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Theranostics 2023Increased methylation of key genes has been observed in kidney diseases, suggesting that the ten-eleven translocation (Tet) methyl-cytosine dioxygenase family as well...
Increased methylation of key genes has been observed in kidney diseases, suggesting that the ten-eleven translocation (Tet) methyl-cytosine dioxygenase family as well as 5mC oxidation may play important roles. As a member of the Tet family, the role of Tet1 in acute kidney injury (AKI) remains unclear. knockout mice, with or without tempol treatment, a scavenger of reactive oxygen species (ROS), were challenged with ischemia and reperfusion (I/R) injury or unilateral ureteral obstruction (UUO) injury. RNA-sequencing, Western blotting, qRT-PCR, bisulfite sequencing, chromatin immunoprecipitation, immunohistochemical staining, and dot blot assays were performed. Tet1 expression was rapidly upregulated following I/R or UUO injury. Moreover, knockout mice showed increased renal injury and renal cell death, increased ROS accumulation, G2/M cell cycle arrest, inflammation, and fibrosis. Severe renal damage in injured knockout mice was alleviated by tempol treatment. Mechanistically, Tet1 reduced the 5mC levels in an enzymatic activity-dependent manner on the promoters of and to promote their expression, thus lowering injury-induced excessive ROS and reducing I/R or UUO injury. Tet1 plays an important role in the development of AKI by promoting SOD expression through a DNA demethylase-dependent mechanism.
Topics: Animals; Mice; Acute Kidney Injury; Kidney; Mice, Knockout; Oxidative Stress; Reactive Oxygen Species; Reperfusion Injury; Superoxide Dismutase; Ureteral Obstruction
PubMed: 37908721
DOI: 10.7150/thno.87416 -
Nutrients Sep 2023It is hypothesized that garlic, , might protect against oxidative stress that causes damage to cells and tissues leading to the development of various health conditions...
It is hypothesized that garlic, , might protect against oxidative stress that causes damage to cells and tissues leading to the development of various health conditions including cancer. However, it is not known whether garlic's potential anticancer benefits differ by form of garlic consumed. This study aimed to quantify and compare the in vitro antioxidant and antiproliferative activity of several garlic forms in water and alcohol extracts including fresh garlic, fresh garlic set aside, heated garlic, heated garlic set aside, garlic powder, black garlic, two commercially available garlic supplements. Antioxidant activity of different garlic forms were measured using three assays: DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) assay, superoxide assay, and hydroxyl assay. In vitro effects of garlic extracts were investigated against the most common lung cancer subtypes: H520, H1975, and A549 cell lines using the sulforhodamine B (SRB) assay. Among free radical scavenging assays, Garlicin, a commercially available supplement, displayed high antioxidant activity in water and alcohol extracts (DPPH assay: 2.02 mg AAE (mg ascorbic acid equivalent)/g garlic and 3.53 mg AAE/g garlic, respectively; superoxide assay: 6.73 mg AAE/g garlic and 7.13 mg AAE/g garlic, respectively). In the hydroxyl assay, water extract of fresh garlic crushed and set aside for 10 min showed the highest antioxidant activity. Garlicin alcohol extract and fresh garlic water extracts strongly inhibited the proliferation of H1975, A549 and H520 cells. Other forms of garlic including garlic powder and black garlic exhibited low antioxidant and antiproliferative activity. Our results demonstrate that the preparation and processing methods of garlic may lead to different antioxidant benefits.
