Free Radical Biology and Medicine最新文献

Enzymatically formed side-chain oxysterols function as signaling molecules regulating cholesterol homeostasis and act as intermediates in the biosynthesis of bile acids. In addition to these physiological functions, an imbalance in oxysterol homeostasis has been implicated in pathophysiology. Cholesterol 25-hydroxylase (CH25H) and its product 25-hydroxycholesterol (25-OHC), also formed by autoxidation, are associated with amyotrophic lateral sclerosis. However, the effects of 25-OHC on cell viability in glial cells remain unclear. This study demonstrates that 25-OHC induces ferroptosis, an iron-dependent programmed cell death, in mouse Schwann IMS32 cells. Mechanistically, 25-OHC suppressed the expression of selenoprotein glutathione peroxidase 4 (GPX4) at both the transcriptional and translational levels by inhibiting the processing of sterol regulatory element-binding proteins (SREBPs). In addition, 25-OHC upregulated the expression of NADH-cytochrome b5 reductase 1 (CYB5R1) and NADPH-cytochrome P450 reductase (POR), enzymes that promote lipid peroxidation. We further found that 25-OHC increases the expression of glutathione-specific gamma-glutamylcyclotransferase 1 (CHAC1) and decreases glutathione levels. Importantly, non-cytotoxic concentrations of 25-OHC enhanced cellular sensitivity to ferroptosis inducers by downregulating GPX4 expression. These findings reveal a multifaceted approach whereby 25-OHC induces ferroptosis through SREBP pathway suppression and redox imbalance in mouse Schwann IMS32 cells.

Oxidation of thiol proteins and redox signaling occur in cells exposed to H₂O₂ but mechanisms are unclear. We used redox proteomics to seek evidence of oxidation of specific proteins either by a mechanism involving reaction of H₂O₂ with CO₂/bicarbonate to give the more reactive peroxymonocarbonate, or via a relay involving peroxiredoxins (Prdxs). Changes in oxidation state of specific Cys-SH residues on treating Jurkat T lymphoma cells with H₂O₂ were measured by isotopically labeling reduced thiols and analysis by mass spectrometry. The effects of bicarbonate and of knocking out either Prdx1 or Prdx2 were examined. Approximately 14,000 Cys-peptides were detected, of which ∼1 % underwent 2-10 fold loss in thiol content with H₂O₂. Those showing the most oxidation were not affected by the presence of bicarbonate or knockout of either Prdx. Consistent with previous evidence that bicarbonate potentiates inactivation of glyceraldehyde-3-phosphate dehydrogenase, the GAPDH active site Cys residues were significantly more sensitive to H₂O₂ when bicarbonate was present. Several other proteins were identified as promising candidates for further investigation. Although we identified some potential protein candidates for Prdx-dependent oxidation, most of the significant differences between KO and WT cells were seen in proteins for which H₂O₂ unexpectedly increased their CysSH content over untreated cells. We conclude that facilitation of protein oxidation by bicarbonate or Prdx-mediated relays is restricted to a small number of proteins and is insufficient to explain the majority of the oxidation of the cell thiols that occured in response to H₂O₂.

Diabetes-associated periodontitis (DP) is recognized as an inflammatory disease that can lead to teeth loss. Uncontrolled chronic low-grade inflammation-induced senescence impairs the stemness of human periodontal stem cells (hPDLSCs). Sirtuin 3 (SIRT3), an NAD⁺-dependent deacetylase, is pivotal in various biological processes and is closely linked to aging and aging-related diseases. This study aims to explore the mechanism of SIRT3-related senescence and osteogenic differentiation of hPDLSCs under DP and explored the novelty therapeutic targets. Our study revealed that SIRT3 expression was markedly inhibited in periodontal ligament stem cells (PDLSCs) stimulated by high glucose and lipopolysaccharide. Both in vitro and in vivo, reduced SIRT3 expression accelerated cell senescence and impaired osteogenic differentiation of hPDLSCs. We demonstrated that SIRT3 binds to and deacetylates leucine-rich pentatricopeptide repeat-containing protein (LRPPRC), thereby modulating senescence. Additionally, we found that LRPPRC regulates senescence by modulating oxidative phosphorylation and oxidative stress. The activation of SIRT3 by honokiol significantly delayed senescence and promoted alveolar bone regeneration in mice after DP. Our findings indicate that the activation of SIRT3 negatively regulates hPDLSCs senescence by deacetylating LRPPRC, suggesting SIRT3 as a promising therapeutic target for DP.

