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Phlorofucofuroeckol Improves Glutamate-Induced Neurotoxicity through Modulation of Oxidative Stress-Mediated Mitochondrial Dysfunction in PC12 Cells

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ABSTRACT

Stroke is a complex neurodegenerative disorder with a clinically high prevalence and mortality. Despite many efforts to protect against ischemic stroke, its incidence and related permanent disabilities continue to increase. In this study, we found that pretreatment with phlorofucofuroeckol (PFF), isolated from brown algae species, significantly increased cell viability in glutamate-stimulated PC12 cells. Additionally, glutamate-stimulated cells showed irregular morphology, but PFF pretreatment resulted in improved cell morphology, which resembled that in cells cultured under normal conditions. We further showed that PFF pretreatment effectively inhibited glutamate-induced apoptotic cell death in a caspase-dependent manner. Reactive oxygen species (ROS) induced by oxidative stress are closely associated with ischemia-induced neurological diseases. Exposure of PC12 cells to glutamate induced abundant production of intracellular ROS and mitochondrial dysfunction, which was attenuated by PFF in a dose-dependent manner. In vivo studies revealed that PFF-mediated prevention was achieved predominantly through inhibition of apoptosis and mitochondrial ROS generation. Taken together, these results suggest the possibility of PFF as a neuroprotective agent in ischemic stroke.

No MeSH data available.


PFF regulated glutamate-induced ROS generation in PC12 cells.PC12 cells were pretreated with PFF (10 μM) for 45 min and then stimulated with glutamate (5 mM) for 24 h. (A–C) Cells were stained with DHE for 30 min to measure intracellular superoxide (O2-) using fluorescence microscopy (A; scale bars = 25 μm) and flow cytometry (B). (D and E) Cells were stained with H2DCFDA for 30 min to measure intracellular hydrogen peroxide (H2O2) using flow cytometry. (F and G) Cells were stained with MitoSOX for 30 min to measure mitochondrial ROS using flow cytometry. (C, E, G) The bar graph presents a quantitative analysis of the generation of intracellular superoxide (for C), H2O2 (for E), or mitochondrial ROS (for G). Data represent the means and SD of three independent experiments. ***p < 0.001 vs. the control group. SC, vehicle control (0.01% DMSO), Glut, glutamate.
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pone.0163433.g004: PFF regulated glutamate-induced ROS generation in PC12 cells.PC12 cells were pretreated with PFF (10 μM) for 45 min and then stimulated with glutamate (5 mM) for 24 h. (A–C) Cells were stained with DHE for 30 min to measure intracellular superoxide (O2-) using fluorescence microscopy (A; scale bars = 25 μm) and flow cytometry (B). (D and E) Cells were stained with H2DCFDA for 30 min to measure intracellular hydrogen peroxide (H2O2) using flow cytometry. (F and G) Cells were stained with MitoSOX for 30 min to measure mitochondrial ROS using flow cytometry. (C, E, G) The bar graph presents a quantitative analysis of the generation of intracellular superoxide (for C), H2O2 (for E), or mitochondrial ROS (for G). Data represent the means and SD of three independent experiments. ***p < 0.001 vs. the control group. SC, vehicle control (0.01% DMSO), Glut, glutamate.

Mentions: Recently, several studies have reported that impaired generation of ROS, including superoxide (O2•-), hydroxyl radicals (HO•), and hydrogen peroxide (H2O2), was closely associated with caspase-dependent and/or -independent neuronal cell death [30, 31]. Thus, we determined whether PFF was involved in the negative regulation of oxidative stress. PC12 cells were stimulated with glutamate in the presence or absence of PFF, and ROS generation was determined using dihydroethidium (DHE; Fig 4A–4C) or 2,7-dichlorodihydrofluorescein-diacetate (DCFH-DA; Fig 4D and 4E) as a probe for confocal microscopy and flow cytometric analyses. Glutamate alone caused the strong intracellular generation of O2 and H2O2, whereas glutamate-induced generation of ROS in PC12 cells was attenuated significantly by PFF pretreatment.


