Figure 1.
Protective effects of 20E against H2O2-induced cytotoxicity in B35 cells.
A: Chemical structure of 20E. B: Concentration-dependent effect of H2O2 on cell viability in B35 neural cells. B35 neural cells were exposed to different concentrations of H2O2, and then cell viability was assessed 12 h later by an MTT assay. C: Inhibition of H2O2-induced decrease in cell viability by 20E in B35 neural cells. Cells were exposed to 20E at the concentrations of 25 to 400 µM for 24 h or were pretreated with 20E (25 to 400 µM) for 24 h prior to a 12 h incubation with H2O2. Cell viability was assessed, as measured by an MTT assay. D: Cell injury was assessed by testing LDH release. Cells were pretreated with 20E (50 or 400 µM) for 24 h before a 12 h incubation with H2O2, and then LDH release (% of total) was calculated as the percentage of LDH in the medium versus total LDH activity in the cells. Data are reported as the means ± S.D. from three independent experiments. Non-treated cells served as controls. # p<0.05 vs. Control, ## p<0.01 vs. Control, * p<0.05 vs. H2O2 alone, ** p<0.01 vs. H2O2 alone.
Figure 2.
Protective effects of 20E against H2O2-induced apoptosis in B35 cells.
Cells were pretreated with 50 or 400 µM of 20E for 24 h before a 12 h incubation with H2O2. a: control; b: H2O2 treatment; c: 50 µM 20E-pretreatment for 24 h before H2O2 exposure; d: 400 µM 20E-pretreatment for 24 h before H2O2 exposure. A: apoptosis was examined by TUNEL analysis with TUNEL staining (green) and propidium iodide (PI, red) counterstaining. Scale bar, 50 µm. B: Histogram showing the percentage of TUNEL-positive cells in the total cell population after different treatments. C: apoptosis was examined by immunocytochemistry for cleaved caspase-3 with cleaved caspase-3 (red) and dihydrochloride (DAPI, blue) counterstaining. Scale bar, 50 µm. D: Histogram showing the percentage of cleaved caspase-3-positive cells in the total cell population after different treatments. Data are reported as the means ± S.D. from three independent experiments. Non-treated cells served as controls. ## p<0.01 vs. Control, * p<0.05 vs. H2O2 alone, ** p<0.01 vs. H2O2 alone.
Figure 3.
Effect of 20E on H2O2-induced oxidative stress in B35 cells.
Cells were pretreated with 50 or 400 µM 20E for 24 h before a 12 h incubation with H2O2. Then these treated cells were loaded with 30 µM DCF-DA for 30 min. A: Intracellular ROS levels were determined based on DCF fluorescence by confocal laser microscopy. a: control; b: H2O2 treatment; c: 50 µM 20E-pretreatment for 24 h before H2O2 exposure; d: 400 µM 20E-pretreatment for 24 h before H2O2 exposure. B: DCF fluorescence was measured by flow cytometry. Histogram showing the intracellular fluorescence intensity of DCF in different treatment cells. Data are reported as the means ± S.D. from three independent experiments. Non-treated cells served as controls. ## p<0.01 vs. Control, * p<0.05 vs. H2O2 alone, ** p<0.01 vs. H2O2 alone. C: Histogram showing cellular T-AOC in different treatment cells. D: Histogram showing cellular SOD activity in different treatment cells. E: Histogram showing cellular GPX levels in different treatment cells. Data are reported as the means ± S.D. from four independent experiments in triplicate. Non-treated cells served as controls. ## p<0.01 vs. Control, * p<0.05 vs. H2O2 alone, ** p<0.01 vs. H2O2 alone.
Figure 4.
Effect of 20E on mitochondrial membrane potential and lipid peroxidation in H2O2-treated B35 cells.
Cells were treated as Figure 3 and then incubated with the membrane potential indicator JC-1. A: Intracellular red and green fluorescence of JC-1 were determined by confocal laser microscopy. a: control; b: H2O2 treatment; c: 50 µM 20E-pretreatment for 24 h before H2O2 exposure; d: 400 µM 20E-pretreatment for 24 h before H2O2 exposure. B: The intracellular red and green fluorescence of JC-1 was measured by flow cytometry. Histogram showing the red/green fluorescence intensity ratio in different treatment cells. Data are reported as the means ± S.D. from three independent experiments. Non-treated cells served as controls. ## p<0.01 vs. Control, * p<0.05 vs. H2O2 alone, ** p<0.01 vs. H2O2 alone. C: Histogram showing MDA level in different treatment cells. Data are reported as the means ± S.D. from four independent experiments in triplicate. Non-treated cells served as controls. ## p<0.01 vs. Control, * p<0.05 vs. H2O2 alone, ** p<0.01 vs. H2O2 alone.
Figure 5.
Effect of 20E on H2O2-induced elevation of [Ca2+]i in B35 cells.
Cells were pretreated with 50 or 400 µM 20E for 1 h. These treated cells were labeled with 5 µM fluo-3-acetoxymethyl (AM) for 40 min and treated with 1.5 mM H2O2 for the indicated time. [Ca2+]i was monitored using a laser scanning confocal microscope as explained in the “Materials and Methods”. All images (120 images per cell) were processed to analyze changes in [Ca2+]i at the single cell level. The results are expressed as the relative fluorescence intensity (RFI). Each trace shows a single cell from three independent experiments.
Figure 6.
Effect of 20E on NO, iNOS and activation of NF-κB in H2O2-treated B35 cells.