Topics: Antioxidants; Garlic; Superoxides; Powders; Plant Extracts; Water
PubMed: 37836382
DOI: 10.3390/nu15194099 -
Antioxidants & Redox Signaling Oct 2023Mitochondrial (mt) reticulum network in the cell possesses amazing ultramorphology of parallel lamellar cristae, formed by the invaginated inner mitochondrial membrane.... (Review)
Review
Mitochondrial (mt) reticulum network in the cell possesses amazing ultramorphology of parallel lamellar cristae, formed by the invaginated inner mitochondrial membrane. Its non-invaginated part, the inner boundary membrane (IBM) forms a cylindrical sandwich with the outer mitochondrial membrane (OMM). Crista membranes (CMs) meet IBM at crista junctions (CJs) of mt cristae organizing system (MICOS) complexes connected to OMM sorting and assembly machinery (SAM). Cristae dimensions, shape, and CJs have characteristic patterns for different metabolic regimes, physiological and pathological situations. Cristae-shaping proteins were characterized, namely rows of ATP-synthase dimers forming the crista lamella edges, MICOS subunits, optic atrophy 1 (OPA1) isoforms and mitochondrial genome maintenance 1 (MGM1) filaments, prohibitins, and others. Detailed cristae ultramorphology changes were imaged by focused-ion beam/scanning electron microscopy. Dynamics of crista lamellae and mobile CJs were demonstrated by nanoscopy in living cells. With tBID-induced apoptosis a single entirely fused cristae reticulum was observed in a mitochondrial spheroid. The mobility and composition of MICOS, OPA1, and ATP-synthase dimeric rows regulated by post-translational modifications might be exclusively responsible for cristae morphology changes, but ion fluxes across CM and resulting osmotic forces might be also involved. Inevitably, cristae ultramorphology should reflect also mitochondrial redox homeostasis, but details are unknown. Disordered cristae typically reflect higher superoxide formation. To link redox homeostasis to cristae ultramorphology and define markers, recent progress will help in uncovering mechanisms involved in proton-coupled electron transfer the respiratory chain and in regulation of cristae architecture, leading to structural determination of superoxide formation sites and cristae ultramorphology changes in diseases. 39, 635-683.
Topics: Mitochondrial Membranes; Superoxides; Homeostasis; Oxidation-Reduction; Adenosine Triphosphate; Mitochondrial Proteins
PubMed: 36793196
DOI: 10.1089/ars.2022.0173 -
HDGF stimulates liver tumorigenesis by enhancing reactive oxygen species generation in mitochondria.The Journal of Biological Chemistry Nov 2023Hepatoma-derived growth factor (HDGF) overexpression and uncontrolled reactive oxygen species (ROS) accumulation are involved in malignant transformation and poor...
Hepatoma-derived growth factor (HDGF) overexpression and uncontrolled reactive oxygen species (ROS) accumulation are involved in malignant transformation and poor prognosis in various types of cancer. However, the interplay between HDGF and ROS generation has not been elucidated in hepatocellular carcinoma. Here, we first analyzed the profile of HDGF expression and ROS production in newly generated orthotopic hepatomas by ultrasound-guided implantation. In situ superoxide detection showed that HDGF-overexpressing hepatomas had significantly elevated ROS levels compared with adjacent nontumor tissues. Consistently, liver tissues from HDGF-deficient mice exhibited lower ROS fluorescence than those from age- and sex-matched WT mice. ROS-detecting fluorescent dyes and flow cytometry revealed that recombinant HDGF (rHDGF) stimulated the production of superoxide anion, hydrogen peroxide, and mitochondrial ROS generation in cultured hepatoma cells in a dose-dependent manner. In contrast, the inactive Ser103Ala rHDGF mutant failed to promote ROS generation or oncogenic behaviors. Seahorse metabolic flux assays revealed that rHDGF dose dependently upregulated bioenergetics through enhanced basal and total oxygen consumption rate, extracellular acidification rate, and oxidative phosphorylation in hepatoma cells. Moreover, antioxidants of N-acetyl cysteine and MitoQ treatment significantly inhibited HDGF-mediated cell proliferation and invasive capacity. Genetic silencing of superoxide dismutase 2 augmented the HDGF-induced ROS generation and oncogenic behaviors of hepatoma cells. Finally, genetic knockdown nucleolin (NCL) and antibody neutralization of surface NCL, the HDGF receptor, abolished the HDGF-induced increase in ROS and mitochondrial energetics. In conclusion, this study has demonstrated for the first time that the HDGF/NCL signaling axis induces ROS generation by elevating ROS generation in mitochondria, thereby stimulating liver carcinogenesis.
Topics: Animals; Mice; Carcinoma, Hepatocellular; Reactive Oxygen Species; Carcinogenesis
PubMed: 37827291
DOI: 10.1016/j.jbc.2023.105335 -
Proceedings of the National Academy of... Aug 2023Amyotrophic lateral sclerosis (ALS) is a fatal disease affecting upper and lower motor neurons. Microglia directly interact with motor neurons and participate in the...
