as a model for further Keap1-NRF2 PPI inhibitor development. Full article

Tripartite motif-containing protein 21 (TRIM21) is involved in signal transduction and antiviral responses through the ubiquitination of protein targets. TRIM21 was reported to be related to the imbalance of host cell homeostasis caused by viral infection. Our studies indicated that H5N1 highly pathogenic avian influenza virus (HPAIV) infection up-regulated TRIM21 expression in A549 cells. Western blot and qPCR results showed that knockdown of TRIM21 alleviated oxidative stress and ferroptosis induced by H5N1 HPAIV and promoted the activation of antioxidant pathways. Co-IP results showed that TRIM21 promoted oxidative stress and ferroptosis by regulating the SQSTM1-NRF2-KEAP1 axis by increasing SQSTM1 K63-linked polyubiquitination under the condition of HPAIV infection. In addition, TRIM21 attenuated the inhibitory effect of antioxidant NAC on HPAIV titers and enhanced the promoting effect of ferroptosis agonist Erastin on HPAIV titers. Our findings provide new insight into the role of TRIM21 in oxidative stress and ferroptosis induced by viral infection. Full article

Currently, the treatment for sepsis-induced acute lung injury mainly involves mechanical ventilation with limited use of drugs, highlighting the urgent need for new therapeutic options. As a pivotal aspect of acute lung injury, the pathologic activation and apoptosis of endothelial cells related to oxidative stress play a crucial role in disease progression, with NOX4 and Nrf2 being important targets in regulating ROS production and clearance. Echinacoside, extracted from the traditional Chinese herbal plant Cistanche deserticola, possesses diverse biological activities. However, its role in sepsis-induced acute lung injury remains unexplored. Moreover, although some studies have demonstrated the regulation of NOX4 expression by SIRT1, the specific mechanisms are yet to be elucidated. Therefore, this study aimed to investigate the effects of echinacoside on sepsis-induced acute lung injury and oxidative stress in mice and to explore the intricate regulatory mechanism of SIRT1 on NOX4. We found that echinacoside inhibited sepsis-induced acute lung injury and oxidative stress while preserving endothelial function. In vitro experiments demonstrated that echinacoside activated SIRT1 and promoted its expression. The activated SIRT1 was competitively bound to p22 phox, inhibiting the activation of NOX4 and facilitating the ubiquitination and degradation of NOX4. Additionally, SIRT1 deacetylated Nrf2, promoting the downstream expression of antioxidant enzymes, thus enhancing the NOX4-Nrf2 axis and mitigating oxidative stress-induced endothelial cell pathologic activation and mitochondrial pathway apoptosis. The SIRT1-mediated anti-inflammatory and antioxidant effects of echinacoside were validated in vivo. Consequently, the SIRT1-regulated NOX4-Nrf2 axis may represent a crucial target for echinacoside in the treatment of sepsis-induced acute lung injury. Full article

Since the ethanol extract of Alisma orientale Juzepzuk (EEAO) suppresses lung inflammation by suppressing Nuclear Factor-kappa B (NF-κB) and activating Nuclear Factor Erythroid 2-related Factor 2 (Nrf2), we set out to identify chemicals constituting EEAO that suppress lung inflammation. Here, we provide evidence that among the five most abundant chemical constituents identified by Ultra Performance Liquid Chromatography (UPLC) and Nuclear Magnetic Resonance (NMR), alismol is one of the candidate constituents that suppresses lung inflammation in a lipopolysaccharide (LPS)-induced acute lung injury (ALI) mouse model and protects mice from ALI-like symptoms. Alismol did not induce cytotoxicity or reactive oxygen species (ROS). When administered to the lung of LPS-induced ALI mice (n = 5/group), alismol decreased the level of neutrophils and of the pro-inflammatory molecules, including Tumor Necrosis Factor-alpha (TNF-α), Interleukin-1 beta (IL-1β), Interleukin-6 (IL-6), Monocyte Chemoattractant Protein-1 (MCP-1), Interferon-gamma (IFN-γ), and Cyclooxygenase-2 (COX-2), suggesting an anti-inflammatory activity of alismol. Consistent with these findings, alismol ameliorated the key features of the inflamed lung of ALI, such as high cellularity due to infiltrated inflammatory cells, the development of hyaline membrane structure, and capillary destruction. Unlike EEAO, alismol did not suppress NF-κB activity but rather activated Nrf2. Consequently, alismol induced the expression of prototypic genes regulated by Nrf2, including Heme Oxygenase-1 (HO-1), NAD(P)H: quinine oxidoreductase-1 (NQO-1), and glutamyl cysteine ligase catalytic units (GCLC). Alismol activating Nrf2 appears to be associated with a decrease in the ubiquitination of Nrf2, a key suppressive mechanism for Nrf2 activity. Together, our results suggest that alismol is a chemical constituent of EEAO that contributes at least in part to suppressing some of the key features of ALI by activating Nrf2. Full article