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Antioxidants
Volume 12
Issue 8
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Antioxidants, Volume 12, Issue 8 (August 2023) – 172 articles

Cover Story (view full-size image): This review heads a Special Issue of Antioxidants on Glutathione (GSH) and Glutaredoxin (Grx). Among its critical roles, GSH serves as a co-substrate for the formation and breakdown of protein mixed disulfides (protein-SSG). This posttranslational modification regulated by Grx acts as a switch, interconverting inactive and active forms of proteins and mediating redox signal transduction and thiol homeostasis. Dysregulation of this system is implicated in various diseases involving oxidative stress. The ten articles in this Special Issue provide current insights into the GSH/Grx system, including potential clinical applications; for example, elevating Grx could be an anti-fibrotic therapy, and mimicking S-glutathionylation could inhibit dimerization of the key proteases of the HIV and SARS-CoV-2 viruses. View this paper
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23 pages, 729 KiB  
Review
Primary Coenzyme Q10 Deficiency: An Update
by David Mantle, Lauren Millichap, Jesus Castro-Marrero and Iain P. Hargreaves
Antioxidants 2023, 12(8), 1652; https://doi.org/10.3390/antiox12081652 - 21 Aug 2023
Cited by 10 | Viewed by 3399
Abstract
Coenzyme Q10 (CoQ10) has a number of vital functions in all cells, both mitochondrial and extra-mitochondrial. In addition to its key role in mitochondrial oxidative phosphorylation, CoQ10 serves as a lipid soluble antioxidant and plays an important role in fatty acid beta-oxidation and [...] Read more.
Coenzyme Q10 (CoQ10) has a number of vital functions in all cells, both mitochondrial and extra-mitochondrial. In addition to its key role in mitochondrial oxidative phosphorylation, CoQ10 serves as a lipid soluble antioxidant and plays an important role in fatty acid beta-oxidation and pyrimidine and lysosomal metabolism, as well as directly mediating the expression of a number of genes, including those involved in inflammation. Due to the multiplicity of roles in cell function, it is not surprising that a deficiency in CoQ10 has been implicated in the pathogenesis of a wide range of disorders. CoQ10 deficiency is broadly divided into primary and secondary types. Primary CoQ10 deficiency results from mutations in genes involved in the CoQ10 biosynthetic pathway. In man, at least 10 genes are required for the biosynthesis of functional CoQ10, a mutation in any one of which can result in a deficit in CoQ10 status. Patients may respond well to oral CoQ10 supplementation, although the condition must be recognised sufficiently early, before irreversible tissue damage has occurred. In this article, we have reviewed clinical studies (up to March 2023) relating to the identification of these deficiencies, and the therapeutic outcomes of CoQ10 supplementation; we have attempted to resolve the disparities between previous review articles regarding the usefulness or otherwise of CoQ10 supplementation in these disorders. In addition, we have highlighted several of the potential problems relating to CoQ10 supplementation in primary CoQ10 deficiency, as well as identifying unresolved issues relating to these disorders that require further research. Full article
(This article belongs to the Section Health Outcomes of Antioxidants and Oxidative Stress)
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<p>The mammalian CoQ10 biosynthetic pathway with genes harbouring known pathogenic mutations associated with primary CoQ10 deficiency shown in red.</p> Full article ">
12 pages, 2702 KiB  
Article
Neuroprotective Potential of Pyranocoumarins from Angelica gigas Nakai on Glutamate-Induced Hippocampal Cell Death
by Nguyen Khoi Song Tran, Tuy An Trinh, Jaesung Pyo, Chang Geon Kim, Jae Gyu Park and Ki Sung Kang
Antioxidants 2023, 12(8), 1651; https://doi.org/10.3390/antiox12081651 - 21 Aug 2023
Cited by 1 | Viewed by 1143
Abstract
Chronic neurodegenerative diseases are typically associated with oxidative stress conditions leading to neuronal cell death. We aimed to investigate the neuroprotective effect of three pyranocoumarins (decursin, decursinol angelate, and decursinol) targeting oxidative stress factors. Decursin (also known as dehydro-8-prenylnaringenin) is a prenylated coumarin [...] Read more.
Chronic neurodegenerative diseases are typically associated with oxidative stress conditions leading to neuronal cell death. We aimed to investigate the neuroprotective effect of three pyranocoumarins (decursin, decursinol angelate, and decursinol) targeting oxidative stress factors. Decursin (also known as dehydro-8-prenylnaringenin) is a prenylated coumarin compound consisting of a coumarin ring system with a prenyl group attached to one of the carbons in the ring. As a secondary metabolite of plants, pyranocoumarin decursin from Angelica gigas Nakai presented protective effects against glutamate-induced oxidative stress in HT22, a murine hippocampal neuronal cell line. Decursinol (DOH) is a metabolite of decursin, sharing same coumarin ring system but a slightly different chemical structure with the prenyl group replaced by a hydroxyl group (-OH). In our findings, DOH was ineffective while decursin was, suggesting that this prenyl structure may be important for compound absorption and neuroprotection. By diminishing the accumulation of intracellular reactive oxygen species as well as stimulating the expression of HO-1, decursin triggers the self-protection system in neuronal cells. Additionally, decursin also revealed an anti-apoptotic effect by inhibiting chromatin condensation and reducing the forming of annexin-V-positive cells. Full article
(This article belongs to the Special Issue Poly-Pharmacological Approaches for Evaluating Targets with Multiple Antioxidative Mechanisms of Phytochemicals)
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Graphical abstract
Full article ">Figure 1
<p>Protective effects of pyranocoumarins extracted from A. gigas on HT22 cell death induced by glutamate. HT22 cells were cultured in 96-well plates at a density of 1 × 10<sup>4</sup> cells/well. After 24 h, cells were treated with test samples at a range of concentrations (3.125–50 μM) to evaluate their protective effects against cell death induced by 5 mM glutamate. The EZ-Cytox assay was employed to determine cell viability 24 h after the treatment. (*) <span class="html-italic">p</span> &lt; 0.05 vs. glutamate-treated group indicates significant difference.</p> Full article ">Figure 2
<p>JP-203 prevented the chromatin condensation in HT22 cells treated with glutamate. (<b>A</b>) Bright field microscope image of morphologies of HT22 cells treated with 5 mM glutamate and 25 μM JP-203 for 24 h. (<b>B</b>) HT22 cells after treatment with glutamate and JP-203 at a dose of 25 μM for 12 h were stained with Hoechst 33342 and the chromatin condensation was observed by fluorescence microscopy. The yellow arrow indicates condensed chromatin. Scale bar indicates 20 μm.</p> Full article ">Figure 3
<p>The protective effect of JP-203 on oxidative stress resulting from excessive glutamate in HT22 cells. (<b>A</b>) JP-203 diminished the accumulation of intracellular ROS in HT22 cells which was stimulated by glutamate. HT22 cells were incubated with 5 mM glutamate and JP-203 compound at concentrations of 25 and 50 μM. After 8 h, cells were stained with 10 μM 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA) to detect the accumulation of ROS. (<b>B</b>) JP-203 stimulated the expression of HO-1. Cells were treated with JP-203 at the indicated concentrations in the presence or absence of glutamate for 6 h and then total protein was collected for Western blotting analysis. The target proteins were detected by conjugation with epitope-specific primary and secondary antibodies. (<b>C</b>) The quantitative graph of relative expression levels of HO-1 to GAPDH from the Western blot analysis. (*) <span class="html-italic">p</span> &lt; 0.05 vs. glutamate-treated group; (#) <span class="html-italic">p</span> &lt; 0.05 vs. non-treated group.</p> Full article ">Figure 4
<p>The anti-apoptotic activity of JP-203 against glutamate-induced HT22 cell death. (<b>A</b>) The microscopy pictures from TALI image-based cytometric analysis of JP-203. After treatment with 5 mM glutamate and 25 µM JP-203 for 12 h, cells were stained with annexin V–Alexa Fluor 488 for apoptotic cell identification or propidium iodide (PI) for dead cell labeling. (<b>B</b>) The comparative graph illustrating the percentage of apoptotic cells between the JP-203 treated and untreated groups was made using counting of annexin-V-stained cells. (*) indicates <span class="html-italic">p</span> &lt; 0.05 vs. glutamate-treated group. Scale bar indicates 20 μm.</p> Full article ">
13 pages, 3230 KiB  
Article
Effect of Purple Sweet Potato Using Different Cooking Methods on Cytoprotection against Ethanol-Induced Oxidative Damage through Nrf2 Activation in HepG2 Cells
by Dagyeong Kim, Yoonjeong Kim and Younghwa Kim
Antioxidants 2023, 12(8), 1650; https://doi.org/10.3390/antiox12081650 - 21 Aug 2023
Cited by 2 | Viewed by 1637
Abstract
The aim of this study was to investigate the effects of different cooking methods on the hepatoprotective effects of purple sweet potatoes against alcohol-induced damage in HepG2 cells. Purple sweet potatoes (Ipomeoea batatas L. Danjami) were subjected to different cooking methods, including [...] Read more.
The aim of this study was to investigate the effects of different cooking methods on the hepatoprotective effects of purple sweet potatoes against alcohol-induced damage in HepG2 cells. Purple sweet potatoes (Ipomeoea batatas L. Danjami) were subjected to different cooking methods, including steaming, roasting, and microwaving. Steaming resulted in a higher cytoprotective effect against alcohol damage than the other cooking methods. Additionally, the highest inhibition of glutathione depletion and production of reactive oxygen species against alcohol-induced stress were observed in raw and/or steamed purple sweet potatoes. Compared to roasted and/or microwaved samples, steamed samples significantly increased the expression of NADPH quinone oxidoreductase 1, heme oxygenase 1, and gamma glutamate-cysteine ligase in alcohol-stimulated HepG2 cells via the activation of nuclear factor erythroid 2-related factor 2. Moreover, ten anthocyanins were detected in the raw samples, whereas five, two, and two anthocyanins were found in the steamed, roasted, and microwaved samples, respectively. Taken together, steaming purple sweet potatoes could be an effective cooking method to protect hepatocytes against alcohol consumption. These results provide useful information for improving the bioactive properties of purple sweet potatoes using different cooking methods. Full article
(This article belongs to the Special Issue Nrf2 Antioxidative Pathway and NF-κB Signaling)
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<p>Cytotoxicity of various concentration of purple sweet potato (<b>a</b>) and cytoprotective effect (<b>b</b>) of various concentration of samples (25, 50, and 100 µg/mL) against 4% ethanol-induced cytotoxicity. The vertical bars represent mean values ± SD and values marked by same letter are not significantly different (<span class="html-italic">p</span> &lt; 0.05). NS, not significant.</p> Full article ">Figure 2
<p>Effect of purple sweet potato with different cooking methods on ethanol-induced ROS in HepG2 cells. The intracellular reactive oxygen species were measured by monitoring fluorescence increase at 120 min. The vertical bars represent mean values ± SD and values marked by same letter are not significantly different (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 3
<p>Effects of purple sweet potato with different cooking methods on ethanol-induced GSH depletion in HepG2 cells. The vertical bars represent mean values ± SD and values marked by same letter are not significantly different (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 4
<p>Effects of purple sweet potato with different cooking methods on the protein expression of HO-1 (<b>a</b>), NQO1 (<b>b</b>), and GCLC (<b>c</b>) in ethanol-induced HepG2 cells. β-actin served as internal control. All data are representative of three independent experiments. The vertical bars represent as mean values ± SD and values marked by same letter are not significantly different (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 5
<p>Effects of purple sweet potato with different cooking methods on Nrf2 protein expression in cytoplasm (<b>a</b>) and nucleus (<b>b</b>). PCNA for nuclear fraction and β-actin for cytoplasmic fraction were used as an internal control. All data are representative of three independent experiments. The vertical bars represent mean values ± SD and values marked by same letter are not significantly different (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 6
<p>Total ion chromatogram (TIC) of anthocyanins in raw (<b>a</b>), steamed (<b>b</b>), roasted (<b>c</b>), and microwaved (<b>d</b>) purple sweet potato by different cooking methods using LC-QTOF/MS analysis. Cyanidin-3-sophoroside-5-glucoside (1), peonidin-3-sophoroside-5-glucoside (2), cyanidin 3-p-hydroxybenzoyl sophoroside-5-glucoside (3), peonidin 3-p-hydroxybenzoyl sophoroside-5-glucoside (4), cyanidin 3-(6′′′-caffeoyl sophoroside)-5-glucoside (5), cyanidin 3-dicaffeoyl sophoroside-5-glucoside (6), cyanidin 3-dicaffeoyl sophoroside-p-hydroxybenzoyl sophoroside-5-glucoside (7), peonidin 3-(6′′′-caffeoyl sophoroside)-5-glucoside (8), peonidin 3-dicaffeoyl sophoroside-5-glucoside (9), peonidin 3-caffeoyl sophoroside-p-hydroxybenzoyl sophoroside-5-glucoside (10).</p> Full article ">
25 pages, 3368 KiB  
Review
Oxidative Stress and Its Regulation in Diabetic Retinopathy
by Cameron D. Haydinger, Genevieve F. Oliver, Liam M. Ashander and Justine R. Smith
Antioxidants 2023, 12(8), 1649; https://doi.org/10.3390/antiox12081649 - 21 Aug 2023
Cited by 11 | Viewed by 6367
Abstract
Diabetic retinopathy is the retinal disease associated with hyperglycemia in patients who suffer from type 1 or type 2 diabetes. It includes maculopathy, involving the central retina and characterized by ischemia and/or edema, and peripheral retinopathy that progresses to a proliferative stage with [...] Read more.
Diabetic retinopathy is the retinal disease associated with hyperglycemia in patients who suffer from type 1 or type 2 diabetes. It includes maculopathy, involving the central retina and characterized by ischemia and/or edema, and peripheral retinopathy that progresses to a proliferative stage with neovascularization. Approximately 10% of the global population is estimated to suffer from diabetes, and around one in 5 of these individuals have diabetic retinopathy. One of the major effects of hyperglycemia is oxidative stress, the pathological state in which elevated production of reactive oxygen species damages tissues, cells, and macromolecules. The retina is relatively prone to oxidative stress due to its high metabolic activity. This review provides a summary of the role of oxidative stress in diabetic retinopathy, including a description of the retinal cell players and the molecular mechanisms. It discusses pathological processes, including the formation and effects of advanced glycation end-products, the impact of metabolic memory, and involvements of non-coding RNA. The opportunities for the therapeutic blockade of oxidative stress in diabetic retinopathy are also considered. Full article
(This article belongs to the Special Issue The Role of Oxidative Stress in Vascular Disease Associated with Diabetes)
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<p>Clinical features of diabetic retinopathy. (<b>A</b>) Color photograph of the posterior pole of a left eye demonstrating optic nerve head, vascular arcades, and macula. Diabetic maculopathy without edema is present, and microaneurysms and dot hemorrhages can be seen at the macula. (<b>B</b>) Color photograph of the posterior pole of a right eye demonstrating clinically significant diabetic macular edema. Hard exudates appear as well-demarcated yellow deposits at the macula (B1). (<b>C</b>) Optical coherence tomography of the right macula that is shown in panel B, demonstrating overall thickening in heat map image on the left and intraretinal fluid on the retinal cross-sectional image on the right. (<b>D</b>,<b>E</b>) Widefield color fundus photographs of proliferative diabetic retinopathy in two left eyes, demonstrating representative signs of diabetic retinopathy: (D1) cotton wool spot, (D2) venous beading, (D3) intraretinal microvascular abnormalities, (D4) neovascularization at the disc, (D5) venous loop, (E1) intraretinal microvascular abnormalities, (E2) a retinal scar from laser photocoagulation, (E3) retinal neovascularization, (E4) pre-retinal hemorrhage, (E5) blot hemorrhage, and (E6) vitreous hemorrhage.</p> Full article ">
26 pages, 3707 KiB  
Review
The Unfolded Protein Response: A Double-Edged Sword for Brain Health
by Magdalena Gebert, Jakub Sławski, Leszek Kalinowski, James F. Collawn and Rafal Bartoszewski
Antioxidants 2023, 12(8), 1648; https://doi.org/10.3390/antiox12081648 - 21 Aug 2023
Cited by 4 | Viewed by 2013
Abstract
Efficient brain function requires as much as 20% of the total oxygen intake to support normal neuronal cell function. This level of oxygen usage, however, leads to the generation of free radicals, and thus can lead to oxidative stress and potentially to age-related [...] Read more.
Efficient brain function requires as much as 20% of the total oxygen intake to support normal neuronal cell function. This level of oxygen usage, however, leads to the generation of free radicals, and thus can lead to oxidative stress and potentially to age-related cognitive decay and even neurodegenerative diseases. The regulation of this system requires a complex monitoring network to maintain proper oxygen homeostasis. Furthermore, the high content of mitochondria in the brain has elevated glucose demands, and thus requires a normal redox balance. Maintaining this is mediated by adaptive stress response pathways that permit cells to survive oxidative stress and to minimize cellular damage. These stress pathways rely on the proper function of the endoplasmic reticulum (ER) and the activation of the unfolded protein response (UPR), a cellular pathway responsible for normal ER function and cell survival. Interestingly, the UPR has two opposing signaling pathways, one that promotes cell survival and one that induces apoptosis. In this narrative review, we discuss the opposing roles of the UPR signaling pathways and how a better understanding of these stress pathways could potentially allow for the development of effective strategies to prevent age-related cognitive decay as well as treat neurodegenerative diseases. Full article
(This article belongs to the Special Issue Oxidative Stress in Brain Function)
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<p>The role of the endoplasmic reticulum (ER) in maintaining neuron cell homeostasis. (<b>A</b>) As the main Ca<sup>2+</sup> reservoir, the ER is crucial for the regulation of cytosolic Ca<sup>2+</sup> concentration using pumps and channels localized in ER membrane. Those include sarco/endoplasmic reticulum Ca<sup>2+</sup> ATPase (SERCA), Ca<sup>2+</sup>-activated ryanodine receptors (RyRs), and inositol-1,4,5-trisphosphate (IP<sub>3</sub>)-gated IP<sub>3</sub> receptors (IP<sub>3</sub>Rs). They cooperate with the cell membrane Ca<sup>2+</sup> transporters that regulate the influx of extracellular Ca<sup>2+</sup>, exemplified by plasma membrane Ca<sup>2+</sup> ATPase (PMCA) and N-methyl-D-aspartate receptor (NMDAR). (<b>B</b>) Ca<sup>2+</sup> homeostasis processes in the ER and mitochondrion are tightly interconnected, primarily by virtue of the regions of mitochondria-associated membranes (MAMs). An increase in Ca<sup>2+</sup> concentration in MAM promotes its influx into the mitochondrion, mainly through voltage-dependent anion channel (VDAC). High Ca<sup>2+</sup> concentration stimulates the activity of the oxidative processes in the mitochondrion, leading to the increased production of reactive oxygen species (ROS). In turn, ROS-dependent modifications of ER Ca<sup>2+</sup> channels increase their permeability for Ca<sup>2+</sup> and the efflux of Ca<sup>2+</sup> from ER, which closes the positive-feedback loop. (<b>C</b>) The ER is a central cell compartment where the synthesis and quality control of secretory and membrane proteins takes place. The properly folded proteins are directed through secretory pathway to the cell membrane, whereas irreversibly unfolded/misfolded proteins are exported and eventually degraded either in lysosomes or proteasomes. (<b>D</b>) ER-based lipid crosstalk between neurons and astrocytes. Fatty acids (FAs) and the products of their oxidation synthesized in astrocytes are delivered to neurons to support their demand for energy and membrane building components. In turn, nonfunctional peroxidated FAs released by neurons are endocytosed by astrocytes and stored in lipid droplets or catabolized by the mitochondrial FA oxidation pathway.</p> Full article ">Figure 2
<p>The unfolded protein response (UPR) pathway. (<b>A</b>) Three UPR sensors—inositol-requiring protein 1α (IRE1α), protein kinase RNA (PKR)-like endoplasmic reticulum kinase (PERK) and activating transcription factor 6 (ATF6)—are localized in endoplasmic reticulum (ER) membrane and share a common activation signal: the dissociation of binding immunoglobulin protein (BiP) chaperone in response to increased level of unfolded/misfolded proteins. Dimerization of IRE1α, followed by its trans-autophosphorylation, activates its RNase domain. The primary target of IRE1α is the unspliced X box-binding protein 1 (XBP1u) transcript. Spliced XBP1 mRNA (XBP1s) encodes transcription factor XBP1s, which activates UPR-associated genes. IRE1α also degrades certain mRNAs through the regulated IRE1-dependent decay (RIDD) process. Upon dimerization and trans-autophosphorylation, PERK phosphorylates eukaryotic translation initiator factor 2α (eIF2α) to attenuate general protein translation. Phosphorylated eIF2α promotes expression of activating transcription factor 4 (ATF4) and nuclear factor erythroid 2-related factor 2 (NRF2), which are involved in the response to ER and oxidative stress, respectively. ER stress triggers the cleavage of disulfide bonds, stabilizing ATF6 oligomers by protein disulfide isomerase family A member 5 (PDIA5), and this is followed by its transport to the Golgi apparatus where it is processed by site 1 and site 2 proteases (S1P, S2P). Cytosolic ATF6 fragment (ATF6f) is released and imported to the nucleus, where it plays the role of an active transcription factor. (<b>B</b>) Under extensive and persistent ER stress, the UPR switches from proadaptive to a proapoptotic character. Oligomerized IRE1α, stabilized by the disulfide bonds formed by protein disulfide isomerase family A member 6 (PDIA6), recruits tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2), which in turn activates the proapoptotic signal-regulating kinase 1/Janus N-terminal kinase (ASK1/JNK) and the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathways. ATF4 promotes the expression of CCAAT/enhancer-binding protein homologous protein (CHOP) and transcription factor targeting apoptotic genes, including growth arrest and DNA damage-inducible 45 alpha (GADD45A), p53 upregulated modulator of apoptosis (PUMA), phorbol-12-myristate-13-acetate-induced protein 1 (NOXA), and growth arrest and DNA damage-inducible 34 (GADD34). GADD34 forms a complex with protein phosphatase 1 (PP1) to dephosphorylate eIF2α and reverse the inhibition of translation.</p> Full article ">Figure 3
<p>Induction of UPR by oxidative stress. (<b>A</b>) Elevated reactive oxygen species (ROS) levels may cause the oxidation of endoplasmic reticulum (ER) calcium transporters, most notably, ryanodine receptors (RyRs), and inositol-1,4,5-trisphosphate (IP<sub>3</sub>) receptors (IP<sub>3</sub>Rs). Elevated ROS levels also promote sulfoxidation of Cys674 of sarco/endoplasmic reticulum Ca<sup>2+</sup> ATPase (SERCA). These modifications lead to efflux of Ca<sup>2+</sup> from ER and impairment of Ca<sup>2+</sup>-dependent chaperons, calnexin and calreticulin. (<b>B</b>) The disturbance of ER Ca<sup>2+</sup> homeostasis may spread through mitochondria-associated membranes and target the mitochondrion, causing the Ca<sup>2+</sup> influx through the voltage-dependent anion channel (VDAC). High Ca<sup>2+</sup> concentrations induce mitochondrial stress, which leads to activation of the mitochondrial unfolded protein response (mUPR) and formation of mitochondrial permeability transition pores (mPTP). Increased leakage of ROS from electron transport chains and depletion of ATP enhances further ER stress and deregulation of Ca<sup>2+</sup> homeostasis. (<b>C</b>) Increased ROS concentrations combined with the production of NO by nNOS (neuronal nitric oxide synthase) leads to the formation of peroxynitrate (ONOO<sup>-</sup>) which reacts with thiol group of proteins. S-nitrosylation inhibits the activity of modified proteins, including protein disulfide isomerases (PDIs). PDIs, accompanied by ER oxidoreductin 1 (ERO1), catalyze the formation and cleavage of disulfide bonds, and are one of the crucial components of the ER proteostasis system. The reduced-to-oxidized ratio of glutathione (GSH/GSSG), which plays a role analogous to PDIs, may also be increased by the oxidative environment in ER. PDIs also directly affect the UPR sensors and activate transcription factor 6 (ATF6) and inositol-requiring protein 1α (IRE1α).</p> Full article ">Figure 4
<p>The crosstalk between ER stress and oxidative stress. The main linkage between endoplasmic reticulum (ER) and mitochondrion homeostasis is the Ca<sup>2+</sup> concentration interdependence.</p> Full article ">
5 pages, 224 KiB  
Editorial
Antioxidant Properties of Bioactive Compounds in Fruit and Vegetable Waste
by Nerea Jiménez-Moreno, Irene Esparza and Carmen Ancín-Azpilicueta
Antioxidants 2023, 12(8), 1647; https://doi.org/10.3390/antiox12081647 - 21 Aug 2023
Cited by 1 | Viewed by 1099
Abstract
In recent years, great interest has arisen in the study of compounds with antioxidant activity present in agri-food residues [...] Full article
(This article belongs to the Special Issue Antioxidant Properties of Bioactive Compounds in Fruit and Vegetable Waste)
27 pages, 12847 KiB  
Article
Effects of Fisetin Treatment on Cellular Senescence of Various Tissues and Organs of Old Sheep
by Charles A. Huard, Xueqin Gao, Maria E. Dey Hazra, Rony-Orijit Dey Hazra, Kimberly Lebsock, Jeremiah T. Easley, Peter J. Millett and Johnny Huard
Antioxidants 2023, 12(8), 1646; https://doi.org/10.3390/antiox12081646 - 21 Aug 2023
Cited by 6 | Viewed by 4112
Abstract
Fisetin has been shown to be beneficial for brain injury and age-related brain disease via different mechanisms. The purpose of this study was to determine the presence of senescent cells and the effects of fisetin on cellular senescence in the brain and other [...] Read more.
