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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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PFF treatment protected against glutamate-induced neurocytotoxic damage in PC12 cells.(A) PC12 cells were stimulated with glutamate (1, 5, 10 mM) or vehicle control for 24 h and cell viability then was measured with the MTT assay. (B and C) Undifferentiated PC12 cells were treated with PFF (10 μM) for 45 min before glutamate stimulation (5 mM). After 24 h, the neuroprotective effects of PFF were assessed with an inverted phase-contrast microscope (for morphological changes, B; scale bars = 25 μm) and the MTT assay (for cell viability, C). (D) Retinoic acid-mediated differentiated PC12 cells were incubated with PFF (10 μM) for 45 min before glutamate stimulation (5 mM). After 24 h, cell viability was analyzed by MTT assay. Data represent the means and SD of three independent experiments. *p < 0.05, ***p < 0.001 vs. the control group. SC, vehicle control (0.01% DMSO), Glut, glutamate.
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pone.0163433.g002: PFF treatment protected against glutamate-induced neurocytotoxic damage in PC12 cells.(A) PC12 cells were stimulated with glutamate (1, 5, 10 mM) or vehicle control for 24 h and cell viability then was measured with the MTT assay. (B and C) Undifferentiated PC12 cells were treated with PFF (10 μM) for 45 min before glutamate stimulation (5 mM). After 24 h, the neuroprotective effects of PFF were assessed with an inverted phase-contrast microscope (for morphological changes, B; scale bars = 25 μm) and the MTT assay (for cell viability, C). (D) Retinoic acid-mediated differentiated PC12 cells were incubated with PFF (10 μM) for 45 min before glutamate stimulation (5 mM). After 24 h, cell viability was analyzed by MTT assay. Data represent the means and SD of three independent experiments. *p < 0.05, ***p < 0.001 vs. the control group. SC, vehicle control (0.01% DMSO), Glut, glutamate.

Mentions: Production of excessive extracellular glutamate and abnormal activation of is receptors are among the main causes of neuronal cell death during the progression of acute or chronic ischemic stroke. Thus, we investigated whether glutamate-induced neurotoxicity occurred in PC12 cells. Following glutamate stimulation, cell viability decreased markedly in a dose-dependent manner. We observed a reduction of 62% at 5 mM and one of 81% at 10 mM in glutamate-stimulated PC12 cells (Fig 2A). We next evaluated changes in cellular morphology and cell viability to examine the protective effects of PFF in glutamate-stimulated PC12 cells. As shown in Fig 2B, glutamate-stimulated PC12 cells exhibited a round, irregular morphology and a marked reduction in the number of neurons. However, PFF pretreatment effectively improved glutamate-mediated neuronal degeneration. Moreover, the glutamate-induced decrease in cell viability was also attenuated by pretreatment with PFF (Fig 2C). To explore whether glutamate-induced neuronal cell death was also attenuated by pre-treatment with PFF in differentiated, as well as undifferentiated, PC12 cells, PC12 cells were differentiated by treatment with retinoic acid (RA). As shown in Fig 2D, the exposure of differentiated PC12 cells to 5 mM glutamate was sufficient to cause neuronal toxicity; however, these effects were improved by pretreatment with PFF. These results indicate that PFF protects against glutamate-induced neurotoxicity in both undifferentiated and differentiated PC12 cells.


Phlorofucofuroeckol Improves Glutamate-Induced Neurotoxicity through Modulation of Oxidative Stress-Mediated Mitochondrial Dysfunction in PC12 Cells
PFF treatment protected against glutamate-induced neurocytotoxic damage in PC12 cells.(A) PC12 cells were stimulated with glutamate (1, 5, 10 mM) or vehicle control for 24 h and cell viability then was measured with the MTT assay. (B and C) Undifferentiated PC12 cells were treated with PFF (10 μM) for 45 min before glutamate stimulation (5 mM). After 24 h, the neuroprotective effects of PFF were assessed with an inverted phase-contrast microscope (for morphological changes, B; scale bars = 25 μm) and the MTT assay (for cell viability, C). (D) Retinoic acid-mediated differentiated PC12 cells were incubated with PFF (10 μM) for 45 min before glutamate stimulation (5 mM). After 24 h, cell viability was analyzed by MTT assay. Data represent the means and SD of three independent experiments. *p < 0.05, ***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.g002: PFF treatment protected against glutamate-induced neurocytotoxic damage in PC12 cells.(A) PC12 cells were stimulated with glutamate (1, 5, 10 mM) or vehicle control for 24 h and cell viability then was measured with the MTT assay. (B and C) Undifferentiated PC12 cells were treated with PFF (10 μM) for 45 min before glutamate stimulation (5 mM). After 24 h, the neuroprotective effects of PFF were assessed with an inverted phase-contrast microscope (for morphological changes, B; scale bars = 25 μm) and the MTT assay (for cell viability, C). (D) Retinoic acid-mediated differentiated PC12 cells were incubated with PFF (10 μM) for 45 min before glutamate stimulation (5 mM). After 24 h, cell viability was analyzed by MTT assay. Data represent the means and SD of three independent experiments. *p < 0.05, ***p < 0.001 vs. the control group. SC, vehicle control (0.01% DMSO), Glut, glutamate.
Mentions: Production of excessive extracellular glutamate and abnormal activation of is receptors are among the main causes of neuronal cell death during the progression of acute or chronic ischemic stroke. Thus, we investigated whether glutamate-induced neurotoxicity occurred in PC12 cells. Following glutamate stimulation, cell viability decreased markedly in a dose-dependent manner. We observed a reduction of 62% at 5 mM and one of 81% at 10 mM in glutamate-stimulated PC12 cells (Fig 2A). We next evaluated changes in cellular morphology and cell viability to examine the protective effects of PFF in glutamate-stimulated PC12 cells. As shown in Fig 2B, glutamate-stimulated PC12 cells exhibited a round, irregular morphology and a marked reduction in the number of neurons. However, PFF pretreatment effectively improved glutamate-mediated neuronal degeneration. Moreover, the glutamate-induced decrease in cell viability was also attenuated by pretreatment with PFF (Fig 2C). To explore whether glutamate-induced neuronal cell death was also attenuated by pre-treatment with PFF in differentiated, as well as undifferentiated, PC12 cells, PC12 cells were differentiated by treatment with retinoic acid (RA). As shown in Fig 2D, the exposure of differentiated PC12 cells to 5 mM glutamate was sufficient to cause neuronal toxicity; however, these effects were improved by pretreatment with PFF. These results indicate that PFF protects against glutamate-induced neurotoxicity in both undifferentiated and differentiated 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.

No MeSH data available.


Related in: MedlinePlus