The Metabolite Urolithin-A Ameliorates Oxidative Stress in Neuro-2a Cells, Becoming a Potential Neuroprotective Agent
<p>Structure of Urolithin A. Synonyms: 3,8-Dihydroxyurolithin; 3,8-Hydroxydibenzo-α-pyrone; 2′,7-Dihydroxy-3,4-benzocoumarin; δ-Lactone 2′,4,4′-Trihydroxy-2-biphenylcarboxylic acid.</p> "> Figure 2
<p>Mitochondrial activity in Neuro-2a cells culture (MTT assay). (<b>A</b>) Cytotoxicity of Neuro-2a cells after exposure to different concentrations of urolithin A. (<b>B</b>) Cytoprotective effects of urolithin A versus hydrogen peroxide (250 μM). Note: * <span class="html-italic">p</span> < 0.05 versus H<sub>2</sub>O<sub>2</sub>; <sup>##</sup> <span class="html-italic">p</span> < 0.01 versus control.</p> "> Figure 3
<p>ROS production in Neuro-2a cells subjected to oxidative stress by hydrogen peroxide (250 μM) and treatments with urolithin A (0.5–4 μM). Data are expressed as percentage over control cells and the assay was carried out for 90 min in order to measure intracellular ROS production. Note: <sup>#</sup> <span class="html-italic">p</span> < 0.005 versus control; <sup>##</sup> <span class="html-italic">p</span> < 0.001 versus control. Significant differences appeared at the starting point for H<sub>2</sub>O<sub>2</sub>-N2a cells over control cells (<span class="html-italic">p</span> < 0.001). However, pre-treatments with 0.5 and 2 μM urolithin A at 0 and 10 min were associated with significant differences (<span class="html-italic">p</span> < 0.01). After 20 min, significant differences were reached at 1 μM (<span class="html-italic">p</span> < 0.01). Finally, 4 μM of the antioxidant exhibited significant differences (<span class="html-italic">p</span> < 0.01) between 40 and 60 min. Urolithin A (2 μM) displayed a greater mitochondrial response than any other co-treatment.</p> "> Figure 4
<p>Thiobarbituric acid reactive species (TBARS) formation in Neuro-2a cells. Note: * <span class="html-italic">p</span> < 0.05 versus H<sub>2</sub>O<sub>2</sub>.</p> "> Figure 5
<p>Neuro-2a cell culture redox status. (<b>A</b>) Catalase activity. (<b>B</b>) Superoxide dismutase activity. (<b>C</b>) Glutathione reductase activity. (<b>D</b>) Glutathione peroxidase activity. Note: * <span class="html-italic">p</span> < 0.05; ** <span class="html-italic">p</span> < 0.01; *** <span class="html-italic">p</span> < 0.005; **** <span class="html-italic">p</span> < 0.001 versus H<sub>2</sub>O<sub>2</sub>; # <span class="html-italic">p</span> < 0.05 versus Control; ### <span class="html-italic">p</span> < 0.005 versus Control.</p> "> Figure 6
<p>Urolithin A-induced cytoprotection of Neuro-2a cells is mediated by induction of peroxiredoxin 1 and 3 (Prx1, Prx3). Prx 1 (<b>A</b>,<b>B</b>) and Prx 3 (<b>C</b>,<b>D</b>) expressions were determined by Western blot in 10 µg of protein extract and expressed as Prx densitometry. Ponceau red staining of membranes prior to blotting was performed to further check protein transfer and loading. Note: # <span class="html-italic">p</span> < 0.05 versus Control; ## <span class="html-italic">p</span> < 0.01 versus Control</p> "> Figure 7
<p>Enzymatic inhibition of urolithin A. IC<sub>50</sub> values were calculated by non-linear regression. (<b>A</b>) Tyrosinase inhibition profiles of urolithin A and kojic acid. (<b>B</b>) Monoamine oxidase A (MAO-A) inhibition profiles of urolithin A and clorgyline.</p> "> Figure 8
<p>Antioxidant activity of urolithin A against physiological (superoxide) and synthetic (DPPH) radicals. IC<sub>50</sub> were calculated by non-linear regression. (<b>A</b>) Urolithin A scavenges superoxide radicals generated by the xanthine/xanthine oxidase system. (<b>B</b>) DPPH inhibition of urolithin A. Gallic acid and ascorbic acid were used as reference antioxidants.</p> "> Figure 8 Cont.
