Acetylated Chitosan Oligosaccharides Act as Antagonists against Glutamate-Induced PC12 Cell Death via Bcl-2/Bax Signal Pathway
<p>Schematic diagram of the repeating saccharide units in chitosan oligosaccharide and its acetylated derivatives. (A–C) Chemical structure of chitosan oligosaccharides (COS) (<b>A</b>), <span class="html-italic">N</span>-acetylated chitosan oligosaccharides (NACO) (<b>B</b>), and peracetylated chitosan oligosaccharides (PACO) (<b>C</b>). The degree of polymerization (DP) of COS, NACO, and PACO is 2~4.</p> "> Figure 2
<p>Effect of glutamate on undifferentiated and fully-differentiated PC12 cells. (A–B) The undifferentiated (<b>A</b>) and fully-differentiated (<b>B</b>) PC12 cells were plated on the cell culture plates at a density of 1 × 10<sup>5</sup> cells/mL, and then treated with glutamate at different concentrations (0.25, 0.5, 1, 2, 4, 8, 16 and 32 mM) for 24 h. Then the cell viability was evaluated by resazurin assay. The results were presented as a percentage of the normal control group. Values are the mean ± SD (<span class="html-italic">n</span> = 3). Significance: * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01 <span class="html-italic">vs.</span> normal control group without glutamate.</p> "> Figure 3
<p>The cytotoxicity of different chitosan oligosaccharides in PC12 cells. PC12 cells were incubated with different chitosan oligosaccharides COS-2~4 (<b>A</b>), peracetylated chitosan oligosaccharides Q-2~4 (<b>B</b>) and <span class="html-italic">N</span>-acetylated chitosan oligosaccharides N-2~4 (<b>C</b>) at indicated concentrations (100, 200, 400 μg/mL) for 24 h. Then the cell viability was evaluated by resazurin assay. The results were presented as a percentage of non drug treated normal control group. Values are the mean ± SD (<span class="html-italic">n</span> = 3).</p> "> Figure 4
<p>The effect of different chitosan oligosaccharides on glutamate-induced PC12 cell damage. PC12 cells were treated with or without different chitosan oligosaccharides COS-2~4 (<b>A</b>) and peracetylated chitosan oligosaccharides Q-2~4 (<b>B</b>) at indicated concentrations for 2 h. Then cells were treated with glutamate for another 24 h before performing a resazurin assay. The untreated normal cells (control) were assigned values of 100 and the results presented as mean ± SD (<span class="html-italic">n</span> = 4). Significance: # <span class="html-italic">p</span> < 0.05 <span class="html-italic">vs.</span> normal control group; * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01 <span class="html-italic">vs.</span> glutamate treated control group.</p> "> Figure 5
<p>The neuroprotective effects of different acetylated neutral oligosaccharides in PC12 cells. (<b>A</b>) PC12 cells were treated with or without different N-acetylated chitosan oligosaccharides N-2~4 at indicated concentrations for 2 h. Then cells were treated with glutamate for another 24 h before performing a resazurin assay. The untreated normal cells (control) were assigned values of 100 and the results presented as mean ± SD (<span class="html-italic">n</span> = 4). Significance: # <span class="html-italic">p</span> < 0.05 <span class="html-italic">vs.</span> normal control group; * <span class="html-italic">p</span> < 0.05 <span class="html-italic">vs.</span> glutamate treated control group. (<b>B</b>) PC12 cells were treated with or without peracetylated chitobiose Q-2, lactose, acetylated lactose, cellobiose, or acetylated cellobiose at indicated concentrations for 2 h. Then cells were treated with glutamate for another 24 h. The untreated normal cells (control) were assigned values of 100 and the results presented as mean ± SD (<span class="html-italic">n</span> = 3). Significance: ## <span class="html-italic">p</span> < 0.01 <span class="html-italic">vs.</span> normal control group; * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01 <span class="html-italic">vs.</span> glutamate treated control group.</p> "> Figure 5 Cont.