Backgrounds: Bruton tyrosine kinase (BTK), which is highly expressed in immune cells, plays a critical role in regulating the function of macrophages. A growing body of evidence has demonstrated that the accumulation of macrophages in cardiac tissue after myocardial infarction (MI) significantly affects wound healing and ventricular remodeling during the early phase of repair after MI. However, the role of BTK in cardiac repair post-MI, especially in macrophage-mediated repair, remains unclear.

Methods: MI was induced by permanent left anterior descending (LAD) artery ligation in wild-type (WT) mice and macrophage-specific BTK-knockout (BTK^MAC-KO) mice. Expression of BTK and phosphorylated BTK were assessed by western blotting. Then, RNA sequencing and ChIP-qPCR assay were performed to explore potential BTK targets and transcriptional regulatory sites.

Results: BTK, which was mainly expressed in macrophages, was upregulated in mice after MI. Compared with WT mice, BTK^MAC-KO mice had significantly greater mortality due to heart rupture, reduced wall thickness and severe impairment of left ventricular (LV) function after MI. In addition, increased matrix metalloproteinase-9 (MMP-9) expression and decreased α-SMA and collagen expression were observed in BTK^MAC-KO mice after MI. Further experiments revealed that BTK deficiency in macrophages reduces the expression of VEGF and impairs angiogenesis after MI. By RNA sequencing, we found that Nf-kB family genes, as well as the urokinase-type plasminogen activator (uPA), were significantly upregulated in BTK-deficient macrophages. By ChIP-qPCR analysis, we confirmed that uPA was transcriptionally activated by the Nf-kB p65 subunit. Finally, the application of plasminogen activator inhibitor-1 (PAI-1), an uPA inhibitor, markedly protected against cardiac rupture, lowered the mortality rate, and improved cardiac function by increasing collagen deposition and promoting tissue healing in BTK^MAC-KO mice after MI.

Conclusions: The present study identifies PAI-1 as a novel cardioprotective agent for cardiac repair post-MI that increases collagen deposition and promotes tissue healing. A therapeutic strategy targeting BTK may be a promising treatment for cardiac repair post-MI.

Dendritic cells (DCs) are specialized antigen-presenting cells crucial for initiating and regulating adaptive immune responses, making them promising candidates for therapeutic interventions in various immune-mediated diseases. Increasing evidence suggests that the microenvironment in which cells are cultured, as well as the milieu in which they perform their functions, significantly impact their immunomodulatory properties. Among these environmental factors, the role of oxygen in DC biology and its significance for both their in vitro generation and in vivo therapeutic application require investigation. Unlike the atmospheric oxygen level of 21 % commonly used in in vitro assays, physiological oxygen levels are much lower (3-9 %), and hypoxia (<1.3 %) is a prevalent condition of both healthy tissues and disease states. This mismatch between laboratory and physiological conditions underscores the critical need to culture and evaluate therapeutic cells under physiologically relevant oxygen levels to improve their translational relevance and clinical outcomes. This review explores the characteristic hallmarks of human DCs that are influenced by oxygen-dependent pathways, including metabolism, phenotype, cytokine secretion, and migration. Furthermore, we discuss the potential of manipulating oxygen levels to refine the generation and functionality of DCs for therapeutic purposes.

