Antioxidants & Redox Signaling

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) have the potential for use in cell-based therapy, disease modeling, and drug toxicity testing. However, under the conventional differentiation protocol, PSC-CMs are immature and differ from adult cardiomyocytes in electrophysiological characteristics, calcium kinetics, cellular morphology, metabolism, and gene expression. MFN2 tethers sarcoplasmic reticulum (SR) and mitochondria and mediates their interaction via mitochondria-associated endoplasmic reticulum membranes, which tunes the cytosolic Ca²⁺ and reactive oxygen species (ROS) signaling. We aim to investigate if MFN2 would regulate murine embryonic stem cell-derived cardiomyocyte (mESC-CM) maturation, and if yes, what are the underlying mechanisms.

MFN2 knockdown caused detrimental effects on mESC-CM maturation in terms of structure, cytosolic calcium kinetics, electrophysiology, and metabolism. Mechanistically, MFN2 knockdown increased proliferative capacity, increased ROS and activated PI3K/AKT/mTOR activity, and these were all reversed by the ROS scavenger N-acetylcysteine. Meanwhile, MFN2 knockdown decreased the IP3R-VDAC coupling mediated by GRP75. Importantly, GRP75 overexpression restored the decreased IP3R-VDAC coupling, reversed the increased cellular ROS, and reversed the increased PI3K/AKT/mTOR activity caused by MFN2 knockdown. Rapamycin, an mTOR inhibitor, reduced the increased proliferative capacity and restored the impaired electrophysiology caused by MFN2 knockdown.

The current study is the first study to reveal that MFN2-mediated SR-mitochondrial interaction is required for the mESC-CM maturation through the ROS/PI3K/AKT/mTOR axis.

MFN2 is required for the maturation of mESC-CMs through GRP75-dependent, mTOR-mediated suppression of proliferative capacity via the ROS/PI3K/AKT pathway. Our findings advance the understanding of PSC-CM maturation and provide novel insight for strategies to promote PSC-CM maturation. Antioxid. Redox Signal. 44, 799–821.

Depression is a prevalent mood disorder with significant suffering and heightened suicide risk. Accumulating evidence points to oxidative stress and impaired energy metabolism of the brain as key contributors to the pathogenesis of depression. Astragaloside IV (AIV) has been demonstrated to alleviate neurological damage. The capacity of AIV for antioxidation has also been gradually discovered. Yet, it still just scratches the surface of the antidepression mechanisms of AIV. Our aim was to explore a novel mechanism of action of AIV from the perspectives of redox homeostasis and energy metabolism via the reactive oxygen species (ROS)/nuclear factor erythroid 2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap1) axis.

AIV ameliorated anxiety and cognitive dysfunction in rats with depression-like behaviors. Furthermore, we illustrated that AIV reversed redox dyshomeostasis of rats with depression-like behaviors in terms of decreasing the level of malondialdehyde while increasing the abundances of catalase, superoxide dismutase, and glutathione peroxidase. In addition, AIV significantly increased the levels of indicators of energy metabolism that were interrupted by depression. Furthermore, in vitro, AIV significantly increased the levels of indicators related to the Nrf2/Keap1 signaling pathway in corticosterone-induced PC12 cells.

For the first time, it demonstrates that AIV alleviates depression by modulating the ROS/Nrf2/Keap1 axis and restoring hippocampal energy metabolism in a chronic unpredictable mild stress model.

The current findings not only offer novel insights into antidepressant-like effects of AIV through the ROS/Nrf2/Keap1 axis but also provide a foundation for enhancing clinical efficiency and enriching drug selection. Antioxid. Redox Signal. 44, 822–842.

Radiation-induced intestinal injury (RIII) significantly impairs the quality of life in patients with abdominal/pelvic cancer undergoing radiotherapy, often necessitating treatment cessation. The ACE2/Ang-(1–7)/MasR axis, a protective pathway within the renin-angiotensin system, represents a potential anti-inflammatory target. This study explored the role of ACE2 activation in mitigating RIII and the underlying mechanisms.

