Fig 1.
Cytotoxic effect of PFF in PC12 cells.
(A) Chemical structure of PFF. (B) PC12 cells were treated with increasing concentrations of PFF or a vehicle control for 24 h. Cell viability was assessed with the MTT assay. Data represent the means and SD of three independent experiments. SC, vehicle control (0.01% DMSO).
Fig 2.
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.
Fig 3.
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.
Fig 4.
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.
Fig 5.
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.
Fig 6.
PFF treatment prevented MCAO-induced brain damage in a rat animal model of ischemic stroke.
(A) In vivo experimental schedule. PFF was injected intracerebroventricularly into the left lateral cerebral ventricle of the rat, as described in the Materials and Methods section. (B and C) TTC staining was used to assess infarction volume in coronal sections. (B) Representative images of coronal sections from rats receiving sham, MCAO, and PFF plus MCAO on day 5. The red region indicates intact tissue, and the white region indicates the infarct area. (C) The bar graph represents the means ± SD of infarct volume under each condition. (D and E) Histological changes in the hippocampus were observed using cresyl violet staining. (D) Representative images of each section. Scale bars = 25 μm. (E) Cell viability was determined in tissue sections from each rat group. ***p < 0.001 vs. the control group.
Fig 7.
A schematic model depicting the neuroprotective roles of PFF in an ischemic stroke model.
Glutamate caused disturbances in the oxidant/antioxidant balance in the cellular system and then resulted in a permeability transition of the mitochondrial membrane. This further altered the translocation of the mitochondrial death-signaling pro/anti-apoptotic proteins, such as Bax, Bcl-2, and cytochrome c, resulting in the activation of the caspase cascade and DNA damage, culminating in cell death. Administration of PFF helped to maintain the oxidant/antioxidant balance and alter the changes in the mitochondria-mediated apoptotic signaling cascade that resulted in neuronal cell damage.