Phlorofucofuroeckol Improves Glutamate-Induced Neurotoxicity through Modulation of Oxidative Stress-Mediated Mitochondrial Dysfunction in PC12 Cells
PFF regulated glutamate-induced ROS generation in PC12 cells.PC12 cells were pretreated with PFF (10 μM) for 45 min and then stimulated with glutamate (5 mM) for 24 h. (A–C) Cells were stained with DHE for 30 min to measure intracellular superoxide (O2-) using fluorescence microscopy (A; scale bars = 25 μm) and flow cytometry (B). (D and E) Cells were stained with H2DCFDA for 30 min to measure intracellular hydrogen peroxide (H2O2) using flow cytometry. (F and G) Cells were stained with MitoSOX for 30 min to measure mitochondrial ROS using flow cytometry. (C, E, G) The bar graph presents a quantitative analysis of the generation of intracellular superoxide (for C), H2O2 (for E), or mitochondrial ROS (for G). Data represent the means and SD of three independent experiments. ***p < 0.001 vs. the control group. SC, vehicle control (0.01% DMSO), Glut, glutamate.
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Related In: Results  -  Collection

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pone.0163433.g004: PFF regulated glutamate-induced ROS generation in PC12 cells.PC12 cells were pretreated with PFF (10 μM) for 45 min and then stimulated with glutamate (5 mM) for 24 h. (A–C) Cells were stained with DHE for 30 min to measure intracellular superoxide (O2-) using fluorescence microscopy (A; scale bars = 25 μm) and flow cytometry (B). (D and E) Cells were stained with H2DCFDA for 30 min to measure intracellular hydrogen peroxide (H2O2) using flow cytometry. (F and G) Cells were stained with MitoSOX for 30 min to measure mitochondrial ROS using flow cytometry. (C, E, G) The bar graph presents a quantitative analysis of the generation of intracellular superoxide (for C), H2O2 (for E), or mitochondrial ROS (for G). Data represent the means and SD of three independent experiments. ***p < 0.001 vs. the control group. SC, vehicle control (0.01% DMSO), Glut, glutamate.
Mentions: Recently, several studies have reported that impaired generation of ROS, including superoxide (O2•-), hydroxyl radicals (HO•), and hydrogen peroxide (H2O2), was closely associated with caspase-dependent and/or -independent neuronal cell death [30, 31]. Thus, we determined whether PFF was involved in the negative regulation of oxidative stress. PC12 cells were stimulated with glutamate in the presence or absence of PFF, and ROS generation was determined using dihydroethidium (DHE; Fig 4A–4C) or 2,7-dichlorodihydrofluorescein-diacetate (DCFH-DA; Fig 4D and 4E) as a probe for confocal microscopy and flow cytometric analyses. Glutamate alone caused the strong intracellular generation of O2 and H2O2, whereas glutamate-induced generation of ROS in PC12 cells was attenuated significantly by PFF pretreatment.

View Article: PubMed Central - PubMed

ABSTRACT

Stroke is a complex neurodegenerative disorder with a clinically high prevalence and mortality. Despite many efforts to protect against ischemic stroke, its incidence and related permanent disabilities continue to increase. In this study, we found that pretreatment with phlorofucofuroeckol (PFF), isolated from brown algae species, significantly increased cell viability in glutamate-stimulated PC12 cells. Additionally, glutamate-stimulated cells showed irregular morphology, but PFF pretreatment resulted in improved cell morphology, which resembled that in cells cultured under normal conditions. We further showed that PFF pretreatment effectively inhibited glutamate-induced apoptotic cell death in a caspase-dependent manner. Reactive oxygen species (ROS) induced by oxidative stress are closely associated with ischemia-induced neurological diseases. Exposure of PC12 cells to glutamate induced abundant production of intracellular ROS and mitochondrial dysfunction, which was attenuated by PFF in a dose-dependent manner. In vivo studies revealed that PFF-mediated prevention was achieved predominantly through inhibition of apoptosis and mitochondrial ROS generation. Taken together, these results suggest the possibility of PFF as a neuroprotective agent in ischemic stroke.

No MeSH data available.