<p>The neuroprotective effects of different acetylated neutral oligosaccharides in PC12 cells. (<b>A</b>) PC12 cells were treated with or without different N-acetylated chitosan oligosaccharides N-2~4 at indicated concentrations for 2 h. Then cells were treated with glutamate for another 24 h before performing a resazurin assay. The untreated normal cells (control) were assigned values of 100 and the results presented as mean ± SD (<span class="html-italic">n</span> = 4). Significance: # <span class="html-italic">p</span> < 0.05 <span class="html-italic">vs.</span> normal control group; * <span class="html-italic">p</span> < 0.05 <span class="html-italic">vs.</span> glutamate treated control group. (<b>B</b>) PC12 cells were treated with or without peracetylated chitobiose Q-2, lactose, acetylated lactose, cellobiose, or acetylated cellobiose at indicated concentrations for 2 h. Then cells were treated with glutamate for another 24 h. The untreated normal cells (control) were assigned values of 100 and the results presented as mean ± SD (<span class="html-italic">n</span> = 3). Significance: ## <span class="html-italic">p</span> < 0.01 <span class="html-italic">vs.</span> normal control group; * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01 <span class="html-italic">vs.</span> glutamate treated control group.</p> "> Figure 6
<p>Effect of peracetylated chitosan oligosaccharides on glutamate-induced LDH release and ROS overproduction. (<b>A</b>) After the treatment of cells with different PACO monomers Q-2, Q-3 and Q-4 at a concentration of 200 μg/mL for 2 h and 4 mM of glutamate for 24 h, the level of LDH in the culture media was measured using an LDH assay kit. The data were normalized to the activity of LDH released from control cells. Values are the mean ± SD (<span class="html-italic">n</span> = 4). Significance: ## <span class="html-italic">P</span> < 0.01 <span class="html-italic">vs.</span> normal control group; * <span class="html-italic">P</span> < 0.05, ** <span class="html-italic">P</span> < 0.01 <span class="html-italic">vs.</span> glutamate treated control group; (<b>B</b>) After the treatment of cells with different PACO monomers Q-2, Q-3 and Q-4 at the concentration of 200 μg/mL for 2 h and 4 mM of glutamate for another 24 h. The fluorescence intensity of DCF was measured in a microplate-reader. Data were expressed as a percentage of non-treated control. Values are the mean ± SD (<span class="html-italic">n</span> = 4). Significance: ## <span class="html-italic">P</span> < 0.01 <span class="html-italic">vs.</span> normal control group; ** <span class="html-italic">P</span> < 0.01 <span class="html-italic">vs.</span> glutamate treated control group.</p> "> Figure 7
<p>Peracetylated chitosan oligosaccharides protect PC12 cells against glutamate-induced loss of MMP and the activation of Caspase-3 and Caspase-9. (<b>A</b>) After being pretreated with 200 μg/mL of different PACO monomers Q-2, Q-3 and Q-4 for 2 h and 4 mM of glutamate for another 24 h, the mitochondrial membrane potential (MMP) in PC12 cells were evaluated with the probe JC-1. Data were expressed as a percentage of non-treated control. Values are the mean ± SD (<span class="html-italic">n</span> = 3). Significance: # <span class="html-italic">p</span> < 0.05 <span class="html-italic">vs.</span> normal control group; * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01 <span class="html-italic">vs.</span> glutamate treated control group; (<b>B</b>) After being pretreated with 200 μg/mL of different PACO monomers Q-2, Q-3 and Q-4 for 2 h and 4 mM of glutamate for another 24 h, the levels of cleaved caspase-3 were measured by western blot. Blots were also probed for β-actin protein as loading controls. The result shown is a representative of three separate experiments with similar results. (<b>C</b>) Quantification of immunoblot for the ratio of caspase-3 to β-actin. The ratio for non-treated normal control cells was assigned values of 1.0 and the data presented as mean ± SD (<span class="html-italic">n</span> = 3). Significance: ## <span class="html-italic">p</span> < 0.01 <span class="html-italic">vs.</span> normal control group; ** <span class="html-italic">p</span> < 0.01 <span class="html-italic">vs.</span> glutamate treated control group. (D–E) After treatment, the activities of caspase-3 (<b>D</b>) and caspase-9 (<b>E</b>) were measured using an ELISA assay kit (Beyotime, China). Data were expressed as a percentage of non-treated control. Values are the mean ± SD (<span class="html-italic">n</span> = 3). Significance: # <span class="html-italic">p</span> < 0.05 <span class="html-italic">vs.</span> normal control group; * <span class="html-italic">p</span> < 0.05 <span class="html-italic">vs.</span> glutamate treated control group.</p> "> Figure 7 Cont.