<p>Antioxidant activity of urolithin A against physiological (superoxide) and synthetic (DPPH) radicals. IC<sub>50</sub> were calculated by non-linear regression. (<b>A</b>) Urolithin A scavenges superoxide radicals generated by the xanthine/xanthine oxidase system. (<b>B</b>) DPPH inhibition of urolithin A. Gallic acid and ascorbic acid were used as reference antioxidants.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Reagents and Chemicals
2.2. Cytoprotective Properties of Urolithin A in Neuro-2a Cells
2.2.1. Neuro-2a Cell Culture and Treatments with Urolithin A and Hydrogen Peroxide
2.2.2. Mitochondrial Activity in Neuro-2a Cells Subjected to Oxidative Stress after Urolithin A Treatment
2.2.3. ROS Production in Neuro-2a Cells Subjected to Oxidative Stress after Exposition to Urolithin A
2.2.4. Lipid Peroxidation in Neuro-2a Cells Subjected to Oxidative Stress after Exposition to Urolithin A (TBARS assay)
2.2.5. Activity of Antioxidant Enzymes in Neuro-2a Cells Subjected to Oxidative Stress after Exposition to Urolithin A
2.2.6. Peroxiredoxin Expression in Neuro-2a Cells by Immunoblotting
2.3. Urolithin A and Its Role as an Inhibitor of CNS Enzymatic Targets
2.3.1. Tyrosinase (TYR) Inhibition
2.3.2. Acetylcholinesterase (AChE) Inhibition
2.3.3. Monoamine Oxidase A (MAO-A) Inhibition
2.4. Urolithin A and Its Role as a Direct Free Radical Scavenger
2.4.1. Oxygen Radical Antioxidant Capacity ORAC Assay
2.4.2. Superoxide Radicals Generated by Xanthine/Xanthine Oxidase (X/XO) System
2.4.3. DPPH Radical Assay
2.5. Statistical Analysis
3. Results
3.1. Cytoprotective Properties of Urolithin A in Neuro-2a Cells
3.1.1. Urolithin A Improves Mitochondrial Activity in Neuro-2a Cells Subjected to Oxidative Stress (MTT Assay)
3.1.2. Urolithin A Decreases Intracellular ROS Production in Neuro-2a Cells Subjected to Oxidative Stress (DCFHA-DA Assay)
3.1.3. Urolithin A Decreases Lipid Peroxidation in Neuro-2a Cells Subjected to Oxidative Stress (Thiobarbituric Acid Reactive Species, TBARS)
3.1.4. Urolithin A Enhanced the Activity of Antioxidant Enzymes in Neuro-2a Cells Subjected to Oxidative Stress (CAT, SOD, GR, GPx)
3.1.5. Peroxiredoxins Expression
3.2. Urolithin A Inhibits Oxidases (Monoamine Oxidase A and Tyrosinase)
3.3. The Role of Urolithin A as a Direct Free Radical Scavenger
4. Discussion
5. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Cásedas, G.; Les, F.; Choya-Foces, C.; Hugo, M.; López, V. The Metabolite Urolithin-A Ameliorates Oxidative Stress in Neuro-2a Cells, Becoming a Potential Neuroprotective Agent. Antioxidants 2020, 9, 177. https://doi.org/10.3390/antiox9020177
Cásedas G, Les F, Choya-Foces C, Hugo M, López V. The Metabolite Urolithin-A Ameliorates Oxidative Stress in Neuro-2a Cells, Becoming a Potential Neuroprotective Agent. Antioxidants. 2020; 9(2):177. https://doi.org/10.3390/antiox9020177
Chicago/Turabian StyleCásedas, Guillermo, Francisco Les, Carmen Choya-Foces, Martín Hugo, and Víctor López. 2020. "The Metabolite Urolithin-A Ameliorates Oxidative Stress in Neuro-2a Cells, Becoming a Potential Neuroprotective Agent" Antioxidants 9, no. 2: 177. https://doi.org/10.3390/antiox9020177