Treatment with diminazene aceturate (DIZE), a selective ACE2 agonist, prior to lethal radiation blocked intestinal stem cell (ISC) death, enhanced crypt regeneration, preserved epithelial barrier integrity, and reduced intestinal inflammation, thereby promoting mice survival. Notably, the radioprotective effect of DIZE was reversed by ACE2 or MasR antagonists, and other ACE2 agonists exhibited similar radioprotective efficacy. DIZE treatment improved the survival of ISCs both in vitro and in vivo postradiation. Mechanistically, DIZE directly targeted intestinal epithelial cells (IECs), preventing the activation of radiation-induced MAPK (p38/JNK) and NF-κB pathways. This effect was abolished by ACE2 knockdown in a human intestinal epithelial cell line (HIECs) in vitro. Intriguingly, DIZE failed to inhibit endothelial cell apoptosis or attenuate MAPK/NF-κB pathway activation in irradiated endothelial cells. Preliminary evidence indicates DIZE did not affect the radiosensitivity of colorectal tumor cells or azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced colorectal tumors in mice.

This study is the first to demonstrate selective ACE2-mediated intestinal protection without compromising tumor radiosensitivity. These findings demonstrate ACE2 activation selectively shields IECs from radiation damage by inhibiting MAPK/NF-κB pathways, offering a novel therapeutic strategy to alleviate RIII without compromising tumor radiosensitivity. Antioxid. Redox Signal. 44, 843–858.

To determine the role of the ubiquitin-activating enzyme UBA1 in macrophage-mediated renal injury during sepsis-associated acute kidney injury (SA-AKI) and to elucidate the underlying molecular mechanism.

Using a cecal ligation and puncture mouse model, we evaluated renal function, inflammation, and survival in myeloid-specific Uba1 knockout mice (Uba1^M-KO) and littermate controls. Transcriptomic, proteomic, and ubiquitinome analyses were integrated with mechanistic studies in bone marrow–derived macrophages and renal tubular epithelial cell co-cultures. A pharmacologic UBA1 inhibitor (PYR-41) was tested for therapeutic efficacy. UBA1 expression was markedly increased in renal macrophages during SA-AKI. Uba1^M-KO mice demonstrated improved survival, preserved renal function, and attenuated inflammatory responses, as evidenced by reduced cytokine production, reactive oxygen species generation, apoptosis, and macrophage infiltration. Mechanistically, UBA1 promoted ubiquitination and degradation of the nuclear pore protein nucleoporin 35 (NUP35), impairing IκBα nuclear import and activating nuclear factor kappa B (NF-κB) signaling. This led to enhanced macrophage inflammatory activation and subsequent renal tubular injury. Pharmacologic inhibition of UBA1 recapitulated the protective effects of genetic deletion in vivo.

This study identifies UBA1-mediated NUP35 ubiquitination as a previously unrecognized checkpoint linking ubiquitin activation to nuclear pore integrity and inflammatory signaling in sepsis. UBA1 drives macrophage-mediated inflammation in SA-AKI by promoting NUP35 degradation and subsequent activation of NF-κB signaling. Targeting UBA1 represents a promising immunomodulatory strategy for the prevention and treatment of SA-AKI. Antioxid. Redox Signal. 44, 859–877.

Nephrolithiasis is a major global health challenge, with oxidative stress and mitochondrial dysfunction emerging as key drivers of renal injury and stone formation. l-ergothioneine (l-Erg), a naturally occurring antioxidant transported by OCTN1, has shown promising effects in cystinuria models, preventing stone formation. Despite evidence supporting an indirect mechanism of action, key mechanistic aspects have yet to be fully clarified. This study aimed to evaluate whether l-Erg can prevent stone progression in cystinuria and in other types of lithiasis, such as calcium oxalate nephrolithiasis, and to further elucidate its mechanistic basis.

