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

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.

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Glutamate-induced mitochondrial dysfunction in PC12 cells was improved by PFF treatment.PC12 cells were stimulated with glutamate (5 mM, 24 h) in the presence or absence of PFF (10 μM). (A) Cells were immunolabelled with a Tomm20 antibody, followed by the addition of Cy3-conjugated secondary antibody. Representative immunofluorescence images (scale bars = 10 μm). (B) Mitochondrial membrane potential (ΔΨm) was measured under the indicated conditions using the ΔΨm-sensitive fluorochrome MitoTracker Red CMXRos and flow cytometric analysis. (C) MitoTracker fluorescence signals for mitochondrial mass were measured using flow cytometric analysis. (Left in B and C) Representative histogram from three independent replicates. (Right in B and C) Bar graph shows ΔΨm (B) or mitochondrial mass (C) mean fluorescence intensities. Data represent the means and SD of three independent experiments. ***p < 0.001 vs. the control group. U, untreated condition. Glut, glutamate.
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pone.0163433.g005: Glutamate-induced mitochondrial dysfunction in PC12 cells was improved by PFF treatment.PC12 cells were stimulated with glutamate (5 mM, 24 h) in the presence or absence of PFF (10 μM). (A) Cells were immunolabelled with a Tomm20 antibody, followed by the addition of Cy3-conjugated secondary antibody. Representative immunofluorescence images (scale bars = 10 μm). (B) Mitochondrial membrane potential (ΔΨm) was measured under the indicated conditions using the ΔΨm-sensitive fluorochrome MitoTracker Red CMXRos and flow cytometric analysis. (C) MitoTracker fluorescence signals for mitochondrial mass were measured using flow cytometric analysis. (Left in B and C) Representative histogram from three independent replicates. (Right in B and C) Bar graph shows ΔΨm (B) or mitochondrial mass (C) mean fluorescence intensities. Data represent the means and SD of three independent experiments. ***p < 0.001 vs. the control group. U, untreated condition. Glut, glutamate.

Mentions: Accumulation of impaired mitochondria is one of the major causes not only of the induction of the intrinsic apoptotic pathway but also of the generation of mitochondrial ROS [32–34]. To investigate whether the glutamate-mediated increase of abnormal mitochondria was rescued by PFF treatment, we first evaluated mitochondrial morphology using Tomm20 (mitochondrial outer membrane) staining (Fig 5A). Mitochondrial morphology was altered at 12 h and sustained at 24 h in glutamate-stimulated PC12 cells. However, the increased mitochondrial damage was attenuated significantly by PFF treatment (Fig 5A).


Phlorofucofuroeckol Improves Glutamate-Induced Neurotoxicity through Modulation of Oxidative Stress-Mediated Mitochondrial Dysfunction in PC12 Cells
Glutamate-induced mitochondrial dysfunction in PC12 cells was improved by PFF treatment.PC12 cells were stimulated with glutamate (5 mM, 24 h) in the presence or absence of PFF (10 μM). (A) Cells were immunolabelled with a Tomm20 antibody, followed by the addition of Cy3-conjugated secondary antibody. Representative immunofluorescence images (scale bars = 10 μm). (B) Mitochondrial membrane potential (ΔΨm) was measured under the indicated conditions using the ΔΨm-sensitive fluorochrome MitoTracker Red CMXRos and flow cytometric analysis. (C) MitoTracker fluorescence signals for mitochondrial mass were measured using flow cytometric analysis. (Left in B and C) Representative histogram from three independent replicates. (Right in B and C) Bar graph shows ΔΨm (B) or mitochondrial mass (C) mean fluorescence intensities. Data represent the means and SD of three independent experiments. ***p < 0.001 vs. the control group. U, untreated condition. Glut, glutamate.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5036853&req=5

pone.0163433.g005: Glutamate-induced mitochondrial dysfunction in PC12 cells was improved by PFF treatment.PC12 cells were stimulated with glutamate (5 mM, 24 h) in the presence or absence of PFF (10 μM). (A) Cells were immunolabelled with a Tomm20 antibody, followed by the addition of Cy3-conjugated secondary antibody. Representative immunofluorescence images (scale bars = 10 μm). (B) Mitochondrial membrane potential (ΔΨm) was measured under the indicated conditions using the ΔΨm-sensitive fluorochrome MitoTracker Red CMXRos and flow cytometric analysis. (C) MitoTracker fluorescence signals for mitochondrial mass were measured using flow cytometric analysis. (Left in B and C) Representative histogram from three independent replicates. (Right in B and C) Bar graph shows ΔΨm (B) or mitochondrial mass (C) mean fluorescence intensities. Data represent the means and SD of three independent experiments. ***p < 0.001 vs. the control group. U, untreated condition. Glut, glutamate.
Mentions: Accumulation of impaired mitochondria is one of the major causes not only of the induction of the intrinsic apoptotic pathway but also of the generation of mitochondrial ROS [32–34]. To investigate whether the glutamate-mediated increase of abnormal mitochondria was rescued by PFF treatment, we first evaluated mitochondrial morphology using Tomm20 (mitochondrial outer membrane) staining (Fig 5A). Mitochondrial morphology was altered at 12 h and sustained at 24 h in glutamate-stimulated PC12 cells. However, the increased mitochondrial damage was attenuated significantly by PFF treatment (Fig 5A).

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.


Related in: MedlinePlus