Fisetin has been shown to be beneficial for brain injury and age-related brain disease via different mechanisms. The purpose of this study was to determine the presence of senescent cells and the effects of fisetin on cellular senescence in the brain and other vital organs in old sheep, a more translational model. Female sheep 6–7 years old (N = 6) were treated with 100 mg/kg fisetin or vehicle alone on two consecutive days a week for 8 weeks. All vital organs were harvested at the time of sacrifice. Histology, immunofluorescence staining, and RT-Q-PCR were performed on different regions of brain tissues and other organs. Our results indicated that fisetin treatment at the current regimen did not affect the general morphology of the brain. The presence of senescent cells in both the cerebral brain cortex and cerebellum and non-Cornu Ammonis (CA) area of the hippocampus was detected by senescent-associated β-galactosidase (SA-β-Gal) staining and GL13 (lipofuscin) staining. The senescent cells detected were mainly neurons in both gray and white matter of either the cerebral brain cortex, cerebellum, or non-CA area of the hippocampus. Very few senescent cells were detected in the neurons of the CA1-4 area of the hippocampus, as revealed by GL13 staining and GLB1 colocalization with NEUN. Fisetin treatment significantly decreased the number of SA-β-Gal+ cells in brain cortex white matter and GL13+ cells in the non-CA area of the hippocampus, and showed a decreasing trend of SA-β-Gal+ cells in the gray matter of both the cerebral brain cortex and cerebellum. Furthermore, fisetin treatment significantly decreased P16+ and GLB1+ cells in neuronal nuclear protein (NEUN)+ neurons, glial fibrillary acidic protein (GFAP)+ astrocytes, and ionized calcium binding adaptor molecule 1 (IBA1)+ microglia cells in both gray and white matter of cerebral brain cortex. Fisetin treatment significantly decreased GLB1+ cells in microglia cells, astrocytes, and NEUN+ neurons in the non-CA area of the hippocampus. Fisetin treatment significantly decreased plasma S100B. At the mRNA level, fisetin significantly downregulated GLB1 in the liver, showed a decreasing trend in GLB1 in the lung, heart, and spleen tissues, and significantly decreased P21 expression in the liver and lung. Fisetin treatment significantly decreased TREM2 in the lung tissues and showed a trend of downregulation in the liver, spleen, and heart. A significant decrease in NRLP3 in the liver was observed after fisetin treatment. Finally, fisetin treatment significantly downregulated SOD1 in the liver and spleen while upregulating CAT in the spleen. In conclusion, we found that senescent cells were widely present in the cerebral brain cortex and cerebellum and non-CA area of the hippocampus of old sheep. Fisetin treatment significantly decreased senescent neurons, astrocytes, and microglia in both gray and white matter of the cerebral brain cortex and non-CA area of the hippocampus. In addition, fisetin treatment decreased senescent gene expressions and inflammasomes in other organs, such as the lung and the liver. Fisetin treatment represents a promising therapeutic strategy for age-related diseases. Full article
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<p>Gross images and H&amp;E staining of brain tissues after fisetin treatment. (<b>A</b>) Cross-sectional view of the cerebellum and left hemisphere of cerebral brain cortex at different axial level. Red arrows indicate white matter, green arrows indicate gray matter, and pink arrows point to hippocampus. (<b>B</b>) H&amp;E staining of cerebral cortex. (<b>C</b>) H&amp;E staining of cerebellum for gray matter and white matter, showing different cellularity. (<b>D</b>) H&amp;E staining of hippocampus at CA1, CA3, and DG areas. Fisetin treatment did not significantly affect the general morphology of different regions. Scale bars = 100 µm.</p> Full article ">Figure 2
<p>Effects of fisetin treatment on senescent cells in different regions of the brain. (<b>A</b>) Brain cortex SA-β-Gal staining of gray matter region showing many blue stained cells that are mainly large neurons. Nuclei were not stained. (<b>B</b>) Quantification of SA-β-Gal-positive cells. (<b>C</b>) Brain cortex SA-β-Gal staining in the white matter region. (<b>D</b>) Quantification of SA-β-Gal-positive cells in the brain cortex white matter. (<b>E</b>) Cerebellum gray matter SA-β-Gal staining. (<b>F</b>) Quantification of SA-β-Gal-positive cells in the cerebellum gray matter. (<b>G</b>) Cerebellum white matter SA-β-Gal staining. (<b>H</b>) Quantification of SA-β-Gal-positive cells in the white matter of the cerebellum. (<b>I</b>) GL13 (SenTraGor) staining for lipofuscin. GL13<sup>+</sup> cells were stained brown in the cytoplasm. (<b>J</b>) Quantification of GL13<sup>+</sup> cells in the CA area neurons. (<b>K</b>) Quantification of GL13<sup>+</sup> cells of large neurons in non-CA area of hippocampus. Red boxes in each image highlighted SA-β-Gal-positive cells or GL13<sup>+</sup> cells. Scale bars = 100 µm. Exact <span class="html-italic">p</span> values are indicated between group bars.</p> Full article ">Figure 3
<p>Identification of senescent cells of brain cortex and effects of fisetin treatment. (<b>A</b>) P16 and NEUN double immunofluorescent staining for neurons in brain cortex gray and white matter. P16 is stained green in the nuclei. NEUN-labeled neurons are stained in red in the nuclei and perinuclear cytoplasm. Nuclei are stained in blue with DAPI. There are many large red-stained neurons in the gray matter and very few in the white matter. The green channel reveals P16<sup>+</sup> cells in the gray matter that are bigger with diffused staining, while there are small cells with strong signals in the white matter. (<b>B</b>) Quantification of P16<sup>+</sup>/NEUN<sup>+</sup>/Total NEUN<sup>+</sup> cells percentage in the gray matter. (<b>C</b>) Quantification of P16<sup>+</sup>/NEUN<sup>−</sup>/Total DAPI<sup>+</sup> cells in the white matter. (<b>D</b>) P16 and GFAP double immunofluorescence staining of astrocytes in the brain cortex gray and white matter. P16<sup>+</sup> cells are stained green in the nuclei, while GFAP<sup>+</sup> cells are stained red for astrocytes with many projections. (<b>E</b>) Quantification of P16<sup>+</sup>/GFAP<sup>+</sup>/Total GFAP<sup>+</sup> cells percentage in the gray matter. (<b>F</b>) Quantification of P16<sup>+</sup>/GFAP<sup>+</sup>/Total GFAP<sup>+</sup> cells percentage in the white matter. (<b>G</b>) P16 and IBA1 double immunofluorescence staining for microglia cells. P16<sup>+</sup> cells are stained green in the nuclei, while IBA1<sup>+</sup>microglia cells are stained in red. (<b>H</b>) Quantification of P16<sup>+</sup>/IBA1<sup>+</sup>/Total IBA1<sup>+</sup>cells percentage in the gray matter. (<b>I</b>) Quantification of P16<sup>+</sup>/IBA1<sup>+</sup>/Total IBA1<sup>+</sup> cells percentage in the white matter. s White boxes highlight positive cells. Scale bars = 100 µm. Exact <span class="html-italic">p</span> values are shown between the group bars.</p> Full article ">Figure 4
<p>Identification of senescent cells in the brain cortex using double IF staining of GLB1 with different neuronal markers. (<b>A</b>) Double IF of GLB1 with NEUN. NEUN<sup>+</sup> neurons are stained in red in the nuclei and perinuclear cytoplasm, while GLB1 is stained in green in the cytoplasm. Nuclei are stained in blue with DAPI. Colocalized cells are stained in orange/yellow. The green channel shows GLB1 staining. (<b>B</b>) Quantification of GLB1<sup>+</sup>NEUN<sup>+</sup>/Total NEUN<sup>+</sup> cells percentage in the gray matter of the brain cortex. (<b>C</b>) Double IF of GLB1 with GFAP. GFAP<sup>+</sup> cells are stained in red, showing many projections. GLB1<sup>+</sup>GFAP<sup>+</sup>cells are shown in orange, highlighted in the insets. (<b>D</b>) Quantification of GLB1<sup>+</sup>GFAP<sup>+</sup>/Total GFAP<sup>+</sup> cells percentage in the gray matter of the brain cortex. (<b>E</b>) Quantification of GLB1<sup>+</sup>GFAP<sup>+</sup>/Total GFAP<sup>+</sup> cells percentage in the white matter of the brain cortex. (<b>F</b>) Double IF for GLB1 and IBA1. IBA1<sup>+</sup> cells are stained in red color, and are small with few projections. (<b>G</b>) Quantification of the GLB1<sup>+</sup>IBA1<sup>+</sup>/total IBA1<sup>+</sup> cells percentage in the gray matter of the brain cortex. (<b>H</b>) Quantification of the GLB1<sup>+</sup>IBA1<sup>+</sup>/total IBA1<sup>+</sup> cells percentage in the white matter of the brain cortex. Scale bars = 100 µm. White boxes in each image highlight positive cells. Exact <span class="html-italic">p</span> values are shown between the group bars.</p> Full article ">Figure 5
<p>Effects of fisetin treatment on the cellular senescence of different cells in the hippocampus. (<b>A</b>) NEUN and P16 double immunofluorescence staining for the hippocampus, focusing on the CA area. NEUN<sup>+</sup> cells are stained in red and many are colocalized with P16 (merged channel). P16<sup>+</sup> cells are stained in green in the nuclei (green channel). (<b>B</b>) Quantification of P16<sup>+</sup>/NEUN<sup>+</sup>/Total NEUN<sup>+</sup> cells in the CA1-4 area. (<b>C</b>) GFAP and P16 double immunofluorescence staining in the hippocampus. GFAP<sup>+</sup> cells are stained in red, showing many projections, and are mainly located in the non-CA area. P16<sup>+</sup> cells are stained in green in the nuclei. (<b>D</b>) Quantification of P16<sup>+</sup>GFAP<sup>+</sup>/Total GFAP<sup>+</sup> cells in the non-CA area of the hippocampus. (<b>E</b>) IBA1 and P16 double immunofluorescence staining in the hippocampus. IBA1<sup>+</sup> cells are stained in red and mainly located in the non-CA area. P16<sup>+</sup> cells are stained in green in the nuclei. (<b>F</b>) Quantification of P16<sup>+</sup>/IBA1<sup>+</sup>/Total IBA1<sup>+</sup> cells in the non-CA area of the hippocampus. White boxes in each image highlight positive cells of each staining. Scale bars = 100 µm. Exact <span class="html-italic">p</span> values are indicated between the group bars.</p> Full article ">Figure 6
<p>Double IF of GLB1 and neuronal marker for the hippocampus and the effects of fisetin. (<b>A</b>) Double IF for GLB1 and NEUN to detect senescent neurons. GLB1<sup>+</sup> cells are stained in green in the cytoplasm (green channel). Very few GLB1<sup>+</sup>NEUN<sup>+</sup> cells are found in the neurons of the CA 1-4 area. Many GLB1<sup>+</sup>NEUN<sup>+</sup> cells can be observed in the non-CA area neurons, shown in a distinct patched cytoplasm yellow color (highlighted in the insets). (<b>B</b>) Quantification of GLB1<sup>+</sup>NEUN<sup>+</sup> cells/total NEUN<sup>+</sup> cells percentage in the CA1-4 area. (<b>C</b>) Quantification of GLB1<sup>+</sup>NEUN<sup>+</sup>/total NEUN<sup>+</sup> cells in the non-CA area (large neurons). (<b>D</b>) Double IF for GLB1 and GFAP. GFAP<sup>+</sup> astrocytes are stained in red, showing several projections in the non-CA area. GLB1<sup>+</sup> cells are stained green in the cytoplasm. GLB1<sup>+</sup>GAFP<sup>+</sup> cells are highlighted in the insets. (<b>E</b>) Quantification of GLB1<sup>+</sup>GFAP<sup>+</sup>/total GFAP<sup>+</sup> cells percentage. (<b>F</b>) Double IF staining of GLB1 and IBA1 to detect senescent microglia. IBA1<sup>+</sup> cells are stained in red, with smaller size and few projections in the non-CA area. GLB1<sup>+</sup>IBA1<sup>+</sup> cells are highlighted in the insets. (<b>G</b>) Quantification of GLB1<sup>+</sup>IBA1<sup>+</sup>/total IBA1<sup>+</sup> cells percentage in non-CA area. Scale bars = 100 µm. White boxes highlight double positive cells in each staining. Exact <span class="html-italic">p</span> values are indicated between the group bars.</p> Full article ">Figure 7
<p>Semi-quantitative and quantitative RT- Q-PCR results in the brain cortex after fisetin treatment. (<b>A</b>) Electrophoresis images of different target genes. (<b>B</b>) Band density quantification relative to GAPDH. (<b>C</b>) RT-Q-PCR analysis of antioxidant genes. (<b>D</b>) RT-Q-PCR analysis of NEFL, NEFH, and GFAP. (<b>E</b>) RT-Q-PCR analysis of senescence related genes. (<b>F</b>) RT-Q-PCR analysis of SASP genes. (<b>G</b>) RT-Q-PCR analysis of inflammasome genes. Exact <span class="html-italic">p</span> values are shown between the group bars.</p> Full article ">Figure 8
<p>RT-Q-PCR analysis of gene expression of sheep heart tissues after fisetin treatment: (<b>A</b>) RT-Q-PCR results of antioxidant genes; (<b>B</b>) RT-Q-PCR analysis of senescence related genes; (<b>C</b>) mRNA expression of SASP genes; (<b>D</b>) mRNA expression of inflammasome genes. Exact <span class="html-italic">p</span> values are shown between the group bars.</p> Full article ">Figure 9
<p>Gene expressions in the spleen tissues after fisetin treatment: (<b>A</b>) ntioxidant genes mRNA expression; (<b>B</b>) senescence-related genes mRNA expression; (<b>C</b>) SASP gene mRNA expression; (<b>D</b>) inflammasome gene expression. Exact <span class="html-italic">p</span> values are shown between the group bars.</p> Full article ">Figure 10
<p>mRNA gene expression of sheep bone marrow after fisetin treatment: (<b>A</b>) antioxidant genes mRNA expression; (<b>B</b>) senescence-related genes; (<b>C</b>) mRNA expression of P53; (<b>D</b>) SASP genes; (<b>E</b>) inflammasome genes. Exact <span class="html-italic">p</span> values are shown between the group bars.</p> Full article ">Figure 11
<p>Effects of fisetin treatment on gene expression in the lung tissue: (<b>A</b>) mRNA expression of antioxidant genes; (<b>B</b>,<b>C</b>) mRNA expression of senescent cells genes; (<b>D</b>) mRNA expression of SASP gene IL10; (<b>E</b>) mRNA expression of inflammasome genes. Exact <span class="html-italic">p</span> values are indicated between the group bars.</p> Full article ">Figure 12
<p>Effects of fisetin treatment on gene expression of the liver: (<b>A</b>) mRNA expression of antioxidant genes; (<b>B</b>) mRNA expression of senescent genes; (<b>C</b>) mRNA expression of SASP gene IL10; (<b>D</b>) mRNA expression of inflammasome genes. Exact <span class="html-italic">p</span> values are indicated between the groups.</p> Full article ">Figure 13
<p>Effects of fisetin treatment on plasma MDA and S100B: (<b>A</b>) plasma MDA concentration and (<b>B</b>) plasma S100B concentration. Exact <span class="html-italic">p</span> values are indicated between the group bars.</p> Full article ">
16 pages, 1624 KiB  
Article
Individualised Exercise Training Enhances Antioxidant Buffering Capacity in Idiopathic Pulmonary Fibrosis
by Tim J. M. Wallis, Magdalena Minnion, Anna Freeman, Andrew Bates, James M. Otto, Stephen A. Wootton, Sophie V. Fletcher, Michael P. W. Grocott, Martin Feelisch, Mark G. Jones and Sandy Jack
Antioxidants 2023, 12(8), 1645; https://doi.org/10.3390/antiox12081645 - 20 Aug 2023
Viewed by 1445
Abstract
Exercise training is recommended for patients with idiopathic pulmonary fibrosis (IPF); however, the mechanism(s) underlying its physiological benefits remain unclear. We investigated the effects of an individualised aerobic interval training programme on exercise capacity and redox status in IPF patients. IPF patients were [...] Read more.
Exercise training is recommended for patients with idiopathic pulmonary fibrosis (IPF); however, the mechanism(s) underlying its physiological benefits remain unclear. We investigated the effects of an individualised aerobic interval training programme on exercise capacity and redox status in IPF patients. IPF patients were recruited prospectively to an 8-week, twice-weekly cardiopulmonary exercise test (CPET)-derived structured responsive exercise training programme (SRETP). Systemic redox status was assessed pre- and post-CPET at baseline and following SRETP completion. An age- and sex-matched non-IPF control cohort was recruited for baseline comparison only. At baseline, IPF patients (n = 15) had evidence of increased oxidative stress compared with the controls as judged by; the plasma reduced/oxidised glutathione ratio (median, control 1856 vs. IPF 736 p = 0.046). Eleven IPF patients completed the SRETP (median adherence 88%). Following SRETP completion, there was a significant improvement in exercise capacity assessed via the constant work-rate endurance time (+82%, p = 0.003). This was accompanied by an improvement in post-exercise redox status (in favour of antioxidants) assessed via serum total free thiols (median increase, +0.26 μmol/g protein p = 0.005) and total glutathione concentration (+0.73 μM p = 0.03), as well as a decrease in post-exercise lipid peroxidation products (−1.20 μM p = 0.02). Following SRETP completion, post-exercise circulating nitrite concentrations were significantly lower compared with baseline (−0.39 μM p = 0.04), suggestive of exercise-induced nitrite utilisation. The SRETP increased both endurance time and systemic antioxidant capacity in IPF patients. The observed reduction in nitrite concentrations provides a mechanistic rationale to investigate nitrite/nitrate supplementation in IPF patients. Full article
(This article belongs to the Special Issue Exercise-Induced Oxidative Stress in Health and Disease)
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Figure 1

Figure 1
<p>Participant flow diagram. FEV1/FVC—ratio of forced expiratory volume in 1 s to forced vital capacity, LTOT/AOT—long-term oxygen therapy/ambulatory oxygen therapy, * Study recruitment and follow-up assessments of patients with IPF were terminated on 17 March 2020 due to the impact of the COVID-19 pandemic.</p> Full article ">Figure 2
<p>Redox biomarker concentrations at baseline pre- and post-cardiopulmonary exercise testing (CPET) in control vs. IPF participants. PRE-CPET (blue circles) and POST-CPET (red circles). (<b>A</b>) GSH/GSSG—ratio of reduced glutathione (GSH) over glutathione disulfide (GSSG)†; (<b>B</b>) total glutathione; (<b>C</b>) total free thiols (TFT)/protein—total free thiols corrected for protein concentration; (<b>D</b>) TBARS—thiobarbituric acid reactive substances; (<b>E</b>) nitrite; (<b>F</b>) nitrate; (<b>G</b>) RXNO (total nitroso species). Control PRE <span class="html-italic">n</span> = 10, Control POST <span class="html-italic">n</span> = 10, IPF PRE <span class="html-italic">n</span> = 15 IPF POST <span class="html-italic">n</span> = 14. Error bars median ± interquartile range * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01. † Control PRE: GSH/GSSG <span class="html-italic">n</span> = 9. IPF PRE: GSH/GSSG <span class="html-italic">n</span> = 14 and RXNO <span class="html-italic">n</span> = 14. Control POST: GSH/GSSG and RXNO <span class="html-italic">n</span> = 9. IPF POST: GSH/GSSG and RXNO <span class="html-italic">n</span> = 13.</p> Full article ">Figure 3
<p>Primary outcome (endurance time) at baseline and Week 9 for IPF patients completing the structured responsive exercise training programme (SRETP). Endurance time (minutes) at baseline and Week 9 on constant work-rate test at 75% of work rate at peak volume of oxygen consumption (<math display="inline"><semantics> <mrow> <mover> <mi mathvariant="normal">V</mi> <mo>·</mo> </mover> </mrow> </semantics></math>O<sub>2</sub>peak) for IPF patients completing the exercise programme (<span class="html-italic">n</span> = 11). (<b>A</b>) Individual dot plot with error bars—median ± interquartile range. (<b>B</b>) Line graph of individual patient responses. ** <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">Figure 4
<p>Secondary clinical outcomes for IPF participants completing the structured responsive exercise training programme (SRETP). (<b>A</b>) Peak work rate (Watts), (<b>B</b>) predicted forced vital capacity percent (FVC% predicted), (<b>C</b>) peak minute ventilation (peak <math display="inline"><semantics> <mrow> <mover> <mi mathvariant="normal">V</mi> <mo>·</mo> </mover> </mrow> </semantics></math>E, L·min⁻<sup>1</sup>), (<b>D</b>) peak volume of oxygen consumption (<math display="inline"><semantics> <mrow> <mover> <mi mathvariant="normal">V</mi> <mo>·</mo> </mover> </mrow> </semantics></math>O<sub>2</sub>peak, mL·kg⁻<sup>1</sup>·min⁻<sup>1</sup>), (<b>E</b>) anaerobic threshold (AT, mL·kg⁻<sup>1</sup>·min⁻<sup>1</sup>) (<b>F</b>) relationship between oxygen uptake and work rate to anaerobic threshold (AT) (<math display="inline"><semantics> <mrow> <mover> <mi mathvariant="normal">V</mi> <mo>·</mo> </mover> </mrow> </semantics></math>O<sub>2</sub>/WR to AT) <span class="html-italic">n</span> = 11. Error bars - median ± interquartile range.* <span class="html-italic">p</span> &lt; 0.05. Note <span class="html-italic">n</span> = 10 AT and <math display="inline"><semantics> <mrow> <mover> <mi mathvariant="normal">V</mi> <mo>·</mo> </mover> </mrow> </semantics></math>O2/WR to AT.</p> Full article ">Figure 5
<p>Redox biomarker concentrations pre- and post-cardiopulmonary exercise test (CPET) for IPF patients completing the structured responsive exercise training programme (SRETP). Baseline (orange circles), Week 4 (purple circles), and Week 9 (green circles). (<b>A</b>) GSH/GSSG ratio of reduced glutathione (GSH) over glutathione disulfide (GSSG), (<b>B</b>) total glutathione, (<b>C</b>) total free thiols (TFT)/protein—total free thiols corrected for protein concentration, (<b>D</b>) TBARS—thiobarbituric acid reactive substances, (<b>E</b>) nitrite, (<b>F</b>) nitrate (<b>G</b>) RXNO (total nitroso species). Baseline PRE <span class="html-italic">n</span> = 11, baseline POST <span class="html-italic">n</span> = 10, Week 4 PRE <span class="html-italic">n</span> = 8, Week 4 POST <span class="html-italic">n</span> = 8, Week 9 PRE <span class="html-italic">n</span> = 11, Week 9 POST <span class="html-italic">n</span> = 11†. Error bars median ± interquartile range * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01. † Baseline PRE: GSH/GSSH ratio and RXNO <span class="html-italic">n</span> = 10. Baseline POST: GSH/GSSG and RXNO <span class="html-italic">n</span> = 9. Week 4 PRE: GSH/GSSG <span class="html-italic">n</span> = 5, total glutathione <span class="html-italic">n</span> = 6, nitrite and nitrate <span class="html-italic">n</span> = 7, RXNO <span class="html-italic">n</span> = 6. Week 4 POST: GSH/GSSG <span class="html-italic">n</span> = 6, TBARS, TFT/protein and RXNO <span class="html-italic">n</span> = 7. Week 9 PRE: GSH/GSSG <span class="html-italic">n</span> = 9 and RXNO <span class="html-italic">n</span> = 10. Week 9 POST: GSH/GSSG <span class="html-italic">n</span> = 9 and RXNO <span class="html-italic">n</span> = 10.</p> Full article ">
15 pages, 1181 KiB  
Review
Regulation of Mitochondrial Respiration by Hydrogen Sulfide
by Dandan Huang, Guangqin Jing and Shuhua Zhu
Antioxidants 2023, 12(8), 1644; https://doi.org/10.3390/antiox12081644 - 20 Aug 2023
Cited by 2 | Viewed by 1650
Abstract
Hydrogen sulfide (H2S), the third gasotransmitter, has positive roles in animals and plants. Mitochondria are the source and the target of H2S and the regulatory hub in metabolism, stress, and disease. Mitochondrial bioenergetics is a vital process that produces [...] Read more.