Amyotrophic lateral sclerosis (ALS) is a fatal disease affecting upper and lower motor neurons. Microglia directly interact with motor neurons and participate in the progression of ALS. Single-cell mass cytometry (CyTOF) analysis revealed prominent expression of α5 integrin in microglia and macrophages in a superoxide dismutase-1 G93A mouse model of ALS (SOD1). In postmortem tissues from ALS patients with various clinical ALS phenotypes and disease duration, α5 integrin is prominent in motor pathways of the central and peripheral nervous system and in perivascular zones associated with the blood-brain barrier. In SOD1 mice, administration of a monoclonal antibody against α5 integrin increased survival compared to an isotype control and improved motor function on behavioral testing. Together, these findings in mice and in humans suggest that α5 integrin is a potential therapeutic target in ALS.
Topics: Mice; Humans; Animals; Amyotrophic Lateral Sclerosis; Superoxide Dismutase-1; Integrin alpha5; Motor Cortex; Mice, Transgenic; Superoxide Dismutase; Macrophages; Disease Models, Animal
PubMed: 37523555
DOI: 10.1073/pnas.2306731120 -
Nature Communications May 2024NADPH oxidase 5 (NOX5) catalyzes the production of superoxide free radicals and regulates physiological processes from sperm motility to cardiac rhythm. Overexpression...
NADPH oxidase 5 (NOX5) catalyzes the production of superoxide free radicals and regulates physiological processes from sperm motility to cardiac rhythm. Overexpression of NOX5 leads to cancers, diabetes, and cardiovascular diseases. NOX5 is activated by intracellular calcium signaling, but the underlying molecular mechanism of which - in particular, how calcium triggers electron transfer from NADPH to FAD - is still unclear. Here we capture motions of full-length human NOX5 upon calcium binding using single-particle cryogenic electron microscopy (cryo-EM). By combining biochemistry, mutagenesis analyses, and molecular dynamics (MD) simulations, we decode the molecular basis of NOX5 activation and electron transfer. We find that calcium binding to the EF-hand domain increases NADPH dynamics, permitting electron transfer between NADPH and FAD and superoxide production. Our structural findings also uncover a zinc-binding motif that is important for NOX5 stability and enzymatic activity, revealing modulation mechanisms of reactive oxygen species (ROS) production.
Topics: Humans; NADPH Oxidase 5; Molecular Dynamics Simulation; Cryoelectron Microscopy; Calcium; NADP; Flavin-Adenine Dinucleotide; Superoxides; Protein Binding; Reactive Oxygen Species; Zinc; Electron Transport; Enzyme Activation; Binding Sites
PubMed: 38734761
DOI: 10.1038/s41467-024-48467-y -
Cell Communication and Signaling : CCS Sep 2023Arginase-1 (ARG1) promotes collagen synthesis and cell proliferation. ARG1 is highly expressed in various tumour cells. The mechanisms of ARG1 in...
BACKGROUND
Arginase-1 (ARG1) promotes collagen synthesis and cell proliferation. ARG1 is highly expressed in various tumour cells. The mechanisms of ARG1 in epithelial-to-mesenchymal transition (EMT)-associated cataracts were studied herein.
METHODS
C57BL/6 mice, a human lens epithelial cell line (HLEC-SRA01/04), and human lens capsule samples were used in this study. The right lens anterior capsule of the mouse eye was punctured through the central cornea with a 26-gauge hypodermic needle. Human lens epithelial cells (HLECs) were transfected with ARG1-targeted (siARG1) or negative control siRNA (siNC). For gene overexpression, HLECs were transfected with a plasmid bearing the ARG1 coding sequence or an empty vector. Medium containing 0.2% serum with or without transforming growth factor beta-2 (TGF-β2) was added for 6 or 24 h to detect mRNA or protein, respectively. The expression of related genes was measured by quantitative real-time polymerase chain reaction (RT-qPCR), western blotting, and immunohistochemical staining. Transwell assays and wound healing assays were used to determine cell migration. Cell proliferation, superoxide levels, nitric oxide (NO) levels, and arginase activity were estimated using Cell Counting Kit-8 assays, a superoxide assay kit, an NO assay kit, and an arginase activity kit.