The positive role of melatonin in obesity control and skeletal muscle (SKM) preservation is well known. We recently showed that melatonin improves vastus lateralis muscle (VL) fiber oxidative phenotype. However, fiber type characterization, mitochondrial function, and molecular mechanisms that underlie VL fiber switching by melatonin are still undefined. Our study aims to investigate whether melatonin induces fiber switching by NRF2/RCAN/MEF2 pathway activation and mitochondrial oxidative metabolism modulation in the VL of both sex Zücker diabetic fatty (ZDF) rats. 5-Weeks-old male and female ZDF rats (N = 16) and their age-matched lean littermates (ZL) were subdivided into two subgroups: control (C) and orally treated with melatonin (M) (10 mg/kg/day) for 12 weeks. Interestingly, melatonin increased oxidative fibers amounts (Types I and IIa) counteracting the decreased levels found in the VL of obese-diabetic rats, and upregulated NRF2, calcineurin and MEF2 expression. Melatonin also restored the mitochondrial oxidative capacity increasing the respiratory control ratio (RCR) in both sex and phenotype rats through the reduction of the proton leak component of respiration (state 4). Melatonin also improved the VL mitochondrial phosphorylation coefficient and modulated the total oxygen consumption by enhancing complex I, III and IV activity, and fatty acid oxidation (FAO) in both sex obese-diabetic rats, decreasing in male and increasing in female the complex II oxygen consumption. These findings suggest that melatonin treatment induces fiber switching in SKM improving mitochondrial functionality by NRF2/RCAN/MEF2 pathway activation.

Urolithin A, an active precursor derived from the metabolism of ellagitanins in rats and humans, is known for its potential health benefits, including stimulating mitophagy and promoting muscular skeletal function. While experimental studies have demonstrated Urolithin A's potential to enhance cellular health, the detailed molecular interactions through which Urolithin A exerts its effects are not fully elucidated. In this study, we investigated the anti-inflammatory, antioxidation and neuroprotective abilities of Urolithin A in selected targets using molecular docking and molecular dynamics simulation methods. Molecular docking studies revealed the strong affinity for receptors involved in inflammation activities, including human p38 MAP kinase (4DLI) with -10.1 kcal/mol interacting with SER252, LYS249, and ASP294 residues. The binding energy in the 5KIR target was -8.6 kcal/mol, interacting with GLN203 through hydrogen bond, and lastly, 1A9U with an affinity of -6.8 with no hydrogen bond formed with Urolithin A and interacts with van der Waals interactions. In oxidant targets, the influence of Urolithin was observed in 1OG5 with -7.9 kcal/mol interacting with GLN185, PHE447. For the 1M17 target, the binding affinity was -7.7 kcal/mol interacting with THR95 residue and 1ZXM target at -7.4 kcal/mol interacting with TYR36, TYR216, and LEU234 residues. The neuroprotective ability of urolithin A was observed in selected targets for acetylcholinesterase; the binding energy was -9.7 kcal/mol interacting with van der Waals and π interactions; for the 1GQR target, the binding energy was -9.9 kcal/mol interacting with van der Waals and π interactions and for β-amylase (1iyt) the binding energy was -5.5 forming hydrogen bond with SER8, GLN15 residues. Molecular Dynamics simulations at 100 ns of Urolithin A compared with reference 4DLI. The Urolithin A-4DLI complex exhibited greater stability than the reference receptor, as confirmed by RMSD, RMSF, Radius of Gyration, Hydrogen bond, and SASA analyses.

Chloroquine (CQ), an autophagy antagonist, has been recently explored as a repurposable medicine for cancer; however the exact mechanism of its action is still not known. The present study investigated the effect of CQ on colorectal cancer cells to elucidate the underlying molecular mechanisms. We report for the first time that CQ suppresses hypoxia-induced growth and survival of HCT-116 cells by reducing glycolytic capacity and NAD⁺ production through inhibition of PDK1. Furthermore, in silico and in vitro studies revealed that CQ induces structural alteration in the PDK1 protein, leading to its destabilization and promotes its enhanced degradation by proteases. This degradation is in turn inhibited by the MG-132 protease inhibitor. Moreover, CQ-induced suppression of PDK1 results in mitochondrial damage through excessive production of ROS, as reflected by the reduction in mitochondrial membrane potential, which in turn triggers apoptosis through PARP cleavage and Caspase activation. These findings advocate CQ as a promising repurposable chemotherapeutic for colorectal cancer and a novel inhibitor of PDK1.