A: 20E inhibited H2O2-induced NO production. Cells were pretreated with 20E (400 µM) for 24 h or 1400W (200 µM) for 1 h or PDTC (100 µM) for 1 h before a 12 h incubation with H2O2. The production of nitrite in the medium was measured. Data are reported as the means ± S.D. from six experiments with 5×105 cells/experiment. Non-treated cells served as controls. ## p<0.01 vs. Control, ** p<0.01 vs. H2O2 alone. B: 20E inhibited H2O2-induced iNOS expression. Cells were pretreated with 20E (400 µM) for 24 h or 1400W (200 µM) for 1 h or PDTC (100 µM) for 1 h before a 12 h incubation with H2O2. The iNOS expression was analyzed by western blot. The relative intensities of the bands were measured using quantity one software. C–D: 20E inhibited H2O2-induced NF-κB activation. Cells were pretreated with 20E (400 µM) for 24 h or PDTC (100 µM) for 1 h before a 12 h incubation with H2O2. The expression of phosphorylated IκBα (cytosolic part) and phosphorylated NF-κB p65 (in the nuclear extract) was analyzed by western blot. The relative intensities of the bands were measured using quantity one software. Data are reported as the means ± S.D. from three independent experiments. Non-treated cells served as controls. ## p<0.01 vs. Control, * p<0.05 vs. H2O2 alone, ** p<0.01 vs. H2O2 alone.
Figure 7.
Effects of 20E on H2O2-induced phosphorylation of ASK1, MKK4/7 and JNK in B35 cells.
A–C: H2O2 induced phosphorylation of ASK1, MKK4/7 and JNK. Cells were treated with 200 µM H2O2 for 1 h, 6 h or 12 h. Western blotting analysis of intracellular phosphorylation level of ASK1, MKK4/7 and JNK. The relative intensities of the bands were measured using quantity one software. D–F: 20E inhibited H2O2-induced phosphorylation of ASK1, MKK4/7 and JNK1/2. Cells were pretreated with 50 or 400 µM 20E for 24 h before a 12 h incubation with H2O2. Western blotting analysis of intracellular phosphorylation level of ASK1, MKK4/7 and JNK. The relative intensities of the bands were measured using quantity one software. Data are reported as the means ± S.D. from three independent experiments. Non-treated cells served as controls. # p<0.05 vs. Control, ## p<0.01 vs. Control, * p<0.05 vs. H2O2 alone, ** p<0.01 vs. H2O2 alone.
Figure 8.
Protective effects of 20E on cerebral ischemia in rats.
A: 20E attenuated transient MCAO-induced infarction assessed by TTC staining. After 24 h of reperfusion after 2 h of MCAO in rats, five consecutive TTC-stained coronal brain slices arranged in cranial to caudal order are shown. The white brain area represents infarcted tissue. B: Percentage of infarct areas in TTC-stained brain sections (n = 8 per group). C: 20E inhibited MCAO-induced neurological deficits in rats. After 24 h of MCAO reperfusion, the neurological status for the animals was evaluated (n = 8 per group). D: Changes in rCBF were not different between operated and 20E-treated groups (n = 4 per group) during or after MCAO in rats. E: Photographs of TUNEL staining in the ipsilateral cerebral cortex. (a) Five random and nonoverlapping regions were selected in the border of the ischemic cortical area. (b) Only densely labeled cells (arrows) were considered as positive apoptotic cells. (c) Sham-operated group; (d) MCAO group; (e) group treated with 20 mg/kg 20E; (f) group treated with 40 mg/kg 20E. TUNEL-positive cells are indicated by black arrows. *, ischemic cortical area; Scale bar, 200 µm in A; Scale bar, 50 µm in b–f. F: The percentage of TUNEL-positive cells described as the number of TUNEL-positive cells/total number of cells in each field (n = 6 per group). Data are reported as the means ± S.D. ## p<0.01 vs. Sham-operated group, * p<0.05 vs. MCAO group, ** p<0.01 vs. MCAO group.
Figure 9.
Effects of 20E on cleaved caspase-3, T-AOC,SOD,GPX and MDA in ischemic cortex in rat.
A: Photographs of immunohistochemistry staining for cleaved caspase-3 in the ipsilateral cerebral cortex. (a) Five random and nonoverlapping regions were selected in the border of the ischemic cortical area. (b) Only densely labeled cells (arrows) were considered as positive apoptotic cells. (c) Sham-operated group; (d) MCAO group; (e) group treated with 20 mg/kg 20E; (f) group treated with 40 mg/kg 20E. Cleaved caspase-3-positive cells are indicated by black arrows. *, ischemic cortical area; Scale bar, 200 µm in A; Scale bar, 50 µm in b–f. B: The percentage of cleaved caspase-3-positive cells described as the number of cleaved caspase-3-positive cells/total number of cells in each field (n = 6 per group). C: Histogram showing cellular T-AOC in different treatment groups (n = 8 per group). D: Histogram showing cellular SOD activities in different treatment groups (n = 8 per group). E: Histogram showing cellular GPX levels in different treatment groups (n = 8 per group). F: Histogram showing MDA levels in different treatment groups (n = 8 per group). Data are reported as the means ± S.D. ## p<0.01 vs. Sham-operated group, * p<0.05 vs. MCAO group, ** p<0.01 vs. MCAO group.
Figure 10.
Multifunctional cytoprotective pathways are involved in the neuroprotection by 20E.
Ischemia induces intracellular ROS accumulation, antioxidant potential descent and lipid peroxidation, which causes Intracellular oxidative stress. Further, oxidative stress leads to elevation of intracellular [Ca2+]i, the increases in NO production by NF-κB/iNOS pathway and the activation of ASK1-MKK4/7 -JNK stress signaling pathway in cells. Finally, the above changes cause cell death. However, 20E markedly attenuates the above changes and protect from cell death.