Hydrogen sulfide (H2S), the third gasotransmitter, has positive roles in animals and plants. Mitochondria are the source and the target of H2S and the regulatory hub in metabolism, stress, and disease. Mitochondrial bioenergetics is a vital process that produces ATP and provides energy to support the physiological and biochemical processes. H2S regulates mitochondrial bioenergetic functions and mitochondrial oxidative phosphorylation. The article summarizes the recent knowledge of the chemical and biological characteristics, the mitochondrial biosynthesis of H2S, and the regulatory effects of H2S on the tricarboxylic acid cycle and the mitochondrial respiratory chain complexes. The roles of H2S on the tricarboxylic acid cycle and mitochondrial respiratory complexes in mammals have been widely studied. The biological function of H2S is now a hot topic in plants. Mitochondria are also vital organelles regulating plant processes. The regulation of H2S in plant mitochondrial functions is gaining more and more attention. This paper mainly summarizes the current knowledge on the regulatory effects of H2S on the tricarboxylic acid cycle (TCA) and the mitochondrial respiratory chain. A study of the roles of H2S in mitochondrial respiration in plants to elucidate the botanical function of H2S in plants would be highly desirable. Full article
(This article belongs to the Special Issue Hydrogen Sulfide Signaling in Biological Systems)
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<p>The synthesis, properties, and reaction of H<sub>2</sub>S.</p> Full article ">Figure 2
<p>The possible pathways through which H<sub>2</sub>S regulates mitochondrial electron transport chain complexes.</p> Full article ">
21 pages, 2208 KiB  
Article
Multi-Omics Approach Reveals Prebiotic and Potential Antioxidant Effects of Essential Oils from the Mediterranean Diet on Cardiometabolic Disorder Using Humanized Gnotobiotic Mice
by María José Sánchez-Quintero, Josué Delgado, Laura Martín Chaves, Dina Medina-Vera, Mora Murri, Víctor M. Becerra-Muñoz, Mario Estévez, María G. Crespo-Leiro, Guillermo Paz López, Andrés González-Jiménez, Juan A. G. Ranea, María Isabel Queipo-Ortuño, Isaac Plaza-Andrades, Jorge Rodríguez-Capitán, Francisco Javier Pavón-Morón and Manuel F. Jiménez-Navarro
Antioxidants 2023, 12(8), 1643; https://doi.org/10.3390/antiox12081643 - 20 Aug 2023
Viewed by 3174
Abstract
Essential oils sourced from herbs commonly used in the Mediterranean diet have demonstrated advantageous attributes as nutraceuticals and prebiotics within a model of severe cardiometabolic disorder. The primary objective of this study was to assess the influences exerted by essential oils derived from [...] Read more.
Essential oils sourced from herbs commonly used in the Mediterranean diet have demonstrated advantageous attributes as nutraceuticals and prebiotics within a model of severe cardiometabolic disorder. The primary objective of this study was to assess the influences exerted by essential oils derived from thyme (Thymus vulgaris) and oregano (Origanum vulgare) via a comprehensive multi-omics approach within a gnotobiotic murine model featuring colonic microbiota acquired from patients diagnosed with coronary artery disease (CAD) and type-2 diabetes mellitus (T2DM). Our findings demonstrated prebiotic and potential antioxidant effects elicited by these essential oils. We observed a substantial increase in the relative abundance of the Lactobacillus genus in the gut microbiota, accompanied by higher levels of short-chain fatty acids and a reduction in trimethylamine N-oxide levels and protein oxidation in the plasma. Moreover, functional enrichment analysis of the cardiac tissue proteome unveiled an over-representation of pathways related to mitochondrial function, oxidative stress, and cardiac contraction. These findings provide compelling evidence of the prebiotic and antioxidant actions of thyme- and oregano-derived essential oils, which extend to cardiac function. These results encourage further investigation into the promising utility of essential oils derived from herbs commonly used in the Mediterranean diet as potential nutraceutical interventions for mitigating chronic diseases linked to CAD and T2DM. Full article
(This article belongs to the Special Issue Antioxidant Foods and Cardiometabolic Health - 2nd Edition)
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Graphical abstract

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Full article ">Figure 1
<p>Bacterial abundance profile at the order level in fecal samples of mice from the treatment groups. The bars depict the relative abundances (%) for each group based on 16S rRNA gene sequencing (Ion S5TM System).</p> Full article ">Figure 2
<p>Analyses of alpha and beta diversity of bacterial microbiota in fecal samples from the treatment groups. (<b>a</b>) Shannon index; (<b>b</b>) Principal components analysis (PCA); (<b>c</b>) Differential analysis of the abundance of <span class="html-italic">Lactobacillus</span>; (<b>d</b>) Differential analysis of the abundance of <span class="html-italic">Colidextribacter</span> in fecal samples of mice from the treatment groups. The bars represent median and IQR. Data from the thyme-10, thyme-20, oregano-10, and oregano-20 groups were analyzed using the Kruskal–Wallis test followed by Wilcoxon rank sum test as post hoc test. (*) <span class="html-italic">p</span> &lt; 0.05 indicates significant differences compared to the control group.</p> Full article ">Figure 3
<p>Levels of short-chain fatty acid (SCFA) species in fecal samples of mice from the treatment groups. (<b>a</b>) Acetic; (<b>b</b>) Propionic; (<b>c</b>) Butyric acid levels. The bars represent means ± SEM of SFCA levels (area in arbitrary units). Data were analyzed using one-way ANOVA or the Kruskal–Wallis test, followed by Dunn’s multiple comparison test as post hoc test. (*) <span class="html-italic">p</span> &lt; 0.05 indicates significant differences compared to the control group. The black arrow indicates control levels.</p> Full article ">Figure 4
<p>Proteomic analysis in cardiac tissue samples from the treatment groups. (<b>a</b>) Gene Ontology (GO) enrichment analysis of higher and lower abundant proteins. Different colors represent each category: biological process, cellular component, and molecular function; the size of each dot indicates the number of discriminant proteins in within that term. (<b>b</b>) <span class="html-italic">Kyoto Encyclopedia of Genes and Genomes</span> (KEGG) pathway enrichment analysis is represented as a diagram showcasing the higher and lower abundance of discriminant proteins.</p> Full article ">Figure 5
<p>Plasma levels of protein carbonyls and pentosidine in mice from the treatment groups. (<b>a</b>) Aminoadipic semialdehyde (AAS); (<b>b</b>) Glutamic semialdehyde (GGS); (<b>c</b>) Pentosidine levels. The bars represent means ± SEM of nmol/mg. Data were analyzed using one-way ANOVA or the Kruskal–Wallis test, followed by Dunn’s multiple comparison test as post hoc test. (*) <span class="html-italic">p</span> &lt; 0.05 and (***) <span class="html-italic">p</span> &lt; 0.001 indicate significant differences as compared to the control group. The black arrow indicates control levels.</p> Full article ">Figure 6
<p>Plasma levels of trimethylamine N-oxide (TMAO) and cholesterol in mice from the treatment groups. (<b>a</b>) TMAO; (<b>b</b>) Total cholesterol; (<b>c</b>) LDL cholesterol levels. The bars represent means ± SEM of µM or mg/dL. Data were analyzed using one-way ANOVA or the Kruskal–Wallis test, followed by Dunn’s multiple comparison test as post hoc test. (*) <span class="html-italic">p</span> &lt; 0.05 indicates significant differences compared to the control group. The black arrow indicates control levels.</p> Full article ">
19 pages, 1641 KiB  
Review
A Critical Review of Kaempferol in Intestinal Health and Diseases
by Jun Chen, Haopeng Zhong, Zhouyin Huang, Xingping Chen, Jinming You and Tiande Zou
Antioxidants 2023, 12(8), 1642; https://doi.org/10.3390/antiox12081642 - 20 Aug 2023
Cited by 3 | Viewed by 2345
Abstract
Kaempferol, a secondary metabolite found in plants, is a naturally occurring flavonoid displaying significant potential in various biological activities. The chemical structure of kaempferol is distinguished by the presence of phenyl rings and four hydroxyl substituents, which make it an exceptional radical scavenger. [...] Read more.
Kaempferol, a secondary metabolite found in plants, is a naturally occurring flavonoid displaying significant potential in various biological activities. The chemical structure of kaempferol is distinguished by the presence of phenyl rings and four hydroxyl substituents, which make it an exceptional radical scavenger. Most recently, an increasing number of studies have demonstrated the significance of kaempferol in the regulation of intestinal function and the mitigation of intestinal inflammation. The focus of the review will primarily be on its impact in terms of antioxidant properties, inflammation, maintenance of intestinal barrier function, and its potential in the treatment of colorectal cancer and obesity. Future research endeavors should additionally give priority to investigating the specific dosage and duration of kaempferol administration for different pathological conditions, while simultaneously conducting deeper investigations into the comprehensible mechanisms of action related to the regulation of aryl hydrocarbon receptor (AhR). This review intends to present novel evidence supporting the utilization of kaempferol in the regulation of gut health and the management of associated diseases. Full article
(This article belongs to the Special Issue Dietary Antioxidants and Gut Health)
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<p>Chemical structure of kaempferol. (<b>A</b>). Two-dimensional structure; (<b>B</b>). Three-dimensional structure (Source from ChemSpider).</p> Full article ">Figure 2
<p>The beneficial effects of kaempferol on the intestine and the proposed mechanism of action based on current knowledge. Abbreviations: BCRP, breast cancer resistance protein; COX-2, cyclooxygenase-2; CRP, C-reaction protein; DR5, death receptor 5; eWAT, epididymal white adipose tissue; FXR, farnesoid X receptor; GSR, glutathione reductase; GSTA4, glutathione S-transferase 4; HO-1, heme oxygenase-1; ICAM-1, intercellular adhesion molecule-1; IFN-γ, interferon-γ; IL-1β, interleukin-1β; IL-6, interleukin-6; IL-10, interleukin-10; iONS, inducible nitric oxide synthase; iWAT, inguinal white adipose tissue; LDH, lactate dehydrogenase; LTB<sub>4</sub>, leukotriene B<sub>4</sub>; MAPK, mitogen-activated protein kinase; MCP-1, monocyte chemoattractant protein-1; MIP-3α, macrophage inflammatory protein-3 alpha; MPO, myeloperoxidase; MRP2, multi-drug resistance-associated protein 2; NF-κB, nuclear factor kappa-B; NLRP3, nucleotide oligomerization domain (NOD)-like receptor 3; Nrf2, nuclear factor-E2-related factor 2; PARP, poly ADPribose polymerase; PGE<sub>2</sub>, prostaglandin E<sub>2</sub>; PKA, protein kinase A; pWAT, perirenal white adipose tissue; RhoA/ROCK, Ras homolog gene family member A/Rho-associated protein kinase; ROS, reactive oxygen species; STAT, signal transducer and activator of transcription; TEER, transepithelial electrical resistance; TFF3, trefoil factor family 3; TLR4, toll-like receptor 4; TNF-α, tumor necrosis factor-α; VCAM-1, vascular cell adhesion molecule-1.</p> Full article ">Figure 3
<p>AhR signaling pathway. Abbreviations: AhR, aryl hydrocarbon receptor; AhRE, aryl hydrocarbon receptor responsive elements; Ahrr, AhR repressor; AIP, AhR-interacting protein; ARNT, aryl hydrocarbon receptor nuclear translocator; Cyp1a1, cytochrome P450 family 1 subfamily A member 1; Cyp1b1; cytochrome P450 family 1 subfamily B member 1; HSP90, heat shock protein 90; Ido1, indoleamine 2,3-dioxygenase 1; P23, co­chaperone P23; SRC, SRC protein kinase; Tdo, tryptophan 2,3-dioxygenase.</p> Full article ">
24 pages, 5534 KiB  
Article
Comprehensive Comparison of Effects of Antioxidant (Astaxanthin) Supplementation from Different Sources in Haliotis discus hannai Diet
by Weiguang Zou, Jiawei Hong, Wenchao Yu, Yaobin Ma, Jiacheng Gan, Yanbo Liu, Xuan Luo, Weiwei You and Caihuan Ke
Antioxidants 2023, 12(8), 1641; https://doi.org/10.3390/antiox12081641 - 19 Aug 2023
Viewed by 1536
Abstract
Dietary antioxidant supplementation, especially astaxanthin, has shown great results on reproductive aspects, egg quality, growth, survival, immunity, stress tolerance, and disease resistance in aquatic animals. However, the effects of dietary astaxanthin supplementation from different sources are still unknown. A comprehensive comparison of survival, [...] Read more.
Dietary antioxidant supplementation, especially astaxanthin, has shown great results on reproductive aspects, egg quality, growth, survival, immunity, stress tolerance, and disease resistance in aquatic animals. However, the effects of dietary astaxanthin supplementation from different sources are still unknown. A comprehensive comparison of survival, growth, immune response, antioxidant activity, thermal resistance, disease resistance, and intestinal microbial structure was conducted in dietary antioxidant supplementation from the sources of Gracilaria lemaneiformis (GL), industrial synthetic astaxanthin (80 mg/kg astaxanthin actual weight, named as group ‘SA80’), Phaffia rhodozyma (80 mg/kg astaxanthin actual weight, named as group ‘PR80’) and Haematococcus pluvialis (120 mg/kg astaxanthin actual weight, named as group ‘HP120’) at their optimal supplementation amounts. Furthermore, the SA80, PR80, and HP120 groups performed better in all aspects, including survival, growth, immune response, antioxidant activity, thermal resistance, and disease resistance, compared with the GL group. The PR80 and HP120 group also had a better growth performance than the SA80 group. In terms of heat stress and bacterial challenge, abalone in the PR80 group showed the strongest resistance. Overall, 80 mg/kg astaxanthin supplementation from Phaffia rhodozyma was recommended to obtain a more effective and comprehensive outcome. This study contributes to the discovery of the optimum dietary astaxanthin supplementation source for abalone, which is helpful to improve the production efficiency and economic benefits of abalone. Future research can further explore the action mechanism and the method of application of astaxanthin to better exploit its antioxidant role. Full article
(This article belongs to the Section Natural and Synthetic Antioxidants)
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<p>Heat adhesion duration (HAD) of <span class="html-italic">H. discus hannai</span> fed diets with astaxanthin from different supplementation sources under heat-resistance evaluation. The values marked by dashed lines were the time when the attachment rate dropped to a half.</p> Full article ">Figure 2
<p>The survival rate of <span class="html-italic">H. discus hannai</span> fed diets with astaxanthin from different supplementation sources under the bacterial challenge experiment. The values marked by dashed lines were the time when the survival rate dropped to a half.</p> Full article ">Figure 3
<p>Total hemocyte counts (THC) of <span class="html-italic">H. discus hannai</span> fed diets with astaxanthin from different supplementation sources under the bacterial challenge. Different letters in different groups indicate significant differences (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 4
<p>The hemocyte mortality rate of <span class="html-italic">H. discus hannai</span> fed diets with astaxanthin from different supplementation sources under bacterial challenge. Different letters in different groups indicate significant differences (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 5
<p>Reactive oxygen species (ROS) of <span class="html-italic">H. discus hannai</span> fed diets with astaxanthin from different supplementation sources under bacterial challenge. Different letters in different groups indicate significant differences (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 6
<p>Phagocytic activity of <span class="html-italic">H. discus hannai</span> fed diets with astaxanthin from different supplementation sources under bacterial challenge. Different letters in different groups indicate significant differences (<span class="html-italic">p</span> &lt; 0.05).</p> Full article ">Figure 7
<p>Beta diversity of the intestinal microbiota of <span class="html-italic">Haliotis discus hannai</span> fed diets with astaxanthin from different supplementation sources. Analyzed by principal component analysis (PCA) using PC1 versus PC2 axes based on UniFrac distance.</p> Full article ">Figure 8
<p>Relative abundance of the top 10 intestinal microbiota of <span class="html-italic">Haliotis discus hannai</span> fed diets with astaxanthin from different supplementation sources at phylum (<b>A</b>) and genus (<b>B</b>) level.</p> Full article ">Figure 9
<p>Linear discriminant analysis of effect size (LEfSe) of intestinal microbiota of <span class="html-italic">Haliotis discus hannai</span> fed diets with astaxanthin from different supplementation sources. (<b>A</b>) Linear discriminant analysis scores of the abundance of taxa. (<b>B</b>) Cladogram showing differences in the abundance of taxa.</p> Full article ">Figure 10
<p>The clustering heat map analysis of the function at level 2 of the intestinal microbiota of <span class="html-italic">Haliotis discus hannai</span> fed diets with astaxanthin from different supplementation sources.</p> Full article ">Figure 11
<p>Analysis of species with differences among groups (the relative abundance of the PR80 group especially was significantly higher than that of the SA80 group). (<b>A</b>) Species with differences in Firmicutes; (<b>B</b>) Species with differences in Proteobacteria. Asterisk among different groups means significant differences (* <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01).</p> Full article ">Figure 11 Cont.
<p>Analysis of species with differences among groups (the relative abundance of the PR80 group especially was significantly higher than that of the SA80 group). (<b>A</b>) Species with differences in Firmicutes; (<b>B</b>) Species with differences in Proteobacteria. Asterisk among different groups means significant differences (* <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01).</p> Full article ">
23 pages, 3827 KiB  
Article
Insight into Physiological and Biochemical Determinants of Salt Stress Tolerance in Tetraploid Citrus
by Marie Bonnin, Bénédicte Favreau, Alexandre Soriano, Nathalie Leonhardt, Julie Oustric, Radia Lourkisti, Patrick Ollitrault, Raphaël Morillon, Liliane Berti and Jérémie Santini
Antioxidants 2023, 12(8), 1640; https://doi.org/10.3390/antiox12081640 - 19 Aug 2023
Cited by 2 | Viewed by 1355
Abstract
Citrus are classified as salt-sensitive crops. However, a large diversity has been observed regarding the trends of tolerance among citrus. In the present article, physiological and biochemical studies of salt stress tolerance were carried out according to the level of polyploidy of different [...] Read more.