Using mouse models, l-Erg significantly reduced cystine stone growth and renal inflammation, and its combination with d-penicillamine enhanced stone dissolution and mitigated drug-related toxicity. In calcium oxalate nephrolithiasis, l-Erg decreased crystal deposition, preserved renal architecture, normalized glutathione levels, and restored mitochondrial respiration. Transcriptomic analysis revealed downregulation of immune pathways and activation of cell cycle genes, suggesting attenuation of inflammation and promotion of tubular repair.

This study is the first to demonstrate that l-Erg exerts renoprotective effects through combined antioxidant and mitochondrial mechanisms in two major forms of nephrolithiasis and introduces a dual therapeutic approach combining an antioxidant with a cystine-solubilizing agent.

By targeting oxidative stress and mitochondrial dysfunction, l-Erg represents a promising therapeutic strategy for nephrolithiasis, either alone or as an adjunct to current treatments. Antioxid. Redox Signal. 44, 878–891.

Hyperhomocysteinemia (HHcy) has been established as a significant risk factor for liver diseases, and its impact on nitrative stress and protein nitration has also been recognized. Nuclear receptor coactivator 4 (NCOA4) is crucial in maintaining iron homeostasis and regulating ferroptosis. However, limited studies are available regarding the post-translational modifications (PTMs) of NCOA4 in liver injury.

Mice fed with a diet containing 2.5% methionine and water with 1.8 g/L DL-Hcy were used in this study. The HHcy-induced liver injury phenotype was assessed through serum aspartate aminotransferase/alanine aminotransferase (AST/ALT), hematoxylin and eosin staining, Masson staining, and real-time quantitative PCR. To further analyze the mechanisms, we conducted tandem mass tag proteomics analysis, Perls blue staining, transmission electron microscopy observation, liver iron content determination, malondialdehyde level, autophagic flux analysis, and detection of indicators related to ferritinophagy–ferroptosis pathway. Additionally, the nitrated NCOA4 was enhanced in HHcy stimulation, especially at sites such as Tyr78, Tyr214, Tyr269, Tyr335, and Tyr608. Peroxynitrite decomposition catalyst (PDC) was used to clear peroxynitrite, while nitrated NCOA4 was inhibited, thereby alleviating liver injury caused by HHcy. We established an HHcy mouse model and confirmed liver injury by elevated serum AST/ALT, histopathology, and fibrosis markers. Mechanistically, HHcy led to hepatic iron overload, lipid peroxidation, and ferroptosis. These effects were associated with upregulated autophagy and NCOA4-mediated ferritinophagy. Importantly, we confirmed in vitro that HHcy enhanced the nitration of NCOA4 at specific tyrosine residues (Tyr78, 214, 269, 335, 608). Scavenging peroxynitrite with a decomposition catalyst inhibited NCOA4 nitration, subsequently suppressing ferritinophagy, reducing iron overload and lipid peroxidation, and ultimately alleviating liver injury.

We find that HHcy induces nitrative modification at Tyr78/214/269/335/608 sites of NCOA4, upregulating ferritinophagy levels, increasing the intracellular labile iron pool, and triggering iron-dependent lipid peroxidation-driven ferroptosis, thereby promoting the occurrence and development of liver injury. These data indicate that inhibiting nitrated NCOA4, thereby curtailing ferritinophagy, represents a potential therapeutic target for the treatment of HHcy-induced liver injury. This study is the first to identify NCOA4 nitration as a critical PTM that amplifies ferritinophagy under conditions of nitrative stress. We pinpoint five specific nitration sites on NCOA4 and establish a novel causal link between HHcy, protein nitration, and ferroptosis-driven liver injury. Inhibiting the nitrated NCOA4 or upstream peroxynitrite formation to restrain pathological ferritinophagy represents a promising therapeutic strategy for HHcy-induced liver injury. Antioxid. Redox Signal. 44, 892–909.

