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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.

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Related in: MedlinePlus

PFF enhanced survival in glutamate-stimulated PC12 cells via regulation of apoptotic cell death.PC12 cells were stimulated with glutamate (5 mM, 24 h) in the presence or absence of PFF (10 μM). (A–B) Cells were subjected to Annexin V/PI staining and analyzed by flow cytometry (A) and fluorescence microscopy (B; scale bars = 25 μm). (C) DNA fragmentation and nuclear condensation were detected by Hoechst 33258 staining under each condition. Scale bars = 25 μm. (D) Cell lysates were collected and then subjected to SDS-PAGE, followed by immunoblot analysis using anti-caspase-3 Abs. Actin was used as a loading control. SC, vehicle control (0.01% DMSO), Glut, glutamate.
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pone.0163433.g003: PFF enhanced survival in glutamate-stimulated PC12 cells via regulation of apoptotic cell death.PC12 cells were stimulated with glutamate (5 mM, 24 h) in the presence or absence of PFF (10 μM). (A–B) Cells were subjected to Annexin V/PI staining and analyzed by flow cytometry (A) and fluorescence microscopy (B; scale bars = 25 μm). (C) DNA fragmentation and nuclear condensation were detected by Hoechst 33258 staining under each condition. Scale bars = 25 μm. (D) Cell lysates were collected and then subjected to SDS-PAGE, followed by immunoblot analysis using anti-caspase-3 Abs. Actin was used as a loading control. SC, vehicle control (0.01% DMSO), Glut, glutamate.

Mentions: Previous studies have found that glutamate exposure resulted in neuronal cell death through necrosis or apoptosis, depending on the neuronal cell type [28, 29]. To examine the inhibitory mechanisms of PFF in glutamate-induced cytotoxicity, PC12 cells were assessed by Annexin V and propidium iodide (PI) staining followed by flow cytometry (Fig 3A) or confocal microscopy (Fig 3B). Glutamate stimulation alone greatly facilitated the induction of apoptosis, including Annexin V+/PI- (24.8%) and Annexin V+/PI+ populations (17.7%); however, AnnexinV-/PI+ populations were not altered. By comparison, pretreatment with PFF significantly inhibited glutamate-induced apoptosis (Annexin V+/PI-, 18.0%; Annexin V+/PI+ populations, 8.4%). Moreover, glutamate-induced DNA fragmentation was effectively attenuated by PFF pretreatment (Fig 3C). We further examined whether PFF regulated caspase-dependent cell death. As shown in Fig 3D, glutamate-induced cleavage of caspase-3, -8, and poly (ADP-ribose) polymerase (PARP) was inhibited in PC12 cells by pretreatment with PFF. These findings suggested that PFF treatment significantly improved neuronal degeneration through attenuation of caspase-dependent apoptosis in glutamate-stimulated PC12 cells.


Phlorofucofuroeckol Improves Glutamate-Induced Neurotoxicity through Modulation of Oxidative Stress-Mediated Mitochondrial Dysfunction in PC12 Cells
PFF enhanced survival in glutamate-stimulated PC12 cells via regulation of apoptotic cell death.PC12 cells were stimulated with glutamate (5 mM, 24 h) in the presence or absence of PFF (10 μM). (A–B) Cells were subjected to Annexin V/PI staining and analyzed by flow cytometry (A) and fluorescence microscopy (B; scale bars = 25 μm). (C) DNA fragmentation and nuclear condensation were detected by Hoechst 33258 staining under each condition. Scale bars = 25 μm. (D) Cell lysates were collected and then subjected to SDS-PAGE, followed by immunoblot analysis using anti-caspase-3 Abs. Actin was used as a loading control. SC, vehicle control (0.01% DMSO), Glut, glutamate.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5036853&req=5

pone.0163433.g003: PFF enhanced survival in glutamate-stimulated PC12 cells via regulation of apoptotic cell death.PC12 cells were stimulated with glutamate (5 mM, 24 h) in the presence or absence of PFF (10 μM). (A–B) Cells were subjected to Annexin V/PI staining and analyzed by flow cytometry (A) and fluorescence microscopy (B; scale bars = 25 μm). (C) DNA fragmentation and nuclear condensation were detected by Hoechst 33258 staining under each condition. Scale bars = 25 μm. (D) Cell lysates were collected and then subjected to SDS-PAGE, followed by immunoblot analysis using anti-caspase-3 Abs. Actin was used as a loading control. SC, vehicle control (0.01% DMSO), Glut, glutamate.
Mentions: Previous studies have found that glutamate exposure resulted in neuronal cell death through necrosis or apoptosis, depending on the neuronal cell type [28, 29]. To examine the inhibitory mechanisms of PFF in glutamate-induced cytotoxicity, PC12 cells were assessed by Annexin V and propidium iodide (PI) staining followed by flow cytometry (Fig 3A) or confocal microscopy (Fig 3B). Glutamate stimulation alone greatly facilitated the induction of apoptosis, including Annexin V+/PI- (24.8%) and Annexin V+/PI+ populations (17.7%); however, AnnexinV-/PI+ populations were not altered. By comparison, pretreatment with PFF significantly inhibited glutamate-induced apoptosis (Annexin V+/PI-, 18.0%; Annexin V+/PI+ populations, 8.4%). Moreover, glutamate-induced DNA fragmentation was effectively attenuated by PFF pretreatment (Fig 3C). We further examined whether PFF regulated caspase-dependent cell death. As shown in Fig 3D, glutamate-induced cleavage of caspase-3, -8, and poly (ADP-ribose) polymerase (PARP) was inhibited in PC12 cells by pretreatment with PFF. These findings suggested that PFF treatment significantly improved neuronal degeneration through attenuation of caspase-dependent apoptosis in glutamate-stimulated 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