Citrus are classified as salt-sensitive crops. However, a large diversity has been observed regarding the trends of tolerance among citrus. In the present article, physiological and biochemical studies of salt stress tolerance were carried out according to the level of polyploidy of different citrus genotypes. We particularly investigated the impact of tetraploidy in trifoliate orange (Poncirus trifoliata (L.) Raf.) (PO4x) and Cleopatra mandarin (Citrus reshni Hort. Ex Tan.) (CL4x) on the tolerance to salt stress compared to their respective diploids (PO2x and CL2x). Physiological parameters such as gas exchange, ions contents in leaves and roots were analyzed. Roots and leaves samples were collected to measure polyphenol, malondialdehyde (MDA), ascorbate and H2O2 contents but also to measure the activities of enzymes involved in the detoxification of active oxygen species (ROS). Under control conditions, the interaction between genotype and ploidy allowed to discriminate different behavior in terms of photosynthetic and antioxidant capacities. These results were significantly altered when salt stress was applied when salt stress was applied. Contrary to the most sensitive genotype, that is to say the diploid trifoliate orange PO2x, PO4x was able to maintain photosynthetic activity under salt stress and had better antioxidant capacities. The same observation was made regarding the CL4x genotype known to be more tolerant to salt stress. Our results showed that tetraploidy may be a factor that could enhance salt stress tolerance in citrus. Full article
(This article belongs to the Section Antioxidant Enzyme Systems)
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<p>Discriminant analysis of biochemical and photosynthetic variables under control conditions. PLS-DA biplot represents discrimination of Cleopatra (CL) and trifoliate orange (PO) genotypes at two ploidy levels (2x, 4x) in roots (<b>a</b>) and leaves (<b>b</b>). Measurements were performed at W4 (90 mM). Colored dots represent biological replicates for each genotype. sPLS-DA selected variables were represented in bold.</p> Full article ">Figure 2
<p>Discriminant analysis of biochemical and photosynthetic variables under control (C) and stress conditions (S). PLS-DA biplot represents discrimination of Cleopatra (CL) and trifoliate orange (PO) genotypes at two ploidy levels (2x, 4x), with (S) and without salt stress (C), in roots (<b>a</b>) and leaves (<b>b</b>). Measurements were performed at W4 (90 mM). Colored dots represent biological replicates for each genotype. sPLS-DA selected variables were represented in bold.</p> Full article ">Figure 3
<p>Mineral uptake. (<b>a</b>) Roots Na<sup>+</sup> content and (<b>b</b>) Roots Cl<sup>−</sup> content (mg/g) with (S) and without salt stress (C). Leaves Na+ and Cl<sup>−</sup> content are represented in (<b>c</b>,<b>d</b>), respectively, with (S) and without salt stress (C). Measurements were performed after four weeks of salt treatment (90 mM). Data represents 3 independent measurements (<span class="html-italic">n</span> = 3). Significance of the values were analyzed using the Kruskal–Wallis test (<span class="html-italic">p</span> &gt; 0.05) and mean comparison using Dunn’s test. Groups are separated by letters. Each group is assigned one or more letters. Groups sharing the same letter are not significantly different. Letter displays a clear and succinct way to present results of multiple comparisons.</p> Full article ">Figure 4
<p>(<b>a</b>) Leaves/roots Cl<sup>−</sup> ratio under control (C) and stress condition (S). (<b>b</b>) Leaves/roots Na<sup>+</sup> ratio under control (C) and stress condition (S). Measurements were performed after four weeks of salt treatment (90 mM). Data represents 3 independent measurements (<span class="html-italic">n</span> = 3). Significance of the values were analyzed using Kruskal–Wallis test (<span class="html-italic">p</span> &gt; 0.05) and mean comparison using Dunn’s test. Groups are separated by letters. Each group is assigned one or more letters. Groups sharing the same letter are not significantly different. Letter displays a clear and succinct way to present results of multiple comparisons.</p> Full article ">Figure 5
<p>Leaves symptoms under Control (C) and Stressed (S) (W4 90 mM). Trifoliate orange (PO) Cleopatra mandarin (CL); Diploid (2x), tetraploid (4x). Bar represents 1 cm.</p> Full article ">Figure 6
<p>Photosynthetic performances under control (C) and salt stress conditions (S). Stomatal conductance (<span class="html-italic">g<sub>s</sub></span>) (<b>a</b>), net photosynthesis (<span class="html-italic">P<sub>net</sub></span>) (<b>b</b>), carboxylation efficiency (<span class="html-italic">P<sub>net</sub></span>/ci) (<b>c</b>) and indicator of electron transport utilized by acceptors other than CO<sub>2</sub> (ETR/<span class="html-italic">P<sub>net</sub></span>) (<b>d</b>). Data represent 3 independent measurements (<span class="html-italic">n</span> = 3) after four weeks of salt treatment (90 mM). Significance of the values were analyzed using Kruskal–Wallis test (<span class="html-italic">p</span> &gt; 0.05) and mean comparison using Dunn’s test. C and S represent Control and stressed plants, respectively. Groups are separated by letters. Each group is assigned one or more letters. Groups sharing the same letter are not significantly different. Letter displays a clear and succinct way to present results of multiple comparisons.</p> Full article ">Figure 7
<p>Ascorbate cycle, antioxidant metabolism and osmoprotection in leaves under control (C) and salt stress conditions (S). (<b>a</b>) Proline content and (<b>b</b>) DHAR specific activity. after four weeks of salt treatment (90 mM). Significance of the values were analyzed using Kruskal–Wallis test (<span class="html-italic">p</span> &gt; 0.05) and mean comparison using Dunn’s test (<span class="html-italic">p</span>-value: &lt; 0.05). C and S represent Control and stressed plants, respectively. Groups are separated by letters. Each group is assigned one or more letters. Groups sharing the same letter are not significantly different. Letter displays a clear and succinct way to present results of multiple comparisons.</p> Full article ">Figure 8
<p>Summary: biological significance of principal variables selected by sPLS-DA in leaves and roots of Trifoliate orange (PO) and Cleopatra (CL) genotypes under control and stress conditions. This figure design was realized using Biorender (biorender.com).</p> Full article ">
17 pages, 3415 KiB  
Article
Integrated Multi-Omics Analysis for Inferring Molecular Players in Inclusion Body Myositis
by Judith Cantó-Santos, Laura Valls-Roca, Ester Tobías, Clara Oliva, Francesc Josep García-García, Mariona Guitart-Mampel, Félix Andújar-Sánchez, Anna Esteve-Codina, Beatriz Martín-Mur, Joan Padrosa, Raquel Aránega, Pedro J. Moreno-Lozano, José César Milisenda, Rafael Artuch, Josep M. Grau-Junyent and Glòria Garrabou
Antioxidants 2023, 12(8), 1639; https://doi.org/10.3390/antiox12081639 - 19 Aug 2023
Cited by 2 | Viewed by 1466
Abstract
Inclusion body myositis (IBM) is an acquired inflammatory myopathy affecting proximal and distal muscles that leads to weakness in patients over 50. It is diagnosed based on clinical and histological findings in muscle related to inflammation, degeneration, and mitochondria. In relation to IBM, [...] Read more.
Inclusion body myositis (IBM) is an acquired inflammatory myopathy affecting proximal and distal muscles that leads to weakness in patients over 50. It is diagnosed based on clinical and histological findings in muscle related to inflammation, degeneration, and mitochondria. In relation to IBM, a shortage of validated disease models and a lack of biomarkers and effective treatments constitute an unmet medical need. To overcome these hurdles, we performed an omics analysis of multiple samples from IBM patients (saliva, fibroblasts, urine, plasma, and muscle) to gain insight into the pathophysiology of IBM. Degeneration was evident due to the presence of amyloid β peptide 1–42 (Aβ1–42) in the saliva of the analyzed IBM patients. The presence of metabolic disarrangements in IBM was indicated by an imbalanced organic acid profile in fibroblasts and urine. Specifically, abnormal levels of L-pyroglutamic and orotic acid were supported by the abnormal expression of related metabolites in plasma and urine (glutathione and pyrimidines) and the aberrant expression of upstream gene regulators (L2HGDH, IDH2, OPLAH, and ASL) in muscle. Combined levels of L-pyroglutamic and orotic acid displayed an outstanding biomarker signature in urine with 100% sensitivity and specificity. The confirmation of systemic metabolic disarrangements in IBM and the identification of novel biomarkers reported herein unveil novel insights that require validation in larger cohorts. Full article
(This article belongs to the Special Issue Antioxidants and Oxidative Stress in the Development of Diseases and Therapy)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Amyloid β peptide 1–42 (Aβ1–42) in saliva samples. (<b>a</b>) Bar graphs of the concentration of Aβ1–42 in inclusion body myositis (IBM) vs. control (CTL) samples. (<b>b</b>) Receiver operating characteristic (ROC) curve and area under the ROC curve (AUC) of Aβ1–42 (AUC = 0.78 ± 0.13. <span class="html-italic">p</span>-value = 0.12), yielding sensitivity and specificity scores of 66.70% and 50.00%, respectively. Higher Aβ1–42 concentrations and high AUC scores in the IBM group suggested that this peptide could aid in discriminating between cohorts.</p> Full article ">Figure 2
<p>Organic acid profiles in inclusion body myositis (IBM) vs. control (CTL) fibroblasts (<span class="html-italic">n</span> = 11 vs. 10). (<b>a</b>) Fold change (FC) of the concentration of organic acids in IBM vs. CTL fibroblasts (* <span class="html-italic">p</span>-value &lt; 0.05). (<b>b</b>) Bar graph of L-pyroglutamic and 2-hydroxyvaleric acids (<span class="html-italic">p</span>-value &lt; 0.05). (<b>c</b>) Receiver operating characteristic (ROC) curve and area under the ROC curve (AUC) of L-pyroglutamic (AUC = 0.79 ± 0.12. <span class="html-italic">p</span>-value = 0.03) and 2-hydroxyvaleric acids (AUC = 0.84 ± 0.09. <span class="html-italic">p</span>-value = 0.01). Their sensitivity and specificity values are 72.7% and 80% for L-pyroglutamic acid and 72.70% and 77.80% for 2-hydroxy glutaric acid. All the organic acids presented increased concentrations in the IBM vs. CTL fibroblasts, among which L-pyroglutamic and 2-hydroxy glutaric acids were the most remarkable, with the former being associated with glutathione synthesis and the latter with the TCA cycle.</p> Full article ">Figure 3
<p>Organic acid profiles in the urine of inclusion body myositis (IBM) vs. control (CTL) samples (<span class="html-italic">n</span> = 6/group). (<b>a</b>) Fold change (FC) of the concentration of organic acids in the urine of lBM vs. CTLs. (<b>b</b>) Bar graphs of L-pyroglutamic and orotic acids (<span class="html-italic">p</span>-value &lt; 0.05). (<b>c</b>) Receiver operating characteristic (ROC) curve and area under the ROC curve (AUC) of L-pyroglutamic (AUC = 0.64 ± 0.17. <span class="html-italic">p</span>-value = 0.42) and orotic acids (AUC = 0.94 ± 0.07. <span class="html-italic">p</span>-value = 0.01). Their sensitivity and specificity values are 50.0% and 83.3% for L-pyroglutamic acid and 100.0% and 83.3% for orotic acid, but these values changed to 100% sensitivity and specificity when both acids were tested together. The presence of altered organic acids in urine corroborated the imbalanced organic acid profile in fibroblasts and highlights L-pyroglutamic and orotic acid as potential fluid biomarkers.</p> Full article ">Figure 4
<p>Nucleotides in urine and glutathione in plasma of inclusion body myositis (IBM) vs. control (CTL) samples (<span class="html-italic">n</span> = 6/group). (<b>a</b>) Fold change (FC) of the concentration of nucleotides in urine of IBM patients vs. CTLs. (<b>b</b>) Bar graphs of orotidine and pseudo-uridine (<span class="html-italic">p</span>-value &lt; 0.05). (<b>c</b>) Receiver operating characteristic (ROC) curve and area under the ROC curve (AUC) of orotidine (AUC = 0.89 ± 0.10. <span class="html-italic">p</span>-value = 0.02) and pseudo-uridine (AUC = 0.94 ± 0.07. <span class="html-italic">p</span>-value = 0.01). Their sensitivity and specificity values are 66.7% and 83.3% for orotidine and 83.3% for both in pseudo-uridine. (<b>d</b>) Glutathione peroxidase (GPX) activity in IBM vs. CTL plasma samples (<span class="html-italic">p</span>-value = 0.05). (<b>e</b>) Receiver Operating Characteristic (ROC) curve and area under the ROC curve (AUC) of GPX (AUC = 0.74 ± 0.11, <span class="html-italic">p</span>-value = 0.05), with 66.7% sensitivity and 75.0% specificity. Almost all nucleotides were present in higher concentrations in the urine isolated from the IBM patients, with orotidine and pseudo-uridine being statistically significant, thereby corroborating the altered orotic acid biosynthesis previously observed. GPX activity was also higher in the IBM patients, suggesting increased glutathione redox system activity associated with oxidative stress.</p> Full article ">Figure 5
<p>Schematic representation of the tricarboxylic acid cycle (TCA) (in blue), purine (in red) and pyrimidine (in green) metabolism (both part of nucleotide metabolism), and oxidative stress (in turquoise). All of these processes are related to the metabolic profile examined in the organic acid, nucleotide, and RNA seq analyses. Abbreviations: IMP: inosine monophosphate; AMP: adenosine monophosphate; OMP: orotate monophosphate; UMP: uridine 5’-monophosphate; SAH: S-adenosylhomocysteine; GSH: glutathione; GSSG: glutathione disulfide; GPX: glutathione peroxidase.</p> Full article ">Figure 6
<p>Metabolic pathways in the muscle RNA-seq of inclusion body myositis (IBM) patients vs. controls (CTLs). Each pathway is represented with a different color with the genes unique to that pathway (domes) and the interactions of genes across pathways (chords). Wider domes and chords represent a higher number of genes. Among them, carbohydrate metabolism and metals and cofactors, with 56.8% and 52.8% of DEGs involved, were the most affected metabolic pathways (<a href="#app1-antioxidants-12-01639" class="html-app">Table S2</a>). Overall, these data support the notion of functional metabolic deregulation at the gene expression level and confirm the relevance of metabolic deregulation in relation to this disease.</p> Full article ">Figure 7
<p>Interactome of the metabolic differentially expressed genes (DEGs) in muscle related to the significantly altered organic acid patterns (in fibroblasts and urine) and their associated pathways. The arrows next to the genes and metabolites refer to their increased or decreased expression in IBM samples. The different colors (blue, green, and pink) indicate the upstream (genes) and downstream (organic acids and nucleotides) effectors of each pathway.</p> Full article ">
17 pages, 2505 KiB  
Article
Exploring the Relationship between Antioxidant Enzymes, Oxidative Stress Markers, and Clinical Profile in Relapsing–Remitting Multiple Sclerosis
by Anna Bizoń, Justyna Chojdak-Łukasiewicz, Sławomir Budrewicz, Anna Pokryszko-Dragan and Agnieszka Piwowar
Antioxidants 2023, 12(8), 1638; https://doi.org/10.3390/antiox12081638 - 19 Aug 2023
Cited by 5 | Viewed by 1427
Abstract
We aimed to investigate the extent of alterations in the pro/antioxidant balance in the blood of patients with relapsing–remitting multiple sclerosis (RRMS) in relation to drug-modified therapy, gender, disability score, and disease duration. 161 patients (67 men and 94 women, aged 24–69 years, [...] Read more.
We aimed to investigate the extent of alterations in the pro/antioxidant balance in the blood of patients with relapsing–remitting multiple sclerosis (RRMS) in relation to drug-modified therapy, gender, disability score, and disease duration. 161 patients (67 men and 94 women, aged 24–69 years, median 43.0) and 29 healthy individuals (9 men and 20 women, aged 25–68 years, median 41.0) were included in the study. We measured the activity of superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) as well as the concentration of interleukin-6 (IL-6), lipid peroxidation parameters (LPO), total oxidant status (TOS), and total antioxidant capacity (TAS). The activity of SOD did not show any significant differences between patients with RRMS and the control group in our study. In contrast, significant decreased GPx activity and increased CAT activity was observed in the blood of patients with RRMS compared to the control group. Additionally, the activity of CAT was influenced by gender and the use of disease-modifying therapies. Disease-modifying therapies also affected the concentration of TOS, TAS, and LPO. Our studies indicated that enhancing GPx activity may be more beneficial to providing potential therapeutic strategies aimed at modulating antioxidant defenses to mitigate oxidative stress in this disease. Full article
(This article belongs to the Special Issue Risk Factors for Neurodegenerative Diseases: Oxidative Stress Footprints as the Common Feature)
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<p>(<b>a</b>) Receiver operating characteristic (ROC) curves evaluating the ability of TOS, TAS, LPO concentration or the value of the OSI ratio to distinguish patients with RRMS and control group. (<b>b</b>) Receiver operating characteristic (ROC) curves evaluating the ability of NO<sub>2</sub>, NO<sub>3</sub> or IL-6 concentration to distinguish patients with RRMS and control group. (<b>c</b>) Receiver operating characteristic (ROC) curves evaluating the ability of SOD, GPx, CAT activity or the value of the SOD/GPx + CAT ratio to distinguish patients with RRMS and control group.</p> Full article ">Figure 1 Cont.
<p>(<b>a</b>) Receiver operating characteristic (ROC) curves evaluating the ability of TOS, TAS, LPO concentration or the value of the OSI ratio to distinguish patients with RRMS and control group. (<b>b</b>) Receiver operating characteristic (ROC) curves evaluating the ability of NO<sub>2</sub>, NO<sub>3</sub> or IL-6 concentration to distinguish patients with RRMS and control group. (<b>c</b>) Receiver operating characteristic (ROC) curves evaluating the ability of SOD, GPx, CAT activity or the value of the SOD/GPx + CAT ratio to distinguish patients with RRMS and control group.</p> Full article ">Figure 1 Cont.
<p>(<b>a</b>) Receiver operating characteristic (ROC) curves evaluating the ability of TOS, TAS, LPO concentration or the value of the OSI ratio to distinguish patients with RRMS and control group. (<b>b</b>) Receiver operating characteristic (ROC) curves evaluating the ability of NO<sub>2</sub>, NO<sub>3</sub> or IL-6 concentration to distinguish patients with RRMS and control group. (<b>c</b>) Receiver operating characteristic (ROC) curves evaluating the ability of SOD, GPx, CAT activity or the value of the SOD/GPx + CAT ratio to distinguish patients with RRMS and control group.</p> Full article ">Figure 2
<p>(<b>a</b>,<b>b</b>) The concentration of TOS, TAS and the value of OSI (<b>a</b>) and LPO and IL-6 (<b>b</b>) in the blood of patients with RRMS divided according to particular DMT with statistical analysis. (<b>c</b>) The concentration of NO<sub>2</sub> and NO<sub>3</sub> in the blood of patients with RRMS divided according to particular DMT with statistical analysis. (<b>d</b>) The activity of SOD, GPx, CAT and the value of the SOD/GPx + CAT ratio in the blood of patients with RRMS divided according to particular DMT with statistical analysis. Legend: □—median value; ᴏ—outliers.</p> Full article ">Figure 2 Cont.
<p>(<b>a</b>,<b>b</b>) The concentration of TOS, TAS and the value of OSI (<b>a</b>) and LPO and IL-6 (<b>b</b>) in the blood of patients with RRMS divided according to particular DMT with statistical analysis. (<b>c</b>) The concentration of NO<sub>2</sub> and NO<sub>3</sub> in the blood of patients with RRMS divided according to particular DMT with statistical analysis. (<b>d</b>) The activity of SOD, GPx, CAT and the value of the SOD/GPx + CAT ratio in the blood of patients with RRMS divided according to particular DMT with statistical analysis. Legend: □—median value; ᴏ—outliers.</p> Full article ">
13 pages, 1245 KiB  
Article
Technologically Driven Approaches for the Integrative Use of Wild Blackthorn (Prunus spinosa L.) Fruits in Foods and Nutraceuticals
by Oana Viorela Nistor, Ștefania Adelina Milea, Bogdan Păcularu-Burada, Doina Georgeta Andronoiu, Gabriela Râpeanu and Nicoleta Stănciuc
Antioxidants 2023, 12(8), 1637; https://doi.org/10.3390/antiox12081637 - 19 Aug 2023
Cited by 2 | Viewed by 1024
Abstract
Different technological approaches were used in this study for the valorization of blackthorn (Prunus spinosa L.) fruits in marmalade, jam, jelly, and nutraceuticals. Marmalade showed the highest concentrations of polyphenols (7.61 ± 0.05 mg gallic acid equivalents/g dry weight (DW)) and flavonoids [...] Read more.
Different technological approaches were used in this study for the valorization of blackthorn (Prunus spinosa L.) fruits in marmalade, jam, jelly, and nutraceuticals. Marmalade showed the highest concentrations of polyphenols (7.61 ± 0.05 mg gallic acid equivalents/g dry weight (DW)) and flavonoids (4.93 ± 0.22 mg catechin equivalents/g DW), whereas jam retained the highest content of anthocyanins (66.87 ± 1.18 mg cyanidin-3-O-glucoside equivalents/g DW). A good correlation between polyphenol and flavonoid contents and antioxidant activity was found, the highest value being 21.29 ± 1.36 mmol Trolox/g DW for marmalade. Alternatively, the fresh pulp was enriched with inulin, followed by inoculation with Lactobacillus acidophilus, and freeze-dried, allowing a powder to be obtained with a viable cell content of 6.27 × 107 CFU/g DW. A chromatographic analysis of blackthorn skin revealed that myricetin (2.04 ± 0.04 mg/g DW) was the main flavonoid, followed by (+)–catechin (1.80 ± 0.08 mg/g DW), (−)-epicatechin (0.96 ± 0.02 mg/g DW), and vanillic acid (0.94 ± 0.09 mg/g DW). The representative anthocyanins were cyanidin 3-O-glucoside, cyanidin 3-O-rutinoside, and peonidin 3-O-glucoside, with an average concentration of 0.75 mg/g DW. The skin extract showed comparable IC50 values for tyrosinase (1.72 ± 0.12 mg/mL), α-amylase (1.17 ± 0.13 mg/mL), and α-glucosidase (1.25 ± 0.26 mg/mL). The possible use of kernels as calorific agents was demonstrated through the evaluation of calorific power of 4.9 kWh/kg. Full article
(This article belongs to the Special Issue Antioxidants in Fruits and Their Health-Promoting Effects)
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Figure 1

Figure 1
<p>HPLC chromatograms of the flavonoids and polyphenols at 280 nm (<b>a</b>) and 320 nm (<b>b</b>) from blackthorn extract. Peaks’ identification: (<b>a</b>) 1—gallic acid; 2—chlorogenic acid; 3-(-)-epicatechin; 4—caffeic acid; 6—vanillic acid; 7—p-coumaric acid; 12—myricetin; 14—isorhamnetin; 5, 8–11, 13, 15–17—unidentified peaks; (<b>b</b>) 1—gallic acid; 2-(+)-catechin; 3—chlorogenic acid; 4—epicatechin; 5—caffeic acid; 17—myricetin; 23—isorhamnetin; 6–16, 18–22—unidentified peaks.</p> Full article ">Figure 2
<p>HPLC chromatogram of the anthocyanins from blackthorn extract at 520 nm. Peaks’ identification: 5—Delphinidin 3-<span class="html-italic">O</span>-β-D-glucoside; 7—Cyanidin 3-<span class="html-italic">O</span>-glucoside; 8—Cyanidin 3-<span class="html-italic">O</span>-rutinoside; 10—Peonidin 3-<span class="html-italic">O</span>-glucoside; 1–4, 6, 9, 11–14—unidentified peaks.</p> Full article ">
15 pages, 1683 KiB  
Article
Development and Characterization of Inula britannica Extract-Loaded Liposomes: Potential as Anti-Inflammatory Functional Food Ingredients
by Chi Rac Hong, Eun Ha Lee, Young Hoon Jung, Ju-Hoon Lee, Hyun-Dong Paik, Sung-Chul Hong and Seung Jun Choi
Antioxidants 2023, 12(8), 1636; https://doi.org/10.3390/antiox12081636 - 18 Aug 2023
Cited by 2 | Viewed by 1442
Abstract
We investigated the potential of Inula britannica extract encapsulated in liposomes as a functional food ingredient with enhanced bioavailability and stability. Inula britannica, known for its anti-inflammatory properties and various health benefits, was encapsulated using a liposome mass production manufacturing method, and [...] Read more.