RESULTS
ARG1, alpha-smooth muscle actin (α-SMA), fibronectin, and Ki67 expression increased after lens capsular injury, while zonula occludens-1 (ZO-1) expression decreased. Fibronectin and collagen type I alpha1 chain (collagen 1A1) expression increased, and cell migration increased significantly in ARG1-overexpressing HLECs compared with those transfected with an empty vector after TGF-β2 treatment. These effects were reversed by ARG1 knockdown. The arginase-related pathway plays an important role in EMT. mRNAs of enzymes of the arginase-related pathway were highly expressed after ARG1 overexpression. ARG1 knockdown suppressed these expression changes. Numidargistat (CB-1158) dihydrochloride (CB-1158), an ARG1 inhibitor, suppressed TGF-β2-induced anterior subcapsular cataract (ASC) by reducing the proliferation of lens epithelial cells (LECs) and decreasing fibronectin, α-SMA, collagen 1A1, and vimentin expression. Compared with that in nonanterior subcapsular cataract (non-ASC) patients, the expression of ARG1, collagen 1A1, vimentin, fibronectin, and Ki67 was markedly increased in ASC patients.
CONCLUSIONS
ARG1 can regulate EMT in EMT-associated cataracts. Based on the pathogenesis of ASC, these findings are expected to provide new therapeutic strategies for patients.
Topics: Animals; Humans; Mice; Arginase; Cataract; Ki-67 Antigen; Mice, Inbred C57BL; Superoxides; Transforming Growth Factor beta2; Vimentin; Epithelial-Mesenchymal Transition
PubMed: 37723490
DOI: 10.1186/s12964-023-01210-4 -
Cell Reports Oct 2023Mutations in SOD1 cause amyotrophic lateral sclerosis (ALS) through gain-of-function effects, yet the mechanisms by which misfolded mutant SOD1 (mutSOD1) protein impairs...
Mutations in SOD1 cause amyotrophic lateral sclerosis (ALS) through gain-of-function effects, yet the mechanisms by which misfolded mutant SOD1 (mutSOD1) protein impairs human motor neurons (MNs) remain unclear. Here, we use induced-pluripotent-stem-cell-derived MNs coupled to metabolic stable isotope labeling and mass spectrometry to investigate proteome-wide degradation dynamics. We find several proteins, including the ALS-causal valosin-containing protein (VCP), which predominantly acts in proteasome degradation and autophagy, that degrade slower in mutSOD1 relative to isogenic control MNs. The interactome of VCP is altered in mutSOD1 MNs in vitro, while VCP selectively accumulates in the affected motor cortex of ALS-SOD1 patients. Overexpression of VCP rescues mutSOD1 toxicity in MNs in vitro and in a C. elegans model in vivo, in part due to its ability to modulate the degradation of insoluble mutSOD1. Our results demonstrate that VCP contributes to mutSOD1-dependent degeneration, link two distinct ALS-causal genes, and highlight selective protein degradation impairment in ALS pathophysiology.
Topics: Animals; Humans; Amyotrophic Lateral Sclerosis; Superoxide Dismutase-1; Proteome; Valosin Containing Protein; Induced Pluripotent Stem Cells; Caenorhabditis elegans; Motor Neurons; Homeostasis; Mutation
PubMed: 37776851
DOI: 10.1016/j.celrep.2023.113160 -
Basic Research in Cardiology Aug 2023In the context of myocardial infarction, the burst of superoxide generated by reverse electron transport (RET) at complex I in mitochondria is a crucial trigger for... (Review)
Review
In the context of myocardial infarction, the burst of superoxide generated by reverse electron transport (RET) at complex I in mitochondria is a crucial trigger for damage during ischaemia/reperfusion (I/R) injury. Here we outline the necessary conditions for superoxide production by RET at complex I and how it can occur during reperfusion. In addition, we explore various pathways that are implicated in generating the conditions for RET to occur and suggest potential therapeutic strategies to target RET, aiming to achieve cardioprotection.
Topics: Humans; Electron Transport; Superoxides; Oxidative Phosphorylation; Mitochondria; Myocardial Infarction; Reperfusion Injury
PubMed: 37639068
DOI: 10.1007/s00395-023-01002-4