Metabolic dysfunction-associated fatty liver disease (MAFLD), a globally prevalent condition, can lead to serious complications, such as liver cirrhosis, hepatocellular carcinoma, and an increased risk of hepatic failure and mortality. MAFLD progression is influenced by various factors, including lipid overload, mitochondrial dysfunction, reactive oxygen species (ROS) production, and inflammation. Corylin, a flavonoid extracted from Psoralea corylifolia L., possesses multiple beneficial properties, including anti-inflammatory, antiproliferative, antiobesity, and antioxidant effects, and can alleviate hyperglycemia, hyperlipidemia, and insulin resistance. However, the beneficial potential of corylin for MAFLD has not been explored. Herein, we investigated the effects of corylin on MAFLD.

Corylin reduced plasma hyperglycemia, ROS levels, lipid accumulation, inflammation, and fibrosis in livers of high-fat diet (HFD)-fed mice; additionally, it improved insulin resistance and enhanced glycogen synthesis. In palmitic acid (PA)-treated HepG2 cells, corylin decreased excessive lipogenesis, ROS production, and mitochondrial dysfunction while promoting glucose uptake, glycogen synthesis, and mitochondrial activation. Furthermore, corylin reduced inflammation in PA-treated RAW264.7 macrophages and decreased fibrotic protein expression in TGF-β-treated LX-2 hepatic stellate cells (HSCs). The regulatory effects of corylin on PA-treated HepG2 cells and RAW264.7 macrophages were mediated through adenosine 5′-monophosphate-activated protein kinase regulation; however, similar effects were not observed in transforming growth factor beta-stimulated LX-2 HSCs.

These findings suggest that corylin has anti-ROS, anti-inflammatory, antilipogenic, and antifibrotic properties.

Consequently, corylin is a promising beneficial candidate for the treatment of MAFLD. Antioxid. Redox Signal. 44, 910–927.

Cisplatin is an effective chemotherapeutic agent, but its clinical use is limited by dose-dependent ototoxicity that leads to irreversible sensorineural hearing loss. Accumulating evidence implicates impaired autophagy–lysosomal homeostasis in cisplatin-induced ototoxicity. Transcription factor EB (TFEB), a master regulator of autophagy and lysosomal biogenesis, represents a promising therapeutic target. Clotrimazole, an FDA-approved antifungal drug with emerging cytoprotective properties, has not been investigated for its potential to mitigate cisplatin-induced ototoxicity.

We evaluated the protective effects of clotrimazole using House Ear Institute-Organ of Corti 1 cells, cochlear explants, and an adult C57BL/6J mouse model of transtympanic cisplatin ototoxicity. Apoptosis, reactive oxygen species (ROS), and autophagy flux were assessed using biochemical assays and imaging. RNA-sequencing was performed to identify transcriptional pathways regulated by clotrimazole. TFEB dependence was verified using small interfering RNA knockdown and pharmacological inhibition of AMP-activated protein kinase (AMPK). Cochlear function was assessed using auditory brainstem responses (ABRs), and hair-cell and synapse survival were quantified by immunofluorescence.

Clotrimazole significantly reduced cisplatin-induced apoptosis, ROS generation, and calcium overload. Transcriptomic profiling and functional assays revealed robust activation of autophagy. Clotrimazole promoted AMPK activation, suppressed mTORC1 signaling, and enhanced TFEB nuclear translocation. TFEB or AMPK inhibition abrogated these protective effects. In vivo, intratympanic clotrimazole preserved hair cell survival, maintained ribbon synapses, and significantly reduced ABR threshold shifts.

Clotrimazole protects against cisplatin-induced ototoxicity by activating the AMPK–mTOR–TFEB axis and restoring autophagy lysosomal homeostasis. These findings support TFEB-targeted autophagy activation as a promising therapeutic strategy for preventing cisplatin-induced hearing loss. Antioxid. Redox Signal. 44, 928–950.