We investigated the potential of Inula britannica extract encapsulated in liposomes as a functional food ingredient with enhanced bioavailability and stability. Inula britannica, known for its anti-inflammatory properties and various health benefits, was encapsulated using a liposome mass production manufacturing method, and the physical properties of liposomes were evaluated. The liposomes exhibited improved anti-inflammatory effects in lipopolysaccharide-activated RAW 264.7 macrophages, suppressing the production of pro-inflammatory mediators such as nitric oxide and prostaglandin E2 and downregulating the expression of iNOS and COX-2 transcription factors. Additionally, we observed reduced production of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β, and modulation of the NF-κB and mitogen-activated protein kinase signaling pathways. These findings suggest that Inula britannica extract encapsulated in liposomes could serve as a valuable functional food ingredient for managing and preventing inflammation-related disorders, making it a promising candidate for incorporation into various functional food products. The enhanced absorption and stability provided by liposomal encapsulation can enable better utilization of the extract’s beneficial properties, promoting overall health and well-being. Full article
(This article belongs to the Section Health Outcomes of Antioxidants and Oxidative Stress)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Characterization of liposomes encapsulating <span class="html-italic">I. britannica</span> extract. (<b>A</b>) The size distribution of liposomes encapsulating <span class="html-italic">I. britannica</span> extract. (<b>B</b>) Transmission electron microscopic analysis of liposomes encapsulating <span class="html-italic">I. britannica</span> extract.</p> Full article ">Figure 2
<p>Anti-inflammatory effects of <span class="html-italic">I. britannica</span> extract encapsulated in liposomes in lipopolysaccharide (LPS)-induced RAW 264.7 cells. (<b>A</b>) Nitric oxide (NO) production (μM); (<b>B</b>) prostaglandin E2 (PGE<sub>2</sub>) production (pg/mL); the relative intensity of (<b>C</b>) iNOS; and (<b>D</b>) COX-2 expression. Data are represented as mean ± SEM (error bar). <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001 versus non-LPS-induced cells. ** <span class="html-italic">p</span> &lt; 0.01, and *** <span class="html-italic">p</span> &lt; 0.001 versus LPS-only treated cells. The data were statistically analyzed using a one-way ANOVA followed by Tukey’s post hoc test.</p> Full article ">Figure 3
<p>Effects of <span class="html-italic">I. britannica</span> extract encapsulated in liposomes on the mRNA expression of proinflammatory cytokines in lipopolysaccharide (LPS)-induced RAW 264.7 cells. (<b>A</b>) TNF-α; (<b>B</b>) IL-6; and (<b>C</b>) IL-1β expression. Data are represented as mean ± SEM (error bar). <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001 versus non-LPS-induced cells. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, and *** <span class="html-italic">p</span> &lt; 0.001 versus LPS-only treated cells. The data were statistically analyzed using a one-way ANOVA followed by Tukey’s post hoc test.</p> Full article ">Figure 4
<p>Comparative analysis of NF-κB protein expression level following <span class="html-italic">I. britannica</span> extract encapsulated in liposomes treatment in LPS-induced RAW 264.7 cells. (<b>A</b>) Western blot comparison of protein expression. Quantitative comparative analysis of phosphorylation of (<b>B</b>) IκB-α, (<b>C</b>) p-65, and (<b>D</b>) p-50. Data are represented as mean ± SEM (error bar). <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001 versus non-LPS-induced cells., ** <span class="html-italic">p</span> &lt; 0.01, and *** <span class="html-italic">p</span> &lt; 0.001 versus LPS-only treated cells. The data were statistically analyzed using a one-way ANOVA followed by Tukey’s post hoc test.</p> Full article ">Figure 5
<p>Comparative analysis of mitogen-activated protein kinase (MAPK) protein expression level following <span class="html-italic">I. britannica</span> extract encapsulated in liposomes treatment in lipopolysaccharide (LPS)-induced RAW 264.7 cells. (<b>A</b>) Western blot comparison of protein expression. Quantitative comparative analysis of phosphorylation of (<b>B</b>) ERK, (<b>C</b>) JNK, and (<b>D</b>) p-38. Data are represented as mean ± SEM (error bar). <sup>###</sup> <span class="html-italic">p</span> &lt; 0.001 versus non-LPS-induced cells. ** <span class="html-italic">p</span> &lt; 0.01, and *** <span class="html-italic">p</span> &lt; 0.001 versus LPS-only treated cells. The data were statistically analyzed using a one-way ANOVA followed by Tukey’s post hoc test.</p> Full article ">
26 pages, 9712 KiB  
Article
SIRT5 Activation and Inorganic Phosphate Binding Reduce Cancer Cell Vitality by Modulating Autophagy/Mitophagy and ROS
by Federica Barreca, Michele Aventaggiato, Laura Vitiello, Luigi Sansone, Matteo Antonio Russo, Antonello Mai, Sergio Valente and Marco Tafani
Antioxidants 2023, 12(8), 1635; https://doi.org/10.3390/antiox12081635 - 18 Aug 2023
Cited by 5 | Viewed by 1558
Abstract
Cancer cells show increased glutamine consumption. The glutaminase (GLS) enzyme controls a limiting step in glutamine catabolism. Breast tumors, especially the triple-negative subtype, have a high expression of GLS. Our recent study demonstrated that GLS activity and ammonia production are inhibited by sirtuin [...] Read more.
Cancer cells show increased glutamine consumption. The glutaminase (GLS) enzyme controls a limiting step in glutamine catabolism. Breast tumors, especially the triple-negative subtype, have a high expression of GLS. Our recent study demonstrated that GLS activity and ammonia production are inhibited by sirtuin 5 (SIRT5). We developed MC3138, a selective SIRT5 activator. Treatment with MC3138 mimicked the deacetylation effect mediated by SIRT5 overexpression. Moreover, GLS activity was regulated by inorganic phosphate (Pi). Considering the interconnected roles of GLS, SIRT5 and Pi in cancer growth, our hypothesis is that activation of SIRT5 and reduction in Pi could represent a valid antitumoral strategy. Treating cells with MC3138 and lanthanum acetate, a Pi chelator, decreased cell viability and clonogenicity. We also observed a modulation of MAP1LC3B and ULK1 with MC3138 and lanthanum acetate. Interestingly, inhibition of the mitophagy marker BNIP3 was observed only in the presence of MC3138. Autophagy and mitophagy modulation were accompanied by an increase in cytosolic and mitochondrial reactive oxygen species (ROS). In conclusion, our results show how SIRT5 activation and/or Pi binding can represent a valid strategy to inhibit cell proliferation by reducing glutamine metabolism and mitophagy, leading to a deleterious accumulation of ROS. Full article
(This article belongs to the Special Issue Autophagy-Mediated Cellular Oxidative Stress Regulations)
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Figure 1

Figure 1
<p>MC3138 and lanthanum acetate treatments increase cell death. (<b>A</b>,<b>B</b>) MDA-MB-231 cells were either left untreated or treated with lanthanum acetate, MC3138 or MC3138 + lanthanum acetate for 24 and 48 h. The percentage of cells in the different phases of the cell cycle was measured after propidium iodide staining and cytofluorimetric analysis. Percentages of sub-G1 cells are also reported and graphed in (<b>C</b>) showing an increase in the following treatments. (<b>D</b>,<b>E</b>) MDA-MB-231 cells silenced for GLS1 were either left untreated or treated with lanthanum acetate, MC3138 or MC3138 + lanthanum acetate for 24 and 48 h. The percentage of cells in the different phases of the cell cycle was measured after propidium iodide staining and cytofluorimetric analysis. Percentage of sub-G1 cells are also reported and graphed in (<b>F</b>) showing the increase following treatments. (<b>G</b>,<b>H</b>) CAL-62 cells were either left untreated or treated with lanthanum acetate, MC3138 or MC3138 + lanthanum acetate for 24 and 48 h. The percentage of cells in the different phases of the cell cycle was measured after propidium iodide staining and cytofluorimetric analysis. Percentage of sub-G1 cells are also reported and graphed in (<b>I</b>) showing an increase in following treatments. (<b>J</b>,<b>K</b>) CAL-62 cells silenced for GLS1 were either left untreated or treated with lanthanum acetate, MC3138 or MC3138 + lanthanum acetate for 24 and 48 h. The percentage of cells in the different phases of the cell cycle was measured after propidium iodide staining and cytofluorimetric analysis. Percentage of sub-G1 cells are also reported and graphed in (<b>L</b>) showing an increase following treatments. Experiments were repeated at least three times. Differences between pairs of groups were analyzed by Student’s <span class="html-italic">t</span>-test. * Significantly increased compared to control untreated cells. * <span class="html-italic">p</span> &lt; 0.05. CTRL, control; lanth. ac., lanthanum acetate.</p> Full article ">Figure 2
<p>Hypoxia influences cell cycle and reduces cell death upon MC3138 and lanthanum acetate treatment. (<b>A</b>,<b>B</b>) MDA-MB-231 cells were either left untreated or treated with lanthanum acetate, MC3138 or MC3138 + lanthanum acetate under hypoxia for 24 and 48 h. The percentage of cells in the different phases of the cell cycle was measured after propidium iodide staining and cytofluorimetric analysis. Percentages of sub-G1 cells are also reported and graphed in (<b>C</b>) showing increases in the following treatments. (<b>D</b>,<b>E</b>) MDA-MB-231 cells silenced for GLS1 were either left untreated or treated with lanthanum acetate, MC3138 or MC3138 + lanthanum acetate under hypoxia for 24 and 48 h. The percentage of cells in the different phases of the cell cycle was measured after propidium iodide staining and cytofluorimetric analysis. Percentages of sub-G1 cells are also reported and graphed in (<b>F</b>) showing the increase in following treatments. (<b>G</b>,<b>H</b>) CAL-62 cells were either left untreated or treated with lanthanum acetate, MC3138 or MC3138 + lanthanum acetate under hypoxia for 24 and 48 h. The percentage of cells in the different phases of the cell cycle was measured after propidium iodide staining and cytofluorimetric analysis. Percentages of sub-G1 cells are also reported and graphed in (<b>I</b>) showing the increase in following treatments. (<b>J</b>,<b>K</b>) CAL-62 cells silenced for GLS1 were either left untreated or treated with lanthanum acetate, MC3138 or MC3138 + lanthanum acetate under hypoxia for 24 and 48 h. The percentage of cells in the different phases of the cell cycle was measured after propidium iodide staining and cytofluorimetric analysis. Percentages of sub-G1 cells are also reported and graphed in (<b>L</b>) showing the increase in following treatments. Experiments were repeated at least three times. CTRL, control; lanth. ac., lanthanum acetate.</p> Full article ">Figure 3
<p>MC3138 reduces basal expression of HIF-1α in MDA-MB-231 cells. (<b>A</b>,<b>B</b>) MDA-MB-231 and MDA-MB-231 GLS1- cells were either left untreated or treated as indicated for 24 and 48 h under normoxic conditions. HIF-1α expression was determined by Western blot as described in Materials and Methods. (<b>C</b>,<b>D</b>) MDA-MB-231 and MDA-MB-231 GLS1- cells were either left untreated or treated as indicated for 24 and 48 h under hypoxic conditions. HIF-1α expression was determined by Western blot as described in Materials and Methods. GAPDH was used as loading control. HIF-1α expression was normalized with GAPDH and plotted as shown. * Significantly increased compared with untreated cells. *, <span class="html-italic">p</span> &lt; 0.05. # Significantly decreased compared with control cells. #, <span class="html-italic">p</span> &lt; 0.05. CTRL, control; lanth. ac., lanthanum acetate.</p> Full article ">Figure 4
<p>HIF-1α expression in CAL-62 cells. (<b>A</b>,<b>B</b>), CAL-62 and CAL-62 GLS1- cells were either left untreated or treated as indicated for 24 and 48 h under normoxic conditions. HIF-1α expression was determined by Western blot as described in Materials and Methods. (<b>C</b>,<b>D</b>), CAL-62 and CAL-62 GLS1- cells were either left untreated or treated as indicated for 24 and 48 h under hypoxic conditions. HIF-1α expression was determined by Western blot as described in Materials and Methods. GAPDH was used as loading control. HIF-1α expression was normalized with GAPDH and plotted as shown. * Significantly increased compared with untreated cells. *, <span class="html-italic">p</span> &lt; 0.05. # Significantly decreased compared with control cells. #, <span class="html-italic">p</span> &lt; 0.05. CTRL, control; lanth. ac., lanthanum acetate.</p> Full article ">Figure 5
<p>MC3138 and lanthanum acetate treatments prevent colony formation. (<b>A</b>) MDA-MB-231 cells were either left untreated or treated with lanthanum acetate, MC3138 or MC3138 + lanthanum acetate for 24 and 48 h. After 10 days, colony formation was obtained as described in Materials and Methods in 100 mm dishes and images taken. The number of colonies was counted and reported in the graph on the right side. (<b>B</b>) MDA-MB-231 GLS1- cells were either left untreated or treated with lanthanum acetate, MC3138 or MC3138 + lanthanum acetate for 24 and 48 h. After 10 days, colony formation was obtained as described in Materials and Methods in 100 mm dishes and images taken. The number of colonies was counted and reported in the graph on the right side. (<b>C</b>) CAL-62 cells were either left untreated or treated with lanthanum acetate, MC3138 or MC3138 + lanthanum acetate for 24 and 48 h. After 7 days, colony formation was obtained as described in Materials and Methods in 100 mm dishes and images taken. The number of colonies was counted and reported in the graph on the right side. (<b>D</b>) CAL-62 GLS1- cells were either left untreated or treated with lanthanum acetate, MC3138 or MC3138 + lanthanum acetate for 24 and 48 h. After 7 days, colony formation was obtained as described in Materials and Methods in 100 mm dishes and images taken. The number of colonies was counted and reported in the graph on the right side. Experiments were repeated three times. Differences between pairs of groups were analyzed by Student’s <span class="html-italic">t</span>-test. # Significantly decreased compared with untreated cells. #, <span class="html-italic">p</span> &lt; 0.05. CTRL, control; lanth. ac., lanthanum acetate.</p> Full article ">Figure 6
<p>MC3138 and lanthanum acetate treatments prevent colony formation under hypoxia. (<b>A</b>) MDA-MB-231 cells were either left untreated or treated with lanthanum acetate, MC3138 or MC3138 + lanthanum acetate under hypoxia for 24 and 48 h. After 10 days, colony formation was obtained as described in Materials and Methods in 100 mm dishes and images taken. The number of colonies was counted and reported in the graph on the right side. (<b>B</b>) MDA-MB-231 GLS1- cells were either left untreated or treated with lanthanum acetate, MC3138 or MC3138 + lanthanum acetate under hypoxia for 24 and 48 h. After 10 days, colony formation was obtained as described in Materials and Methods in 100 mm dishes and images taken. The number of colonies was counted and reported in the graph on the right side. (<b>C</b>) CAL-62 cells were either left untreated or treated with lanthanum acetate, MC3138 or MC3138 + lanthanum acetate under hypoxia for 24 and 48 h. After 7 days, colony formation was obtained as described in Materials and Methods in 100 mm dishes and images taken. The number of colonies was counted and reported in the graph on the right side. (<b>D</b>) CAL-62 GLS1- cells were either left untreated or treated with lanthanum acetate, MC3138 or MC3138 + lanthanum acetate under hypoxia for 24 and 48 h. After 7 days, colony formation was obtained as described in Materials and Methods in 100 mm dishes and images taken. The number of colonies was counted and reported in the graph on the right side. Experiments were repeated three times. Differences between pairs of groups were analyzed by Student’s <span class="html-italic">t</span>-test. # Significantly decreased compared with untreated cells. #, <span class="html-italic">p</span> &lt; 0.05. CTRL, control; lanth. ac., lanthanum acetate.</p> Full article ">Figure 7
<p>MC3138 reduces the expression of phosphate transporters. (<b>A</b>) MDA-MB-231 cells were treated as indicated in the figure. Expression levels of plasma membrane (SLC20A1 and SLC20A2 and mitochondrial (SLC25A3)) Pi transporters were determined by Western blot. Densitometric analysis of the gels was performed as described in Materials and Methods and results graphed below the blots. (<b>B</b>) MDA-MB-231 GLS1- cells were treated as indicated in the figure. Expression levels of plasma membrane (SLC20A1 and SLC20A2 and mitochondrial (SLC25A3)) Pi transporters were determined by Western blot. Densitometric analysis of the gels was performed as described in Materials and Methods and results graphed below the blots. (<b>C</b>) CAL-62 cells were treated as indicated in the figure. Expression levels of plasma membrane (SLC20A1 and SLC20A2 and mitochondrial (SLC25A3)) Pi transporters were determined by Western blot. Densitometric analysis of the gels was performed as described in Materials and Methods and results graphed below the blots. (<b>D</b>) CAL-62 GLS1- cells were treated as indicated in the figure. Expression levels of plasma membrane (SLC20A1 and SLC20A2 and mitochondrial (SLC25A3)) Pi transporters were determined by Western blot. Densitometric analysis of the gels was performed as described in Materials and Methods and results graphed below the blots. Data are representative of three separate experiments with GAPDH used as a loading control. Differences between pairs of groups were analyzed by Student’s <span class="html-italic">t</span>-test. * Significantly increased compared with untreated cells. *, <span class="html-italic">p</span> &lt; 0.05. # Significantly decreased compared with untreated cells. #, <span class="html-italic">p</span> &lt; 0.05. CTRL, control; lanth. ac., lanthanum acetate.</p> Full article ">Figure 8
<p>MC3138 and lanthanum acetate treatments reduce the expression of autophagy and mitophagy markers in breast cancer cells. (<b>A</b>) MDA-MB-231 cells were treated as indicated in the figure. Expression levels of autophagy proteins ULK1, phosphorylated ULK1 (pULK1), BECN1 and MAP1LC3B were determined by Western blot. Densitometric analysis of the gels was performed as described in Materials and Methods and results graphed on the right side. (<b>B</b>) MDA-MB-231 cells were treated as indicated in the figure. Expression levels of mitophagy proteins PRKN and BNIP3 were determined by Western blot. Densitometric analysis of the gels was performed as described in Materials and Methods and results graphed on the right side. (<b>C</b>) MDA-MB-231 GLS1- cells were treated as indicated in the figure. Expression levels of autophagy proteins ULK1, phosphorylated ULK (pULK1), BECN1 and MAP1LC3B were determined by Western blot. Densitometric analysis of the gels was performed as described in Materials and Methods and results graphed on the right side. (<b>D</b>) MDA-MB-231 GLS1- cells were treated as indicated in the figure. Expression levels of mitophagy proteins PRKN and BNIP3 were determined by Western blot. Densitometric analysis of the gels was performed as described in Materials and Methods and results graphed on the right side. Data are representative of three separate experiments with GAPDH used as a loading control. Differences between pairs of groups were analyzed by Student’s <span class="html-italic">t</span>-test. * Significantly increased compared with untreated cells. *, <span class="html-italic">p</span> &lt; 0.05. # Significantly decreased compared with untreated cells. #, <span class="html-italic">p</span> &lt; 0.05. CTRL, control; lanth. ac., lanthanum acetate.</p> Full article ">Figure 9
<p>MC3138 and lanthanum acetate treatments reduce the expression of autophagy and mitophagy markers in thyroid cancer cells. (<b>A</b>) CAL-62 cells were treated as indicated in the figure. Expression levels of autophagy proteins ULK1, phosphorylated ULK1 (pULK1), BECN1 and MAP1LC3B were determined by Western blot. Densitometric analysis of the gels was performed as described in Materials and Methods and results graphed on the right side. (<b>B</b>) CAL-62 cells were treated as indicated in the figure. Expression levels of mitophagy proteins PRKN and BNIP3 were determined by Western blot. Densitometric analysis of the gels was performed as described in Materials and Methods and results graphed on the right side. (<b>C</b>) CAL-62 GLS1- cells were treated as indicated in the figure. Expression levels of autophagy proteins ULK1, phosphorylated ULK1 (pULK1), BECN1 and MAP1LC3B were determined by Western blot. Densitometric analysis of the gels was performed as described in Materials and Methods and results graphed on the right side. (<b>D</b>) CAL-62 GLS1- cells were treated as indicated in the figure. Expression levels of mitophagy proteins PRKN and BNIP3 were determined by Western blot. Densitometric analysis of the gels was performed as described in Materials and Methods and results graphed on the right side. Data are representative of three separate experiments with GAPDH used as a loading control. Differences between pairs of groups were analyzed by Student’s <span class="html-italic">t</span>-test. * Significantly increased compared with untreated cells. *, <span class="html-italic">p</span> &lt; 0.05. # Significantly decreased compared with untreated cells. #, <span class="html-italic">p</span> &lt; 0.05. CTRL, control; lanth. ac., lanthanum acetate.</p> Full article ">Figure 10
<p>Modulation of autophagy and mitophagy markers in breast cancer cells treated with MC3138 and lanthanum acetate under hypoxia. (<b>A</b>) MDA-MB-231 cells were treated under hypoxia as indicated in the figure. Expression levels of autophagy proteins ULK1, phosphorylated ULK1 (pULK1), BECN1 and MAP1LC3B were determined by Western blot. Densitometric analysis of the gels was performed as described in Materials and Methods and results graphed on the right side. (<b>B</b>) MDA-MB-231 cells were treated under hypoxia as indicated in the figure. Expression levels of mitophagy proteins PRKN and BNIP3 were determined by Western blot. Densitometric analysis of the gels was performed as described in Materials and Methods and results graphed on the right side. (<b>C</b>) MDA-MB-231 GLS1- cells were treated under hypoxia as indicated in the figure. Expression levels of autophagy proteins ULK1, phosphorylated ULK1 (pULK1), BECN1 and MAP1LC3B were determined by Western blot. Densitometric analysis of the gels was performed as described in Materials and Methods and results graphed on the right side. (<b>D</b>) MDA-MB-231 GLS1- cells were treated under hypoxia as indicated in the figure. Expression levels of mitophagy proteins PRKN and BNIP3 were determined by Western blot. Densitometric analysis of the gels was performed as described in Materials and Methods and results graphed on the right side. Data are representative of three separate experiments with GAPDH used as a loading control. Differences between pairs of groups were analyzed by Student’s <span class="html-italic">t</span>-test. * Significantly increased compared with untreated cells. *, <span class="html-italic">p</span> &lt; 0.05. # Significantly decreased compared with untreated cells. #, <span class="html-italic">p</span> &lt; 0.05. CTRL, control; lanth. ac., lanthanum acetate.</p> Full article ">Figure 11
<p>Modulation of autophagy and mitophagy markers in thyroid cancer cells treated with MC3138 and lanthanum acetate under hypoxia. (<b>A</b>) CAL-62 cells were treated under hypoxia as indicated in the figure. Expression levels of autophagy proteins ULK1, phosphorylated ULK1 (pULK1), BECN1 and MAP1LC3B were determined by Western blot. Densitometric analysis of the gels was performed as described in Materials and Methods and results graphed on the right side. (<b>B</b>) CAL-62 cells were treated under hypoxia as indicated in the figure. Expression levels of mitophagy proteins PRKN and BNIP3 were determined by Western blot. Densitometric analysis of the gels was performed as described in Materials and Methods and results graphed on the right side. (<b>C</b>) CAL-62 GLS1- cells were treated under hypoxia as indicated in the figure. Expression levels of autophagy proteins ULK1, phosphorylated ULK1 (pULK1), BECN1 and MAP1LC3B were determined by Western blot. Densitometric analysis of the gels was performed as described in Materials and Methods and results graphed on the right side. (<b>D</b>) CAL-62 GLS1- cells were treated under hypoxia as indicated in the figure. Expression levels of mitophagy proteins PRKN and BNIP3 were determined by Western blot. Densitometric analysis of the gels was performed as described in Materials and Methods and results graphed on the right side. Data are representative of three separate experiments with GAPDH used as a loading control. Differences between pairs of groups were analyzed by Student’s <span class="html-italic">t</span>-test. * Significantly increased compared with untreated cells. *, <span class="html-italic">p</span> &lt; 0.05. # Significantly decreased compared with untreated cells. #, <span class="html-italic">p</span> &lt; 0.05. CTRL, control; lanth. ac., lanthanum acetate.</p> Full article ">Figure 12
<p>MC3138 and lanthanum acetate treatments increase cytosolic and mitochondrial ROS in cancer cells under normoxic and hypoxic conditions (<b>A</b>) MDA-MB-231, CAL-62 and GLS1- clones were either left untreated or treated with lanthanum acetate, MC3138 or MC3138 + lanthanum acetate for 24 (<b>upper left</b>) and 48 h (<b>lower left</b>). Total ROS content was measured using DCFH-DA as indicated in Materials and Methods and graphed as mean fluorescence intensity (MFI). (<b>B</b>) MDA-MB-231, CAL-62 and GLS1- clones were either left untreated or treated with lanthanum acetate, MC3138 or MC3138 + lanthanum acetate for 24 (<b>upper right</b>) and 48 h (<b>lower right</b>). Mitochondrial ROS content was measured using MitoSOX Red as indicated in Materials and Methods and graphed as mean fluorescence intensity (MFI). (<b>C</b>) MDA-MB-231, CAL-62 and GLS1- clones were either left untreated or treated with lanthanum acetate, MC3138 or MC3138 + lanthanum acetate for 24 (<b>upper left</b>) and 48 h (<b>lower left</b>) under hypoxia. Total ROS content was measured using DCFH-DA as indicated in Materials and Methods and graphed as median fluorescence intensity (MFI). (<b>D</b>) MDA-MB-231, CAL-62 and GLS1- clones were either left untreated or treated with lanthanum acetate, MC3138 or MC3138 + lanthanum acetate for 24 (<b>upper right</b>) and 48 h (<b>lower right</b>) under hypoxia. Mitochondrial ROS content was measured using MitoSOX Red as indicated in Materials and Methods and graphed as median fluorescence intensity (MFI). Data are representative of three separate experiments. Differences between pairs of groups were analyzed by Student’s <span class="html-italic">t</span>-test. * Significantly increased compared with untreated cells. *, <span class="html-italic">p</span> &lt; 0.05. # Significantly decreased compared with untreated cells. #, <span class="html-italic">p</span> &lt; 0.05. CTRL, control; lanth. ac., lanthanum acetate.</p> Full article ">
26 pages, 5849 KiB  
Article
Fibroblast Upregulation of Vitamin D Receptor Represents a Self-Protective Response to Limit Fibroblast Proliferation and Activation during Pulmonary Fibrosis
by Juan Wei, Junhui Zhan, Hui Ji, Yitong Xu, Qingfeng Xu, Xiaoyan Zhu and Yujian Liu
Antioxidants 2023, 12(8), 1634; https://doi.org/10.3390/antiox12081634 - 18 Aug 2023
Cited by 1 | Viewed by 1695
Abstract
Dysregulation of vitamin D receptor (VDR) is implicated in chronic obstructive pulmonary disease. However, whether VDR dysregulation contributes to the development of pulmonary fibrosis remains largely unknown. Analysis of bulk and single-cell RNA profiling datasets revealed VDR upregulation in lung fibroblasts from patients [...] Read more.
Dysregulation of vitamin D receptor (VDR) is implicated in chronic obstructive pulmonary disease. However, whether VDR dysregulation contributes to the development of pulmonary fibrosis remains largely unknown. Analysis of bulk and single-cell RNA profiling datasets revealed VDR upregulation in lung fibroblasts from patients with pulmonary fibrosis or fibrotic mice, which was validated in lung fibroblasts from bleomycin-exposed mice and bleomycin-treated fibroblasts. Stable VDR knockdown promoted, whereas the VDR agonist paricalcitol suppressed lung fibroblast proliferation and activation. Gene set enrichment analysis (GSEA) showed that the JAK/STAT pathway and unfolded protein response (UPR), a process related to endoplasmic reticulum (ER) stress, were enriched in lung fibroblasts of fibrotic lungs. Stable VDR knockdown stimulated, but paricalcitol suppressed ER stress and JAK1/STAT3 activation in lung fibroblasts. The STAT3 inhibitor blocked bleomycin- or stable VDR knockdown-induced ER stress. Paricalcitol inhibited the bleomycin-induced enrichment of STAT3 to the ATF6 promoter, thereby suppressing ATF6 expression in fibroblasts. Paricalcitol or intrapulmonary VDR overexpression inactivated JAK1/STAT3 and suppressed ER stress in bleomycin-treated mice, thus resulting in the inhibition of fibroblast proliferation and activation. Collectively, this study suggests that fibroblast VDR upregulation may be a self-protective response to limit fibroblast proliferation and activation during pulmonary fibrosis by suppressing the JAK1/STAT3/ER stress pathway. Full article
(This article belongs to the Section Health Outcomes of Antioxidants and Oxidative Stress)
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Figure 1

Figure 1
<p>VDR was specifically upregulated in lung fibroblasts during pulmonary fibrosis. (<b>A</b>,<b>B</b>) Published GEO datasets were analyzed for VDR expression in lung fibroblasts isolated from UIP/IPF patients (<b>A</b>) or bleomycin-treated mice (<b>B</b>). (<b>C</b>) Published GEO datasets were analyzed for VDR expression in primary cultured lung fibroblasts treated with TGF-β in vitro for the indicated exposure times. (<b>D</b>–<b>F</b>) A published scRNA-seq dataset (GSE132771) was analyzed in this study. (<b>D</b>) shows UMAP plot of all cells acquired by scRNA-seq of human samples. Cells were obtained from normal lungs (blue) and IPF lungs (red). When all cell types were analyzed as a pool, no significant difference was found in VDR levels between control and IPF groups (<b>E</b>). However, VDR expression was significantly elevated in fibroblasts from the lungs of patients with IPF (<b>F</b>). (<b>G</b>) Mice were intratracheally instilled with bleomycin (3 mg/kg) or saline. Lung tissues or fibroblasts were obtained at day 14 after bleomycin instillation. The mouse lung fibroblast cell line Mlg was treated with bleomycin (5 μg/mL) for 48 h. VDR protein expression in lung tissues, lung fibroblasts, and Mlg cells was assessed by Western blot. Representative protein bands are presented on the left side of corresponding histograms. Lung tissue samples were collected from 7 control mice and 7 bleomycin-treated mice. All cell samples were collected from four independent experiments. Data are expressed as means ± SEM. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01 vs. Control, ns represents non-significant. BLM represents bleomycin.</p> Full article ">Figure 2
<p>Stable knockdown of VDR promotes lung fibroblast proliferation and activation. Mlg cells were transfected with shRNA lentivirus targeting VDR (Lv-ShRNA-VDR) to establish a fibroblast cell line with stable VDR knockdown. Control cells were transfected with scrambled shRNA lentivirus (Lv-ShRNA-NC). (<b>A</b>) The knockdown efficiency of stable transfection of Lv-ShRNA-VDR or Lv-ShRNA-NC was confirmed by measuring mRNA and protein levels of VDR by qPCR and Western blot analysis, respectively. (<b>B</b>) Cell proliferation was assessed using the MTT assay at the indicated time point. (<b>C</b>), Cell proliferation was also assessed using the EdU incorporation assay. The percentage of proliferative cells is shown on the right of the representative images as a ratio of EdU<sup>+</sup> cells to the total cell number. Scale bars correspond to 50 μm. (<b>D</b>,<b>E</b>) qPCR was used to determine mRNA levels of the pro-proliferative genes (<b>D</b>) and fibrosis-specific genes (<b>E</b>). (<b>F</b>) Western blot analysis was used to determine the protein expression of α-SMA. Representative protein bands are shown on the top of corresponding histograms. Data are expressed as means ± SEM (n = 4). * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01 vs. Lv-ShRNA-NC.</p> Full article ">Figure 3
<p>VDR agonist suppresses lung fibroblast proliferation and activation in vitro. (<b>A</b>) Mlg and MLE-12 cells were treated with bleomycin (5 μg/mL) in the presence of increasing concentrations of paricalcitol (0~10 μM) for 48 h. Cell proliferation was detected by MTT Assay. (<b>B</b>–<b>E</b>) Mlg cells were treated with saline or bleomycin (5 μg/mL) in the presence of vehicle or paricalcitol (2.5 μM) for 48 h. (<b>B</b>,<b>C</b>) qPCR was used to determine mRNA levels of the pro-proliferative genes (<b>B</b>) and fibrosis-specific genes (<b>C</b>). (<b>D</b>) Protein levels of α-SMA and fibronectin were determined by Western blot analysis. Representative protein bands are presented on the left of corresponding histograms. (<b>E</b>) Mlg cells were stained with primary antibody against the myofibroblast marker α-SMA (green). Nuclei were stained with blue using 4′,6-diamidino-2-phenylindole (DAPI). Quantification of the α-SMA mean intensity in fibroblasts are presented on the right of the representative images. Scale bars correspond to 50 μm. Data are expressed as means ± SEM (n = 4). ** <span class="html-italic">p</span> &lt; 0.01 vs. Control, &amp;&amp; <span class="html-italic">p</span> &lt; 0.01 vs. BLM. BLM represents bleomycin.</p> Full article ">Figure 4
<p>VDR agonist suppresses while stable knockdown of VDR stimulates endoplasmic reticulum stress in lung fibroblasts. (<b>A</b>–<b>C</b>) Following Hallmark analysis of GSEA, the gene set of unfolded protein response (UPR) was found to be enriched in lung fibroblasts obtained from IPF patients (<b>A</b>), UIP patients (<b>B</b>), and bleomycin-treated mice (<b>C</b>). (<b>D</b>) Mlg cells were treated with saline or bleomycin (5 μg/mL) in the presence of vehicle or paricalcitol (2.5 μM) for 48 h. Protein levels of Grp78 and CHOP were determined by Western blot analysis. Representative protein bands are presented on the left of corresponding histograms. Data are expressed as means ± SEM (n = 4). ** <span class="html-italic">p</span> &lt; 0.01 vs. Control. &amp;&amp; <span class="html-italic">p</span> &lt; 0.01 vs. BLM. BLM represents bleomycin. (<b>E</b>) Mlg cells were stably transfected with Lv-ShRNA-NC or Lv-ShRNA-VDR. Protein levels of Grp78 and CHOP were determined by Western blot analysis. Representative protein bands are presented on the left of corresponding histograms. Data are expressed as means ± SEM (n = 4). ** <span class="html-italic">p</span> &lt; 0.01 vs. Lv-ShRNA-NC.</p> Full article ">Figure 5
<p>VDR agonist suppresses endoplasmic reticulum stress, while stable knockdown of VDR stimulates endoplasmic reticulum stress through regulation of JAK/STAT3 signaling pathway in lung fibroblasts. (<b>A</b>,<b>B</b>) Hallmark analysis of GSEA reveals that the IL6-JAK-STAT3 signaling pathway is enriched in lung fibroblasts obtained from IPF patients (<b>A</b>) and bleomycin-treated mice (<b>B</b>). (<b>C</b>) Mlg cells were treated with saline or bleomycin (5 μg/mL) in the presence of vehicle or paricalcitol (2.5 μM) for 48 h. Protein levels of phosphorylated JAK1, JAK1, phosphorylated STAT3, and STAT3 were determined by Western blot analysis. Representative protein bands were presented on the left of the histograms. Data are expressed as means ± SEM (n = 4). ** <span class="html-italic">p</span> &lt; 0.01 vs. Control, &amp;&amp; <span class="html-italic">p</span> &lt; 0.01 vs. BLM. (<b>D</b>) Mlg cells were stably transfected with Lv-ShRNA-NC or Lv-ShRNA-VDR. Protein levels of phosphorylated JAK1, JAK1, phosphorylated STAT3, and STAT3 were determined by Western blot analysis. Representative protein bands are presented on the left of the histograms. Data are expressed as means ± SEM (n = 4). ** <span class="html-italic">p</span> &lt; 0.01 vs. Lv-ShRNA-NC. (<b>E</b>) Mlg cells were treated with saline or bleomycin (5 μg/mL) in the presence of vehicle or STAT3 inhibitor C188-9 (4 μM) for 48 h. Protein levels of phosphorylated STAT3, STAT3, Grp78, and CHOP were determined by Western blot analysis. Representative protein bands were presented on the left of the histograms. Data are expressed as means ± SEM (n = 4). * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01 vs. Control, &amp; <span class="html-italic">p</span> &lt; 0.05, &amp;&amp; <span class="html-italic">p</span> &lt; 0.01 vs. BLM. (<b>F</b>) Mlg cells stably transfected with Lv-ShRNA-NC or Lv-ShRNA-VDR were treated with vehicle or STAT3 inhibitor C188-9 (4 μM) for 48 h. Protein levels of phosphorylated STAT3, STAT3, Grp78, and CHOP were determined by Western blot analysis. Representative protein bands were presented on the left of the histograms. Data are expressed as means ± SEM (n = 4). * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01 vs. Lv-ShRNA-NC. &amp; <span class="html-italic">p</span> &lt; 0.05, &amp;&amp; <span class="html-italic">p</span> &lt; 0.01 vs. Lv-ShRNA-VDR. BLM represents bleomycin.</p> Full article ">Figure 6
<p>VDR agonist inhibits STAT3-dependent ATF6 transcription and endoplasmic reticulum stress in human lung fibroblasts. MRC-5 cells were treated with saline or bleomycin (5 μg/mL) in the presence of vehicle or paricalcitol (2.5 μM) for 48 h. (<b>A</b>) Protein levels of phosphorylated JAK1, JAK1, phosphorylated STAT3, and STAT3 were determined by Western blot analysis. Representative protein bands were presented on the left of the corresponding histograms. (<b>B</b>) Protein levels of Grp78 and CHOP were determined by Western blot analysis. Representative protein bands were presented on the left of corresponding histograms. (<b>C</b>) qPCR and Western blot analysis were used to determine mRNA and the protein levels of ATF6, respectively. Representative protein bands were presented on the top of corresponding histograms. (<b>D</b>) A schematic diagram shows two predicted conservative consensus STAT3-binding motifs in the proximal region of both human and mouse ATF6 promoters. (<b>E</b>) Chromatin immunoprecipitation (ChIP) assay showed enrichment of STAT3 on two consensus STAT3-binding motifs in human ATF6 promoter. Data are expressed as means ± SEM (n = 4). ** <span class="html-italic">p</span> &lt; 0.01 vs. Control. &amp;&amp; <span class="html-italic">p</span> &lt; 0.01 vs. BLM. BLM represents bleomycin.</p> Full article ">Figure 7
<p>VDR agonist inactivates JAK/STAT3 signaling pathway and suppresses endoplasmic reticulum stress during bleomycin-induced pulmonary fibrosis. Mice were randomly divided into four groups: Control, Paricalcitol, BLM, and BLM + Paricalcitol. (<b>A</b>) Protein levels of phosphorylated JAK1, JAK1, phosphorylated STAT3, and STAT3 in lung tissues were determined in the Control, Paricalcitol, BLM, and BLM + Paricalcitol groups. Representative protein bands were presented on the left of the histograms. (<b>B</b>), Protein levels of Grp78 and CHOP in lung tissues were determined in the Control, Paricalcitol, BLM, and BLM + Paricalcitol groups. Representative protein bands were presented on the left of the histograms. (<b>C</b>) Lung sections were stained with fluorophore-labeled antibodies against the cell proliferation marker Ki67 (Alexa Fluor 647, red) and the fibroblast marker FSP-1 (Alexa Fluor 488, green). DAPI staining was used to detect nuclei (blue). The merge image represents double positive staining for FSP-1 and Ki67. Areas in white boxes were shown enlarged. Scale bars correspond to 50 μm. Quantification of the percentage of ki67<sup>+</sup>/FSP-1<sup>+</sup> cells in total FSP-1<sup>+</sup> cells in lung tissue sections is presented on the right of the representative images. Data are expressed as means ± SEM (n = 7). ** <span class="html-italic">p</span> &lt; 0.01 vs. Control. &amp;&amp; <span class="html-italic">p</span> &lt; 0.01 vs. BLM. BLM represents bleomycin.</p> Full article ">Figure 8
<p>VDR agonist inhibits fibroblast differentiation into myofibroblast during bleomycin-induced pulmonary fibrosis. Mice were randomly divided into four groups: Control, Paricalcitol, BLM, and BLM + Paricalcitol. (<b>A</b>), Lung sections were stained with an anti-α-SMA antibody (green). DAPI staining was used to detect nuclei (blue). Scale bars correspond to 50 μm. (<b>B</b>) Quantification of the mean fluorescent intensity of α-SMA in lung tissue sections. (<b>C</b>) Protein levels of α-SMA in lung tissues were determined in the Control, Paricalcitol, BLM, and BLM + Paricalcitol groups. Representative protein bands were presented on the left of the histograms. Data are expressed as mean ± SEM (n = 7). ** <span class="html-italic">p</span> &lt; 0.01 vs. Control. &amp;&amp; <span class="html-italic">p</span> &lt; 0.01 vs. BLM. BLM represents bleomycin.</p> Full article ">Figure 9
<p>VDR agonist attenuates pulmonary fibrosis and improves pulmonary function in mice exposed to bleomycin. Mice were randomly divided into four groups: Control, Paricalcitol, BLM, and BLM + Paricalcitol. (<b>A</b>) Collagen deposition in lung tissues were examined by Masson’s trichrome staining in the Control, Paricalcitol, BLM, and BLM + Paricalcitol groups. Scale bars correspond to 50 μm. (<b>B</b>) Changes in the ratio of collagen-deposited areas to lung substance areas (a morphometric measure of pulmonary fibrosis). (<b>C</b>) Protein levels of fibronectin and collagen-1 in lung tissues were determined in the Control, Paricalcitol, BLM, and BLM + Paricalcitol groups. Representative protein bands were presented on the top of the histograms. (<b>D</b>,<b>E</b>) The forced vital capacity (FVC) and dynamic lung compliance (Cydn) were measured by Pressure Volume Test and Resistance &amp; Compliance Test, respectively. Data are expressed as mean ± SEM (n = 7). ** <span class="html-italic">p</span> &lt; 0.01 vs. Control. &amp; <span class="html-italic">p</span> &lt; 0.05, &amp;&amp; <span class="html-italic">p</span> &lt; 0.01 vs. BLM. BLM represents bleomycin.</p> Full article ">Figure 10
<p>Intrapulmonary VDR overexpression inhibits JAK/STAT3 activation, endoplasmic reticulum stress, fibroblast proliferation and differentiation into myofibroblasts. Mice were randomly divided into four groups: Lv-NC, Lv-VDR, Lv-NC + BLM, and Lv-VDR + BLM. (<b>A</b>) The protein levels of phosphorylated JAK1, JAK1, phosphorylated STAT3, and STAT3 in lung tissues were determined by Western blot analysis. Representative protein bands were presented on the left of the histograms. (<b>B</b>) Protein levels of Grp78, CHOP and ATF6 in lung tissues were determined by Western blot analysis. Representative protein bands were presented on the left of the histograms. (<b>C</b>) Lung sections were stained with fluorophore-labeled antibodies against cell proliferation marker Ki67 (Alexa Fluor 647, red) and fibroblast marker FSP-1 (Alexa Fluor 488, green). DAPI staining was used to detect nuclei (blue). The merge image represents double positive staining for FSP-1 and Ki67. Areas in white boxes are shown enlarged. Scale bars correspond to 50 μm. Quantification of the percentage of Ki67<sup>+</sup>/FSP-1<sup>+</sup> cells in total FSP-1<sup>+</sup> cells in lung tissue sections are presented on the right of the representative images. (<b>D</b>) Lung sections were stained with anti-α-SMA antibody (red). DAPI staining was used to detect nuclei (blue). The mean fluorescent intensity of the α-SMA in lung tissue sections was quantified and presented on the right of the representative images. Scale bars correspond to 50 μm. Data are expressed as means ± SEM (n = 7). ** <span class="html-italic">p</span> &lt; 0.01 vs. Control. &amp;&amp; <span class="html-italic">p</span> &lt; 0.01 vs. BLM. BLM represents bleomycin.</p> Full article ">Figure 11
<p>VDR overexpression attenuates pulmonary fibrosis in mice exposed to bleomycin. Mice were randomly divided into four groups: Lv-NC, Lv-VDR, Lv-NC + BLM, and Lv-VDR + BLM. (<b>A</b>) Collagen deposition in lung tissues was examined by Masson’s trichrome staining. Scale bars correspond to 50 μm. (<b>B</b>) Changes in the ratio of collagen-deposited areas to lung substance areas (a morphometric measure of pulmonary fibrosis). (<b>C</b>) Protein levels of α-SMA, fibronectin and collagen-1 in lung tissues were determined by Western blot analysis. Representative protein bands are presented on the left of the histograms. Data are expressed as mean ± SEM (n = 7). ** <span class="html-italic">p</span> &lt; 0.01 vs. Control. &amp;&amp; <span class="html-italic">p</span> &lt; 0.01 vs. BLM. BLM represents bleomycin.</p> Full article ">Figure 12
<p>Schematic diagram of the mechanism by which fibroblast upregulation of VDR suppresses lung fibroblast proliferation and activation during pulmonary fibrosis. VDR is specifically upregulated in lung fibroblasts during pulmonary fibrosis. Upon activation, VDR attenuates lung fibroblast proliferation and activation. The anti-fibrotic effects of vitamin D/VDR signaling may be at least partly due to suppression of ER stress in lung fibroblasts. In this study, we have identified JAK1/STAT3 as an upstream regulator of ER stress in fibroblasts. VDR activation inhibits enrichment of STAT3 to ATF6 promoter, thus suppressing ATF6 expression in fibroblasts. Collectively, this study suggests that fibroblast VDR upregulation may be a self-protective response to limit fibroblast proliferation and activation during pulmonary fibrosis by suppressing JAK1/STAT3/ER stress pathway.</p> Full article ">
20 pages, 3302 KiB  
Article
Phytochemical Constituents and Biological Activity of Wild and Cultivated Rosmarinus officinalis Hydroalcoholic Extracts
by Rosaria Francolino, Mara Martino, Lucia Caputo, Giuseppe Amato, Giuseppina Chianese, Ernesto Gargiulo, Carmen Formisano, Benedetta Romano, Giuseppe Ercolano, Angela Ianaro, Laura De Martino and Vincenzo De Feo
Antioxidants 2023, 12(8), 1633; https://doi.org/10.3390/antiox12081633 - 18 Aug 2023
Cited by 4 | Viewed by 1700
Abstract
Rosmarinus officinalis L. is an aromatic evergreen plant from the Lamiaceae family. The purpose of this study was to compare the chemical profile and bioactivities of hydroalcoholic extracts derived from wild and cultivated R. officinalis. The chemical composition of the extracts was [...] Read more.
Rosmarinus officinalis L. is an aromatic evergreen plant from the Lamiaceae family. The purpose of this study was to compare the chemical profile and bioactivities of hydroalcoholic extracts derived from wild and cultivated R. officinalis. The chemical composition of the extracts was evaluated via LC–MS analysis, which revealed the presence of a wide range of phenolic compounds, including flavonoids, phenolic and terpenes. Both extracts showed a similar interesting antioxidant activity, probably related to their content of phenol and flavonoids. The analysis of anti-acetylcholinesterase (AChE), anti-butyrylcholinesterase (BChE), and anti-α-amylase activities showed analogous inhibition, except for AChE, in which the wild type was more active than the cultivated one. Finally, in vitro studies were performed using the J774A.1 murine macrophage cell line, to characterize the anti-inflammatory and the antioxidant effects of the extracts. As expected, pretreatment with the extracts significantly reduced the production proinflammatory cytokines and ROS through modulation of the nitric oxide pathway and the mitochondrial activity. Importantly, it is observed that the anti-inflammatory effect of the extracts was explicated through the inhibition of NF-kB and its downstream mediator COX-2. Collectively, these results demonstrated that these extracts could represent a starting point for developing novel therapeutic strategies for the treatment of inflammation-based diseases. Moreover, since no significant changes were observed in terms of composition and activity, both wild and cultivated R. officinalis extracts can be recommended for food and pharmaceutical purposes. Full article
(This article belongs to the Topic Biological Activity of Plant Extracts)
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Figure 1

Figure 1
<p>Full scan LC-MS chromatograms (positive-ion HRESIMS) of hydroalcoholic extracts of wild (WRO, upper) and cultivated <span class="html-italic">R. officinalis</span> (CRO, lower).</p> Full article ">Figure 2
<p>(<b>A</b>) J774 A.1 cells were treated with increasing concentrations of WRO and CRO. Cytotoxicity was evaluated via MTT assay 24 h after treatment. (<b>B</b>) Representative plot and (<b>C</b>) frequency of dead cells after treatment for 24 h with both CRO and WRO at 10 μg/mL.</p> Full article ">Figure 3
<p>(<b>A</b>,<b>B</b>) J774A.1 cells were treated with extracts (10 μg/mL) for 1 h before stimulation with LPS (100 ng/mL) and INF-γ (20 ng/mL) for 6 h. Subsequently, mRNA levels of the pro-inflammatory cytokines IL-6 (<b>A</b>) and TNF-α (<b>B</b>) were determined. (<b>C</b>) Representative example of flow cytometry analysis of FLUO3-AM staining. (<b>D</b>) FLUO3-AM quantification in terms of mean fluorescence intensity (MFI). Values were expressed as mean ± SEM from at least three independent experiments. °° <span class="html-italic">p</span> &lt; 0.01, °°°° <span class="html-italic">p</span> &lt; 0.0001 indicate a significant effect of LPS/INFγ compared with unstimulated cells (CTRL); * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, **** <span class="html-italic">p</span> &lt; 0.0001 indicated a significant effect of CRO and WRO compared with LPS/INF-γ-stimulated cells.</p> Full article ">Figure 4
<p>(<b>A</b>) Relative mRNA levels of NOS2 in J774 macrophages were determined using qPCR analysis after 6 h. (<b>B</b>) Representative plot and (<b>C</b>) frequency of intracellular NOS2 expression in J774 macrophages evaluated via flow cytometry after 24 h. (<b>D</b>) Levels of NO measured in the cell culture medium of J774 cells after 24 h via the Greiss reaction. Values were expressed as mean ± SEM from at least three independent experiments. °°°° <span class="html-italic">p</span> &lt; 0.0001 indicates a significant effect of LPS/INFγ compared with unstimulated cells (CTRL); * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001 indicated a significant effect of WRO and CRO compared with LPS/INF-γ-stimulated cells.</p> Full article ">Figure 5
<p>(<b>A</b>) Nuclear extracts were prepared using a nuclear extract kit. NF-κB-P65 activity was measured using an ELISA kit. (<b>B</b>) Representative images of p65 and IkB-α proteins detected via Western blot analysis, respectively, in cytosolic (<b>C</b>) and in nuclear (N) extract after 1 h of pre-treatment with 10 µg/mL of WRO and CRO and 30 min of LPS/IFN-γ stimulation. α-tubulin and GAPDH were used as an internal control. (<b>C</b>) mRNA levels of PTGS2 after treatment with WRO and CRO (10 μg/mL) for 1 h and stimulation with LPS (100 ng/mL) and INF-γ (20 ng/mL) for 6 h. (<b>D</b>) Representative images of COX-2 proteins detected via Western blot analysis in total extract. GAPDH was used as an internal control. (<b>E</b>) PGE2 concentrations in J774 cell culture supernatants after 24 h of treatment with the extracts (10 µg/mL) and stimulation with LPS/IFN-γ. Values were expressed as mean ± SEM from at least three independent experiments. °° <span class="html-italic">p</span> &lt; 0.01, °°° <span class="html-italic">p</span> &lt; 0.001, °°°° <span class="html-italic">p</span> &lt; 0.0001 indicate a significant effect of LPS/INFγ compared with unstimulated cells (CTRL); * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, indicate a significant effect of CRO and WRO compared with LPS/INF-γ-stimulated cells.</p> Full article ">Figure 6
<p>(<b>A</b>) Representative example of flow cytometry analysis of DCF-DHA probe. (<b>B</b>) DCF-DHA quantification in terms of mean fluorescence intensity (MFI). (<b>C</b>) Representative examples of flow cytometric analysis of MitoTracker Green (<b>C</b>) with its quantification in terms of mean fluorescence intensity (MFI) (<b>D</b>). Values were express as mean ± SEM from three independent experiments. ° <span class="html-italic">p</span> &lt; 0.05, °° <span class="html-italic">p</span> &lt; 0.01, indicate significant effect of LPS/INFγ-stimulated cells compared with unstimulated cells (CTRL); * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01 indicated significant effect of WRO and CRO extracts compared with stimulated cells.</p> Full article ">
24 pages, 2776 KiB  
Article
Copper(II) Complexes with Carnosine Conjugates of Hyaluronic Acids at Different Dipeptide Loading Percentages Behave as Multiple SOD Mimics and Stimulate Nrf2 Translocation and Antioxidant Response in In Vitro Inflammatory Model
by Francesco Bellia, Valeria Lanza, Irina Naletova, Barbara Tomasello, Valeria Ciaffaglione, Valentina Greco, Sebastiano Sciuto, Pietro Amico, Rosanna Inturri, Susanna Vaccaro, Tiziana Campagna, Francesco Attanasio, Giovanni Tabbì and Enrico Rizzarelli
Antioxidants 2023, 12(8), 1632; https://doi.org/10.3390/antiox12081632 - 18 Aug 2023
Cited by 2 | Viewed by 1513
Abstract
A series of copper(II) complexes with the formula [Cu2+Hy(x)Car%] varying the molecular weight (MW) of Hyaluronic acid (Hy, x = 200 or 700 kDa) conjugated with carnosine (Car) present at different loading were synthesized and characterized via different spectroscopic [...] Read more.
A series of copper(II) complexes with the formula [Cu2+Hy(x)Car%] varying the molecular weight (MW) of Hyaluronic acid (Hy, x = 200 or 700 kDa) conjugated with carnosine (Car) present at different loading were synthesized and characterized via different spectroscopic techniques. The metal complexes behaved as Cu, Zn-superoxide dismutase (SOD1) mimics and showed some of the most efficient reaction rate values produced using a synthetic and water-soluble copper(II)-based SOD mimic reported to date. The increase in the percentage of Car moieties parallels the enhancement of the I50 value determined via the indirect method of Fridovich. The presence of the non-functionalized Hy OH groups favors the scavenger activity of the copper(II) complexes with HyCar, recalling similar behavior previously found for the copper(II) complexes with Car conjugated using β-cyclodextrin or trehalose. In keeping with the new abilities of SOD1 to activate protective agents against oxidative stress in rheumatoid arthritis and osteoarthritis diseases, Cu2+ interaction with HyCar promotes the nuclear translocation of erythroid 2-related factor that regulates the expressions of target genes, including Heme-Oxigenase-1, thus stimulating an antioxidant response in osteoblasts subjected to an inflammatory/oxidative insult. Full article
(This article belongs to the Special Issue Nrf2 Antioxidative Pathway and NF-κB Signaling)
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<p>Chemical structures of HyCar conjugates. The <span class="html-italic">x</span> indicates the molecular weight of Hy in kDa, and Car% indicates the Car loading percentage. The Car units represent the average number of Car units conjugated to Hy.</p> Full article ">Figure 2
<p>Comparison between FTIR−ATR spectra of Hy(200)Car35% (blue) and its complex with Cu<sup>2+</sup> (orange).</p> Full article ">Figure 3
<p>ESR spectra (150 K) of copper(II) complexes with Hy(200)Car35% and Hy(200)Car14% at different pH values. Simulated spectra are represented in red and experimental spectra are in black.</p> Full article ">Figure 4
<p>SOD1-like activity of Cu<sup>2+</sup>Hy(200)Car complexes at different Car loading percentages. The I<sub>50</sub> values of different HyCar conjugates were normalized in respect to the I<sub>50</sub> value of SOD1 [<a href="#B65-antioxidants-12-01632" class="html-bibr">65</a>].</p> Full article ">Figure 5
<p>Catalytic cycle of SOD1: (<b>A</b>) a tentative structure of multiple copper(II) binding sites in Cu-HyCar, and (<b>B</b>) a hypothetical (but based on our results) scheme detailing the O<sub>2</sub><sup>●−</sup> scavenger abilities of copper(II) complexes with HyCar.</p> Full article ">Figure 6
<p>Cu<sup>2+</sup>’s interactions with Car, Hy, and HyCar stimulate hFOB proliferation under MCM stress. Cells were pre-treated with HyCar, Hy, Car, or Hy and Car in the presence or absence of BCS (50 μM) for 1 h and exposed to MCM for 20 h. After the treatments, the plates were imaged via the IncuCyte instrument. Total occupied area of hFOB cells was calculated via IncuCyte Base Analysis using the “Artificial Intelligence (AI)” mask for cell detection; for other parameters, refer to <a href="#antioxidants-12-01632-t001" class="html-table">Table 1</a> in the <a href="#sec2-antioxidants-12-01632" class="html-sec">Section 2</a>. The instrument software generated the percentage of cell confluence (as indicated in the <a href="#sec2-antioxidants-12-01632" class="html-sec">Section 2</a>) for each well. The data points and error bars represent the mean ± SD of two experiments in quadruplicates. Statistical significance is indicated as * <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 7
<p>Hy, Car, and their conjugate interaction with Cu<sup>2+</sup> present in culture medium reduce ROS levels in hFOB cells stressed with MCM. hFOB cells were pre-treated with HyCar, Hy, Car, or Hy and Car in the presence or absence of BCS (50 µM) for 1 h and exposed to MCM for 20 h. Fluorescence measurement was performed via the IncuCyte system using the “Artificial Intelligence (AI)” mask for cell detection; for other parameters, refer to <a href="#antioxidants-12-01632-t002" class="html-table">Table 2</a> in the <a href="#sec2-antioxidants-12-01632" class="html-sec">Section 2</a>. Data are expressed as the total green fluorescence area of cells per image. Statistical significance is indicated as * <span class="html-italic">p</span> &lt; 0.05 or *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 8
<p>The nuclear localization of Nrf2 in MCM-stressed hFOB cells is significantly promoted by HyCar in a Cu<sup>2+</sup>-dependent mode. hFOB cells were pre-treated with HyCar, Hy, Car, or Hy and Car in the presence or absence of BCS (50 µM) for 1 h and exposed to MCM for 48 h. Ratio of fluorescence intensity between nucleus and cytoplasm represents the Nrf2 expression level. All values are mean ± SD of five random field images (see <a href="#app1-antioxidants-12-01632" class="html-app">Figure S7</a>) derived from three independent experiments. Significant differences were indicated using * <span class="html-italic">p</span> ≤ 0.05.</p> Full article ">Figure 9
<p>HyCar affects the Nrf2 signaling pathway in MCM-stressed hFOB cells in a copper-dependent manner. Densitometric analysis (<b>A</b>,<b>B</b>) and representative Western blot images (<b>C</b>) of HO-1 and SOD1 expression in hFOB pre-treated with HyCar, Hy, Car, or Hy and Car in the presence or absence of BCS (50 µM) for 1 h and exposed to MCM for 48 h. The protein expression levels are reported as the ratio over actin. Data are expressed as mean ± SD. Significant differences were indicated using the following methods: * <span class="html-italic">p</span> ≤ 0.05, ** <span class="html-italic">p</span> ≤ 0.01, *** <span class="html-italic">p</span> ≤ 0.001.</p> Full article ">
16 pages, 2833 KiB  
Article
Microbiome–Metabolomic Analysis Reveals Beneficial Effects of Dietary Kelp Resistant Starch on Intestinal Functions of Hybrid Snakeheads (Channa maculata ♀ × Channa argus ♂)
by Shaodan Wang, Zhiheng Zuo, Bin Ye, Li Zhang, Yanbo Cheng, Shaolin Xie, Jixing Zou and Guohuan Xu
Antioxidants 2023, 12(8), 1631; https://doi.org/10.3390/antiox12081631 - 18 Aug 2023
Cited by 1 | Viewed by 1665
Abstract
The benefits of resistant starch on hypoglycemia, obesity prevention, antioxidant status and the alleviation of metabolic syndrome have received considerable attention. In this study, we explored how dietary kelp resistant starch (KRS) enhances intestinal morphology and function through a microbiome–metabolomic analysis. Hybrid snakeheads [...] Read more.
The benefits of resistant starch on hypoglycemia, obesity prevention, antioxidant status and the alleviation of metabolic syndrome have received considerable attention. In this study, we explored how dietary kelp resistant starch (KRS) enhances intestinal morphology and function through a microbiome–metabolomic analysis. Hybrid snakeheads (initial weight: 11.4 ± 0.15 g) were fed experimental diets for 60 days. Fish were fed a basic wheat starch diet and the KRS diet. Dietary KRS improved intestinal morphology and enhanced intestinal antioxidant and digestive capabilities, as evidenced by decreased intestinal damage and upregulated intestinal biochemical markers. The microbiome analysis showed that KRS administration elevated the proportion of butyrate-producing bacteria and the abundance of beneficial bacteria that increases insulin sensitivity. Furthermore, significant alterations in metabolic profiles were observed to mainly associate with the amino acid metabolism (particularly arginine production), the metabolism of cofactors and vitamins, fat metabolism, glutathione metabolism, and the biosynthesis of other secondary metabolites. Additionally, alterations in intestinal microbiota composition were significantly associated with metabolites. Collectively, changes in intestinal microbiota and metabolite profiles produced by the replacement of common starch with dietary KRS appears to play an important role in the development of intestinal metabolism, thus leading to improved intestinal function and homeostasis. Full article
(This article belongs to the Special Issue Antioxidants Benefits in Aquaculture 2.0)
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<p>Regulation of KRS on serum glycolipid levels in hybrid snakeheads. (<b>A</b>) Serum glucose levels. (<b>B</b>) Serum total cholesterol content. (<b>C</b>) Serum triglyceride content. Values are expressed as means ± SD. * Indicates significant difference (* <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01).</p> Full article ">Figure 2
<p>Intestinal villi growth and intestinal damage. (<b>A</b>) H&amp;E staining of the midintestinal portion of the intestine. Upper scale bar = 500 μm, below scale bar = 200 μm. (<b>B</b>) Intestinal villus length statistics. The red circle highlights the shedding of the intestinal villus epithelium. Values are expressed as means ± SD. * Indicates significant difference (** <span class="html-italic">p</span> &lt; 0.01).</p> Full article ">Figure 3
<p>KRS affects intestinal biochemical activity. (<b>A</b>) Intestinal GSH-Px activity. (<b>B</b>) Intestinal SOD activity. (<b>C</b>) Intestinal CAT activity. (<b>D</b>) Intestinal MDA activity. (<b>E</b>) Intestinal α-Ams activity. (<b>F</b>) Intestinal α-Chmo activity. (<b>G</b>) Intestinal Lip activity. (<b>H</b>) Intestinal AKP activity. (<b>I</b>) Intestinal MCT-1 activity. Values are expressed as means ± SD. * Indicates significant difference (* <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01).</p> Full article ">Figure 4
<p>Dietary KRS modifies the composition of microbial population in the intestinal. (<b>A</b>) Venn diagram of intestinal differential microbes. (<b>B</b>) Shannon index of OTU levels. (<b>C</b>) Partial least squares discriminant analysis (PLS-DA) for differential grouping. (<b>D</b>) Genus-level species clustering heatmap for differential grouping. (<b>E</b>) Differential microbiome based on LEfSe analysis. Values are expressed as means ± SD. * Indicates significant difference (* <span class="html-italic">p</span> &lt; 0.05, *** <span class="html-italic">p</span> &lt; 0.001).</p> Full article ">Figure 5
<p>Characterization of intestinal metabolites in different groups. (<b>A</b>) Partial least squares discriminant analysis (PLS-DA) of intestinal metabolites in positive and negative ion mode. (<b>B</b>) Volcano plot of differential metabolites. (<b>C</b>) Cluster heatmap and VIP value analysis of differential metabolites. (<b>D</b>) The importance of differential metabolites was identified based on random forest plots. (<b>E</b>) Radar plots were used to visualize the expression profiles of various groups of differential metabolites. (<b>F</b>) KEGG analysis was used to examine the primary functions of differential metabolites. * indicates significant difference (* <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001).</p> Full article ">Figure 6
<p>Heatmap of associations between intestinal microbial and metabolite profiles. (<b>A</b>) Pearson correlation analysis of phylum-level species and differential metabolites. (<b>B</b>) Pearson correlation analysis of genus-level species and differential metabolites. * indicates significant difference (* <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01).</p> Full article ">
22 pages, 1546 KiB  
Review
Melatonin as a Therapy for Preterm Brain Injury: What Is the Evidence?
by Silke Häusler, Nicola J. Robertson, Klervi Golhen, John van den Anker, Katie Tucker and Thomas K. Felder
Antioxidants 2023, 12(8), 1630; https://doi.org/10.3390/antiox12081630 - 17 Aug 2023
Cited by 4 | Viewed by 1709
Abstract
Despite significant improvements in survival following preterm birth in recent years, the neurodevelopmental burden of prematurity, with its long-term cognitive and behavioral consequences, remains a significant challenge in neonatology. Neuroprotective treatment options to improve neurodevelopmental outcomes in preterm infants are therefore urgently needed. [...] Read more.
Despite significant improvements in survival following preterm birth in recent years, the neurodevelopmental burden of prematurity, with its long-term cognitive and behavioral consequences, remains a significant challenge in neonatology. Neuroprotective treatment options to improve neurodevelopmental outcomes in preterm infants are therefore urgently needed. Alleviating inflammatory and oxidative stress (OS), melatonin might modify important triggers of preterm brain injury, a complex combination of destructive and developmental abnormalities termed encephalopathy of prematurity (EoP). Preliminary data also suggests that melatonin has a direct neurotrophic impact, emphasizing its therapeutic potential with a favorable safety profile in the preterm setting. The current review outlines the most important pathomechanisms underlying preterm brain injury and correlates them with melatonin’s neuroprotective potential, while underlining significant pharmacokinetic/pharmacodynamic uncertainties that need to be addressed in future studies. Full article
(This article belongs to the Section Health Outcomes of Antioxidants and Oxidative Stress)
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<p>Melatonin signaling mechanisms: Melatonin shows receptor-mediated and receptor-independent activity and interacts with a variety of cellular signaling pathways. Both MT1 and MT2 are members of the GPCR receptor family. Melatonin binding and signaling involve several effectors, including PKC, PLC, and PKA, as well as downstream signaling pathways. Melatonin can also enter the cell through passive diffusion and transporters, where it activates mitochondrial receptors or cytosolic quinone reductase 2 (MT3), which in turn activates nuclear receptors ROR/RZR. Melatonin causes a shift towards an anti-inflammatory and antioxidant response and the preservation of mitochondrial and neuronal integrity. (Abbreviations: 5-OH-Trp, 5-Hydroxytryptophan; AMK, <span class="html-italic">N</span><sup>1</sup>-acetyl-5-methoxykynuramine; AFMK, <span class="html-italic">N</span><sup>1</sup>-acetyl-<span class="html-italic">N</span><sup>2</sup>-formyl-5-methoxykynuramine; Akt, protein kinase B; COX, cyclooxygenase; CREB, cAMP response element-binding protein; Cyt, cytochrome c; ETC, electron transport chain; GPX, glutathione peroxidase; HO, heme oxygenase; IκB, Inhibitory-kappa B; iNOS, inducible nitric oxide synthase; KEAP1, Kelch-like ECH-associated protein 1; LOX, lipoxygenase; MAPK, mitogen-activated protein kinase; MPO, myeloperoxidase; mPTP, mitochondrial permeability transition pore; MT1/2/3, melatonin receptors 1/2/3; mtDNA, mitochondrial DNA; NF-κB, nuclear factor- kappa B; Nrf2, nuclear factor erythroid 2-related factor 2; PI3K, phosphoinositide 3-kinase; PKA, protein kinase A; PGC-1α, peroxisome proliferator-activated receptor gamma coactivator 1-alpha; PKC, protein kinase C; PLC, phospholipase C; RNS, reactive nitrogen species; ROR/RZR, retinoid acid receptor-related orphan receptor/retinoid Z receptor (RZR); ROS, reactive oxygen species SIRT1, sirtuin-1; SOD, superoxide dismutase; Trp, tryptophan).</p> Full article ">Figure 2
<p>Neuroprotective and neurorestorative effects of melatonin: Prenatal and postnatal risk factors are associated with increased inflammation, which causes injury to the developing brain. Melatonin exerts its neuroprotective effects by reducing excitotoxicity, oxidative stress, and inflammation (acute and chronic), as well as by promoting oligodendroglial maturation and myelination. (Abbreviations: GMD, gestational diabetes mellitus; RDS, respiratory distress syndrome; PDA, patent ductus arteriosus; BPD, bronchopulmonary dysplasia; IVH, intraventricular hemorrhage; PHVD, posthemorrhagic ventricular dilatation).</p> Full article ">
19 pages, 1287 KiB  
Review
The NOS/NO System in Renal Programming and Reprogramming
by You-Lin Tain and Chien-Ning Hsu
Antioxidants 2023, 12(8), 1629; https://doi.org/10.3390/antiox12081629 - 17 Aug 2023
Cited by 3 | Viewed by 1329
Abstract
Nitric oxide (NO) is a gaseous signaling molecule with renoprotective properties. NO can be produced in NO synthase (NOS)-dependent or -independent manners. NO deficiency plays a decisive role in chronic kidney disease (CKD). Kidney development can be affected in response to adverse intrauterine [...] Read more.
Nitric oxide (NO) is a gaseous signaling molecule with renoprotective properties. NO can be produced in NO synthase (NOS)-dependent or -independent manners. NO deficiency plays a decisive role in chronic kidney disease (CKD). Kidney development can be affected in response to adverse intrauterine conditions that induce renal programming, thereby raising the risk of developing CKD in adulthood. Conversely, detrimental programming processes could be postponed or halted prior to the onset of CKD by early treatments, namely reprogramming. The current review provides an overview of the NOS/NO research performed in the context of renal programming and reprogramming. NO deficiency has been increasingly found to interact with the different mechanisms behind renal programming, such as oxidative stress, aberrant function of the renin–angiotensin system, disturbed nutrient-sensing mechanisms, dysregulated hydrogen sulfide signaling, and gut microbiota dysbiosis. The supplementation of NOS substrates, the inhibition of asymmetric dimethylarginine (ADMA), the administration of NO donors, and the enhancement of NOS during gestation and lactation have shown beneficial effects against renal programming in preclinical studies. Although human data on maternal NO deficiency and offspring kidney disease are scarce, experimental data indicate that targeting NO could be a promising reprogramming strategy in the setting of renal programming. Full article
(This article belongs to the Special Issue Advances for the NO/NOS System)
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<p>The NOS/NO system and the potential effects of NO on kidney functions: NO is generated from the NOS-dependent or -independent pathways. NO bioactivity has been associated with several effects in kidney function, mainly via cGMP-dependent mechanisms, although cGMP-independent mechanisms have also been reported. NOS can be inhibited by asymmetric dimethylarginine (ADMA). ADMA is generated by protein arginine methyl transferases (PRMTs). Dimethylarginine dimethylaminohydrolase-1 (DDAH-1) and -2 (DDAH-2), and alanine-glyoxylate aminotransferase 2 (AGXT2) can metabolize ADMA. NOS uncoupling contributes to reactive oxygen species (ROS) generation. Both ROS and reactive nitrogen species (RNS) can inhibit NO-mediated kidney effects.</p> Full article ">Figure 2
<p>Schema outlining the putative mechanisms of the renal programming linked to the NOS/NO system.</p> Full article ">
23 pages, 2674 KiB  
Review
Roles of Oxidative Stress in Synaptic Dysfunction and Neuronal Cell Death in Alzheimer’s Disease
by Germán Plascencia-Villa and George Perry
Antioxidants 2023, 12(8), 1628; https://doi.org/10.3390/antiox12081628 - 17 Aug 2023
Cited by 12 | Viewed by 3443
Abstract
Alzheimer’s disease (AD) is a brain disorder that progressively undermines memory and thinking skills by affecting the hippocampus and entorhinal cortex. The main histopathological hallmarks of AD are the presence of abnormal protein aggregates (Aβ and tau), synaptic dysfunction, aberrant proteostasis, cytoskeletal abnormalities, [...] Read more.
Alzheimer’s disease (AD) is a brain disorder that progressively undermines memory and thinking skills by affecting the hippocampus and entorhinal cortex. The main histopathological hallmarks of AD are the presence of abnormal protein aggregates (Aβ and tau), synaptic dysfunction, aberrant proteostasis, cytoskeletal abnormalities, altered energy homeostasis, DNA and RNA defects, inflammation, and neuronal cell death. However, oxidative stress or oxidative damage is also evident and commonly overlooked or considered a consequence of the advancement of dementia symptoms. The control or onset of oxidative stress is linked to the activity of the amyloid-β peptide, which may serve as both antioxidant and pro-oxidant molecules. Furthermore, oxidative stress is correlated with oxidative damage to proteins, nucleic acids, and lipids in vulnerable cell populations, which ultimately lead to neuronal death through different molecular mechanisms. By recognizing oxidative stress as an integral feature of AD, alternative therapeutic or preventive interventions are developed and tested as potential or complementary therapies for this devastating neurodegenerative disease. Full article
(This article belongs to the Special Issue Oxidative Stress in Alzheimer's Disease)
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<p>Hallmarks of neurodegenerative diseases. The main histopathological hallmarks identified in Alzheimer’s disease and related dementia are (1) pathological protein aggregation (Aβ and tau), (2) synaptic and neuronal network dysfunction, (3) aberrant proteostasis, (4) cytoskeletal abnormalities, (5) altered energy homeostasis, (6) DNA and RNA defects, (7) neuroinflammation, and (8) neuronal cell death. Reproduced from [<a href="#B6-antioxidants-12-01628" class="html-bibr">6</a>].</p> Full article ">Figure 2
<p>Structure of amyloid-β (1–42) peptide in fibrillar aggregates. (<b>A</b>) Atomic-resolution structure of Aβ<sub>1–42</sub> superimposed with cryo-electron microscopy density map. (<b>B</b>) Inset shows a detailed view of the interactions between the -<span class="html-italic">N</span> and -<span class="html-italic">C</span> terminus (Asp1 and Ala42) with the side chains of Lys28. (<b>C</b>) Side view of the opposing subunits showing the relative orientation of the nonplanar subunits; the cross-β sheets are tilted by 10° with respect to the plane perpendicular to the fibril axis. (<b>D</b>) Side view of Aβ<sub>1–42</sub> fibrils showing the staggered arrangement of the nonplanar subunits. (<b>E</b>) Surface representation of Aβ<sub>1–42</sub> fibrils according to hydrophobicity (Kyte–Doolittle scale), orange–brown areas (hydrophobic, 4.5) to white (neutral, 0.0). (<b>F</b>) Surface view of the “ridge” fibril ends. (<b>G</b>) Surface views of the “groove” fibril ends. Reproduced with permission from [<a href="#B9-antioxidants-12-01628" class="html-bibr">9</a>].</p> Full article ">Figure 3
<p>Oxidative damage to neurons in Alzheimer’s disease.</p> Full article ">Figure 4
<p>Diverse programmed mechanisms related to neuronal failure and death in Alzheimer’s disease.</p> Full article ">Figure 5
<p>Interventions targeting oxidative stress in Alzheimer’s disease. The therapeutic compounds are organized into compounds under clinical trials, bioactive metabolites, natural compounds, and antioxidant vitamins.</p> Full article ">
12 pages, 4148 KiB  
Article
Biomarkers of Oxidative Stress in Systemic Lupus Erythematosus Patients with Active Nephritis
by Lu Liu, Karina de Leeuw, Suzanne Arends, Berber Doornbos-van der Meer, Marian L. C. Bulthuis, Harry van Goor and Johanna Westra
Antioxidants 2023, 12(8), 1627; https://doi.org/10.3390/antiox12081627 - 17 Aug 2023
Cited by 2 | Viewed by 1287
Abstract
Oxidative stress plays an important role in systemic lupus erythematosus (SLE) and especially in lupus nephritis (LN). The aim of this study was to compare redox-related biomarkers between patients with active LN, quiescent SLE (Q-SLE) and healthy controls (HC) and to explore their [...] Read more.
Oxidative stress plays an important role in systemic lupus erythematosus (SLE) and especially in lupus nephritis (LN). The aim of this study was to compare redox-related biomarkers between patients with active LN, quiescent SLE (Q-SLE) and healthy controls (HC) and to explore their association with clinical characteristics such as disease activity in patients. We investigated levels of plasma free thiols (R-SH, sulfhydryl groups), levels of soluble receptor for advanced glycation end products (sRAGE) and levels of malondialdehyde (MDA) in SLE patients with active LN (n = 23), patients with quiescent SLE (n = 47) and HC (n = 23). Data of LN patients who previously participated in Dutch lupus nephritis studies and longitudinal samples up to 36 months were analyzed. Thiol levels were lower in active LN at baseline and Q-SLE patients compared to HC. In generalized estimating equation (GEE) modelling, free thiol levels were negatively correlated with the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) over time (p < 0.001). sRAGE and MDA were positively correlated with the SLEDAI over time (p = 0.035 and p = 0.016, respectively). These results indicate that oxidative stress levels in LN patients are increased compared to HC and associated with SLE disease activity. Therefore, interventional therapy to restore redox homeostasis may be useful as an adjunctive therapy in the treatment of oxidative damage in SLE. Full article
(This article belongs to the Special Issue Oxidative Stress in Rheumatic Diseases)
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Graphical abstract
Full article ">Figure 1
<p>Levels of thiols, sRAGE and MDA at baseline in active LN, quiescent SLE and HC. Violin plots showing levels at baseline: (<b>A</b>) plasma free thiol levels, (<b>B</b>) sRAGE levels, (<b>C</b>) MDA levels. Abbreviations: A-LN: active lupus nephritis, Q-SLE: quiescent systemic lupus erythematosus, HC: healthy controls, sRAGE: soluble receptor for advanced glycation end products, MDA: malondialdehyde, ns: not significant, statistical significance: * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.002, *** <span class="html-italic">p</span> &lt; 0.0002.</p> Full article ">Figure 2
<p>Correlation between oxidative stress biomarkers in active LN patients (<b>A</b>,<b>B</b>), quiescent SLE patients (<b>C</b>,<b>D</b>) and HC (<b>E</b>,<b>F</b>). rho: correlation coefficient; Spearman’s rank correlation was used. <span class="html-italic">p</span> &lt; 0.05: statistical significance.</p> Full article ">Figure 2 Cont.
<p>Correlation between oxidative stress biomarkers in active LN patients (<b>A</b>,<b>B</b>), quiescent SLE patients (<b>C</b>,<b>D</b>) and HC (<b>E</b>,<b>F</b>). rho: correlation coefficient; Spearman’s rank correlation was used. <span class="html-italic">p</span> &lt; 0.05: statistical significance.</p> Full article ">Figure 3
<p>Median levels of oxidative stress biomarkers and SLEDAI scores during 36 months follow-up of LN patients: (<b>A</b>) median plasma-free thiol levels, (<b>B</b>) median sRAGE levels, (<b>C</b>) median MDA levels, (<b>D</b>) median SLEDAI levels. Green dots represent remission group, red dots represent relapse group; each dot with vertical lines indicates median with error. Abbreviations: SLEDAI: Systemic Lupus Erythematosus Disease Activity Index.</p> Full article ">
17 pages, 1662 KiB  
Review
On Males, Antioxidants and Infertility (MOXI): Certitudes, Uncertainties and Trends
by Manuel Alfaro Gómez, María del Rocío Fernández-Santos, Alejandro Jurado-Campos, Pedro Javier Soria-Meneses, Vidal Montoro Angulo, Ana Josefa Soler, José Julián Garde and Virginia Rodríguez-Robledo
Antioxidants 2023, 12(8), 1626; https://doi.org/10.3390/antiox12081626 - 17 Aug 2023
Cited by 3 | Viewed by 1786
Abstract
Male infertility (MI) involves various endogenous and exogenous facts. These include oxidative stress (OS), which is known to alter several physiological pathways and it is estimated to be present at high levels in up to 80% of infertile men. That is why since [...] Read more.
Male infertility (MI) involves various endogenous and exogenous facts. These include oxidative stress (OS), which is known to alter several physiological pathways and it is estimated to be present at high levels in up to 80% of infertile men. That is why since the late 20th century, the relationship between OS and MI has been widely studied. New terms have emerged, such as Male Oxidative Stress Infertility (MOSI), which is proposed as a new category to define infertile men with high OS levels. Another important term is MOXI: Male, Antioxidants, and Infertility. This term refers to the hypothesis that antioxidants could improve male fertility without the use of assisted reproductive technology. However, there are no evidence-based antioxidant treatments that directly improve seminal parameters or birth ratio. In this regard, there is controversy about their use. While certain scientists argue against their use due to the lack of results, others support this use because of their safety profile and low price. Some uncertainties related to the use of antioxidants for treating MI are their questionable efficacy or the difficulties in knowing their correct dosage. In addition, the lack of quality methods for OS detection can lead to excessive antioxidant supplementation, resulting in “reductive stress”. Another important problem is that, although the inflammatory process is interdependent and closely linked to OS, it is usually ignored. To solve these uncertainties, new trends have recently emerged. These include the use of molecules with anti-inflammatory and antioxidant potential, which are also able to specifically target the reproductive tissue; as well as the use of new methods that allow for reliable quantification of OS and a quality diagnosis. This review aims to elucidate the main uncertainties about MOXI and to outline the latest trends in research to develop effective therapies with clinically relevant outcomes. Full article
(This article belongs to the Special Issue Antioxidants in Male Human and Animal Reproduction: In Vitro and In Vivo Studies)
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<p>Worldwide incidence of MOSI in infertile men. <sup>a</sup> National Institutes of Health (NIH). Reproduced with permission from Figure 3, Agarwal, A., et al., World J Mens Health 2019 [<a href="#B5-antioxidants-12-01626" class="html-bibr">5</a>].</p> Full article ">Figure 2
<p>Mechanisms to explain ‘antioxidant paradox’ pertaining to male infertility, both by the induction of reductive stress and the failure to address the interconnected link of oxidative stress (OS). Reproduced with permission from Figure 1 of Dutta, S., Antioxidants 2022.</p> Full article ">Figure 3
<p>A schematic outline of all the protocols here reviewed in both bottom-up and top-down untargeted MS approaches for the proteomic analysis of human SP. Reproduced with permission from Preianò, M. et al., Int. J. Mol. Sci., 2023.</p> Full article ">
21 pages, 7034 KiB  
Article
Ozonated Sunflower Oil Exerted Potent Anti-Inflammatory Activities with Enhanced Wound Healing and Tissue Regeneration Abilities against Acute Toxicity of Carboxymethyllysine in Zebrafish with Improved Blood Lipid Profile
by Kyung-Hyun Cho, Ji-Eun Kim, Ashutosh Bahuguna and Dae-Jin Kang
Antioxidants 2023, 12(8), 1625; https://doi.org/10.3390/antiox12081625 - 17 Aug 2023
Cited by 4 | Viewed by 2808
Abstract
Ozonated sunflower oil (OSO) is an established therapeutic agent and nutraceutical harboring various therapeutic values, including antiallergic, derma-protective, and broad-spectrum antimicrobial activity. Conversely, the medicinal aspects of OSO for wound healing, tissue regeneration, and treatment of inflammation in dyslipidemia have yet to be [...] Read more.
Ozonated sunflower oil (OSO) is an established therapeutic agent and nutraceutical harboring various therapeutic values, including antiallergic, derma-protective, and broad-spectrum antimicrobial activity. Conversely, the medicinal aspects of OSO for wound healing, tissue regeneration, and treatment of inflammation in dyslipidemia have yet to be fully elucidated. Herein, a comparative effect of OSO and sunflower oil (SO) was investigated to heal cutaneous wound and tissue regeneration of zebrafish impediment by carboxymethyllysine (CML) toxicity, following impact on hepatic inflammation and blood lipid profile. After OSO (final 2%, 1 μL) and SO’s (final 2%, 1 μL) treatment, substantial healing was elicited by OSO in the cutaneous wound of zebrafish impaired by CML (final 25 μg). As an important event of wound healing, OSO scavenges the reactive oxygen species (ROS), rescues the wound from oxidative injury, and triggers the essential molecular events for the wound closer. Furthermore, the intraperitoneal injection of OSO was noted to counter the CML-induced adversity and prompt tissue regeneration in the amputated tail fin of zebrafish. Additionally, OSO counters the CML-induced neurotoxicity and rescues the zebrafish from acute mortality and paralysis, along with meticulous recovery of hepatic inflammation, fatty liver changes, and diminished ROS and proinflammatory interleukin (IL)-6 production. Moreover, OSO efficiently ameliorated CML-induced dyslipidemia by alleviating the total blood cholesterol (TC), triglyceride (TG), and increasing high-density lipoproteins cholesterol (HDL-C). The outcome of multivariate assessment employing principal component analysis and hierarchical cluster analysis supports a superior therapeutic potential of OSO over SO against the clinical manifestation of CML. Conclusively, OSO owing to its antioxidant and anti-inflammatory potential, counters CML-induced toxicity and promotes wound healing, tissue regeneration, hepatoprotection, improved blood lipid profile, and survivability of zebrafish. Full article
(This article belongs to the Section Natural and Synthetic Antioxidants)
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<p>The comparative healing effect of sunflower oil (SO) and Raydel ozonated sunflower oil (OSO) against carboxymethyllysine (CML) exposed cutaneous wound in adult zebrafish. (<b>A</b>) Pictorial representation of wound stained with methylene blue (0.1% <span class="html-italic">w</span>/<span class="html-italic">v</span>) and pigment formation during 264 h post-treatment with 6.7× magnifications. The red arrow implies the pigment formation in the wounded area, while the blue arrow represents the wound scar. (<b>B</b>) Percentage wound closer during 264 h post-treatment. The percentage of the wound closer was computed by comparing the stained wound area measured at different times with respect to the wound area at 0 h. The PBS control group received a topical dose of PBS only, CML+PBS groups received a topical dose of CML (25 μg) dissolved in PBS, while the CML+SO2% and CML+OSO2% groups were co-treated with CML (25 μg) with SO2% and OSO2%, respectively. The letters (a,b) above the graphs indicate the statistical difference (<span class="html-italic">p</span> &lt; 0.05) among the groups at the given time.</p> Full article ">Figure 2
<p>Comparative effect of sunflower oil (SO) and Raydel ozonated sunflower oil (OSO) on skin morphology and reactive oxygen species (ROS) production on carboxymethyllysine (CML) treated cutaneous wound in adult zebrafish. (<b>A</b>,<b>C</b>) Portray hematoxylin and eosin (H&amp;E) staining at 72 h- and 168 h post-treatment with 400× magnification The black and blue arrows represent the granulation and muscular tissue, respectively. (<b>B</b>,<b>D</b>) Portray dihydroethidium (DHE) staining for ROS production at 72 h and 168 h post-treatment 400× magnification. The PBS (control) group revied a topical application of PBS only, CML+PBS groups received a topical dose of CML (25 μg) dissolved in PBS, while the CML+SO2% and CML+OSO2% groups were co-treated with CML (25 μg) with SO2% and OSO2%, respectively. The letters (a–d) above the graphs indicate a statistical difference (<span class="html-italic">p</span> &lt; 0.05) among the groups at the given time.</p> Full article ">Figure 3
<p>A comparative tail fin regenerative effect of sunflower oil (SO) and Raydel ozonated sunflower oil (OSO) in carboxymethyllysine (CML) injected adult zebrafish. (<b>A</b>) Morphology of tail fin amid 7 days post-treatment. The red dotted line indicates the regenerated tissue at the proximal end. (<b>B</b>) kinetics of tail fin regenerated area. The PBS group was microinjected with PBS (vehicle), and the CML+PBS group was microinjected with CML (3 mM) dissolved in PBS. CML+SO2% and CML+OSO2% groups were microinjected with CML (3 mM) together with SO2% and OSO2%, respectively. The letters (a–c) above the graphs indicate the statistical difference (<span class="html-italic">p</span> &lt; 0.05) among the groups at the given time.</p> Full article ">Figure 4
<p>A comparative swimming behavior and survivability of zebrafish injected with carboxymethyllysine (CML) and subsequently treated with sunflower oil (SO) and Raydel ozonated sunflower oil (OSO). (<b>A</b>) Zebrafish survivability at 60 min post-injection. (<b>B</b>) Mean value of percentage swimming activity and (<b>C</b>) snapshots of swimming activity at 30 min and 60 min post-injection. The CML+PBS group was injected with CML (250 μg) dissolved in PBS, whereas CML+(SO1%, SO2%, OSO1%, and OSO2%) groups were co-injected with CML (250 μg) along with either SO1% or SO2% or OSO1% or OSO2%. The letters (a,b) above the graphs indicate the statistical difference (<span class="html-italic">p</span> &lt; 0.05) among the groups at the given time.</p> Full article ">Figure 5
<p>Histology of hepatic tissue deciphering a comparative effect of sunflower oil (SO) and Raydel ozonated sunflower oil (OSO) on hepatic morphology, fatty liver changes, oxidative stress, and IL-6 production in carboxymethyllysine (CML) injected adult zebrafish at 60 min post-injection. (<b>A</b>) Hematoxylin and eosin (H&amp;E) staining. The black, blue, and red arrows represent the bile duct, arterial area, and portal vein. (<b>B</b>) Oil red O staining. (<b>C</b>) DHE staining for reactive oxygen species detection. (<b>D</b>) IL-6 production [detected by immunocytochemistry (IHC)]. The depicted images are 400× magnified [graphic scale = 0.1 mm], and the stained area was computed employing ImageJ software (version 1.53, <a href="http://rsb.info.nih.gov/ij/" target="_blank">http://rsb.info.nih.gov/ij/</a> retrieved on 16 May 2022). The CML+PBS group was injected with CML (3 mM) dissolved in PBS, whereas CML+(SO1%, SO2%, OSO1%, and OSO2%) groups were co-injected with CML (3 mM) along with either SO1% or SO2% or OSO1% or OSO2%. <span class="html-italic">p</span> value documented the pairwise statistical variation retrieved from the ANOVA employing the Turkey’s test for post hoc analysis.</p> Full article ">Figure 6
<p>Effect of sunflower oil (SO) and Raydel ozonated sunflower oil (OSO) on the liver function biomarkers (<b>A</b>) aspartate aminotransferase (AST) and (<b>B</b>) alanine aminotransferase (ALT) in carboxymethyllysine (CML) injected adult zebrafish. The CML+PBS group was injected with CML (3 mM) dissolved in PBS, whereas CML+(SO1%, SO2%, OSO1%, and OSO2%) groups were co-injected with CML (3 mM) along with either SO1% or SO2% or OSO1% or OSO2%. The letters (a,b) above the graphs indicate the statistical difference (<span class="html-italic">p</span> &lt; 0.05) among the groups at the given time.</p> Full article ">Figure 7
<p>Comparative analysis of sunflower oil (SO) and Raydel ozonated sunflower oil (OSO) on blood lipid profile of the carboxymethyllysine (CML) injected adult zebrafish. The CML+PBS group was injected with CML (3 mM) dissolved in PBS, whereas CML+(SO1%, SO2%, OSO1%, and OSO2%) groups were co-injected with CML (3 mM) along with either SO1% or SO2% or OSO1% or OSO2%. The letters (a–c) above the graphs indicate the statistical difference (<span class="html-italic">p</span> &lt; 0.05) among the groups at the given time. The TC, HDL-C, and TG are acronyms for total cholesterol, triglyceride, and high-density lipoprotein cholesterol.</p> Full article ">Figure 8
<p>Multivariate analysis based on tested parameters (survivability, swimming, liver function biomarkers, histology, and blood lipid profile) of zebrafish stimulated by carboxymethyllysine (CML) and subsequently treated with sunflower oil (SO) and Raydel ozonated sunflower oil (OSO). (<b>A</b>,<b>B</b>) are the scoring plot and loading plot, respectively, obtained from principal component analysis (PCA). (<b>C</b>) Hierarchical cluster analysis (HCA). The PCA and HCA were performed using Minitab statistical software version 21.4. The CML+PBS group was injected with CML (3 mM) dissolved in PBS, whereas CML+(SO1%, SO2%, OSO1%, and OSO2%) groups were co-injected with CML (3 mM) and SO1% or SO2% or OSO1% or OSO2%. AST, aspartate aminotransferase; ALT, alanine aminotransferase; HDL-C, high-density lipoprotein cholesterol; TC, total cholesterol; TG, triglyceride.</p> Full article ">Figure 9
<p>Summary of Raydel ozonated sunflower (OSO) mediated events that counters carboxymethyllysine (CML) impelled hazards leads to cutaneous wound healing, tissue regeneration and hepatoprotection in zebrafish.</p> Full article ">
15 pages, 1670 KiB  
Review
Energy (and Reactive Oxygen Species Generation) Saving Distribution of Mitochondria for the Activation of ATP Production in Skeletal Muscle
by Alejandra Espinosa, Mariana Casas and Enrique Jaimovich
Antioxidants 2023, 12(8), 1624; https://doi.org/10.3390/antiox12081624 - 17 Aug 2023
Cited by 3 | Viewed by 1891
Abstract
Exercise produces oxidants from a variety of intracellular sources, including NADPH oxidases (NOX) and mitochondria. Exercise-derived reactive oxygen species (ROS) are beneficial, and the amount and location of these ROS is important to avoid muscle damage associated with oxidative stress. We discuss here [...] Read more.
Exercise produces oxidants from a variety of intracellular sources, including NADPH oxidases (NOX) and mitochondria. Exercise-derived reactive oxygen species (ROS) are beneficial, and the amount and location of these ROS is important to avoid muscle damage associated with oxidative stress. We discuss here some of the evidence that involves ROS production associated with skeletal muscle contraction and the potential oxidative stress associated with muscle contraction. We also discuss the potential role of H2O2 produced after NOX activation in the regulation of glucose transport in skeletal muscle. Finally, we propose a model based on evidence for the role of different populations of mitochondria in skeletal muscle in the regulation of ATP production upon exercise. The subsarcolemmal population of mitochondria has the enzymatic and metabolic components to establish a high mitochondrial membrane potential when fissioned at rest but lacks the capacity to produce ATP. Calcium entry into the mitochondria will further increase the metabolic input. Upon exercise, subsarcolemmal mitochondria will fuse to intermyofibrillar mitochondria and will transfer the mitochondria membrane potential to them. These mitochondria are rich in ATP synthase and will subsequentially produce the ATP needed for muscle contraction in long-term exercise. These events will optimize energy use and minimize mitochondria ROS production. Full article
(This article belongs to the Special Issue Skeletal Muscle Redox Signaling and Metabolism)
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<p>Physical interactions between SS and IMF mitochondria. Transmission electron micrograph of myofibers in the transverse plane. SS and IMF mitochondria are distinct organelles. Some SS and IMF mitochondria form continuous organelles (arrowheads) that coexist in both subcellular compartments. SS, Subsarcolemmal; IMF, intermyofibrillar; PM, plasma membrane (sarcolemma); Myofibr, myofibrils; Cap, capillary (Reproduced with permission from M. Picard (J. Appl. Physiol., published by American Physiological Society, 2013) [<a href="#B7-antioxidants-12-01624" class="html-bibr">7</a>]).</p> Full article ">Figure 2
<p>Energy efficient distribution of mitochondria that saves energy and minimizes mitochondria ROS production. Under resting conditions, skeletal muscle has different pools of mitochondria, SSM have a high mitochondrial potential (ΔΨ) and a basal rate of ROS generation from superoxide anions generated by the electron transport chain. Thus, the respiratory chain may allow the “leakage of electrons” and generate ROS. IMF mitochondria have a high expression of MICU1, which prevents calcium from massively entering the mitochondrial space. A condition of exercise implies that the SSM and the IMF mitochondria fuse, generating a network characterized by a lower density of MICU1, a high density of MCU, and an extended proton gradient (enough ΔΨ) needed to generate ATP and to minimize ROS generation. Colors of mitochondria represent diversity (red and green), and yellow represents fused mitochondria.</p> Full article ">Figure 3
<p>Altered dynamics and fusion capacity of mitochondria in disease. A common factor in many pathological conditions affecting skeletal muscle is an imbalance in mitochondrial dynamics. This imbalance is both a cause and consequence of inflammation, increased ROS production, and cell death, leading to muscle deterioration characterized by dysfunctional mitochondria. Different colors of mitochondria refer to SSM, IFM and fused mitochondria as pictures in <a href="#antioxidants-12-01624-f002" class="html-fig">Figure 2</a>.</p> Full article ">
3 pages, 192 KiB  
Editorial
Oxidative DNA Damage and Repair: Mechanisms, Mutations, and Relation to Diseases
by Marina Roginskaya and Yuriy Razskazovskiy
Antioxidants 2023, 12(8), 1623; https://doi.org/10.3390/antiox12081623 - 17 Aug 2023
Cited by 3 | Viewed by 1191
Abstract
Oxidative DNA damage (ODD) by reactive oxygen species (ROS) or reactive nitrogen species (RNS) is an inevitable tradeoff for using oxidation processes by living cells as a source of energy [...] Full article
(This article belongs to the Special Issue Oxidative DNA Damage and Repair: Mechanisms, Mutations, and Relation to Diseases)
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