<p>Peracetylated chitosan oligosaccharides protect PC12 cells against glutamate-induced loss of MMP and the activation of Caspase-3 and Caspase-9. (<b>A</b>) After being pretreated with 200 μg/mL of different PACO monomers Q-2, Q-3 and Q-4 for 2 h and 4 mM of glutamate for another 24 h, the mitochondrial membrane potential (MMP) in PC12 cells were evaluated with the probe JC-1. Data were expressed as a percentage of non-treated control. Values are the mean ± SD (<span class="html-italic">n</span> = 3). Significance: # <span class="html-italic">p</span> < 0.05 <span class="html-italic">vs.</span> normal control group; * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01 <span class="html-italic">vs.</span> glutamate treated control group; (<b>B</b>) After being pretreated with 200 μg/mL of different PACO monomers Q-2, Q-3 and Q-4 for 2 h and 4 mM of glutamate for another 24 h, the levels of cleaved caspase-3 were measured by western blot. Blots were also probed for β-actin protein as loading controls. The result shown is a representative of three separate experiments with similar results. (<b>C</b>) Quantification of immunoblot for the ratio of caspase-3 to β-actin. The ratio for non-treated normal control cells was assigned values of 1.0 and the data presented as mean ± SD (<span class="html-italic">n</span> = 3). Significance: ## <span class="html-italic">p</span> < 0.01 <span class="html-italic">vs.</span> normal control group; ** <span class="html-italic">p</span> < 0.01 <span class="html-italic">vs.</span> glutamate treated control group. (D–E) After treatment, the activities of caspase-3 (<b>D</b>) and caspase-9 (<b>E</b>) were measured using an ELISA assay kit (Beyotime, China). Data were expressed as a percentage of non-treated control. Values are the mean ± SD (<span class="html-italic">n</span> = 3). Significance: # <span class="html-italic">p</span> < 0.05 <span class="html-italic">vs.</span> normal control group; * <span class="html-italic">p</span> < 0.05 <span class="html-italic">vs.</span> glutamate treated control group.</p> "> Figure 8
<p>Peracetylated chitosan oligosaccharides protect PC12 cells against glutamate-induced Cyto C release from mitochondria. (<b>A</b>) After being pretreated with 200 μg/mL of different PACO monomers Q-2, Q-3 and Q-4 for 2 h and 4 mM of glutamate for another 24 h, the protein levels of Cyto C were evaluated by western blot. Blots were also probed for β-actin as loading controls. The result shown is a representative of three separate experiments with similar results; (<b>B</b>) Quantification of immunoblot for the ratio of Cyto C to β-actin. The ratio for non-treated control cells was assigned values of 1.0 and the data presented as mean ± SD (<span class="html-italic">n</span> = 3). Significance: ## <span class="html-italic">p</span> < 0.01 <span class="html-italic">vs.</span> normal control group; * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01 <span class="html-italic">vs.</span> glutamate treated control group. (<b>C</b>–<b>H</b>) After being pretreated with 200 μg/mL of PACO monomers Q-2, Q-3 and Q-4 for 2 h, PC12 cells were exposed to 4 mM glutamate for 24 h. Then the levels of Cyto C in the cytoplasm were detected by immunofluorescence assay using anti-Cyto C antibody. <b>C</b>: Normal control, <b>D</b>: Glu, <b>E</b>: Glu + HupA, <b>F</b>: Glu + Q2, G: Glu + Q3, <b>H</b>: Glu + Q4. Scale bar represents 20 μm.</p> "> Figure 9
<p>Effect of PACOs on the expression of Bax and Bcl-2 in PC12 cells. (A–B) After being pretreated with 200 μg/mL of different PACO monomers Q-2, Q-3 and Q-4 for 2 h and 4 mM of glutamate for another 24 h, the levels of Bax (<b>A</b>) and Bcl-2 (<b>B</b>) were measured by western blot. Blots were also probed for β-actin as loading controls. The result shown is a representative of three separate experiments with similar results; (<b>C</b>) Quantification of immunoblot for the ratio of Bax and Bcl-2. The ratio for non-treated normal control cells was assigned values of 1.0. Values are the mean ± SD (<span class="html-italic">n</span> = 3). Significance: ## <span class="html-italic">p</span> < 0.01 <span class="html-italic">vs.</span> normal control group; * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01 <span class="html-italic">vs.</span> glutamate treated control group.</p> "> Figure 10
<p>Proposed schemas of the mechanisms by which PACO suppressed glutamate-induced apoptosis in PC12 cells. Glutamate induces intracellular ROS generation in PC12 cells. The resultant oxidative stress triggers the activation of Bax protein, thereby reducing ΔΨm, releasing cytochrome c, activating caspase 3, and finally inducing apoptosis. PACO inhibits ROS and suppresses the activity of downstream molecules, such as Bax, Cyto c, and caspase 3, for promoting neuronal survival.</p> ">
Abstract
:1. Introduction
2. Results and Discussion
2.1. Characterization of Chitosan Oligosaccharides and Its Acetylated Derivatives
2.2. Glutamate-Induced PC12 Cell Death
2.3. Oligosaccharides as Antagonists against Glutamate-Induced PC12 Cell Death
2.4. The Structure-Activity Relationship of Acetylated Chitosan Oligosaccharides
2.5. Effect of Peracetylated Chitosan Oligosaccharides (PACO) on LDH Release and ROS Production
2.6. Effects of PACO on Mitochondrial Membrane Potential and the Activation of Caspase-3 and Caspase-9
2.7. Effects of PACO on the Cytochrome c (Cyto C) Release from Mitochondria
2.8. Effects of PACO on the Protein Expression of Bcl-2 and Bax
3. Experimental Section
3.1. Reagents
3.2. COSs Production and Purification
3.3. PACOs and NACOs Preparation and Purification
3.4. MS and NMR Spectroscopy of Isolated PACOs and NACOs
3.5. Cell Culture
3.6. Determination of Cell Viability
3.7. LDH Release Assay
3.8. Measurement of Intracellular ROS
3.9. Measurement of the Mitochondrial Membrane Potential (MMP)
3.10. Immunofluorescence Assay
3.11. Western Blot Analysis
3.12. Statistical Analysis
4. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Hao, C.; Gao, L.; Zhang, Y.; Wang, W.; Yu, G.; Guan, H.; Zhang, L.; Li, C. Acetylated Chitosan Oligosaccharides Act as Antagonists against Glutamate-Induced PC12 Cell Death via Bcl-2/Bax Signal Pathway. Mar. Drugs 2015, 13, 1267-1289. https://doi.org/10.3390/md13031267
Hao C, Gao L, Zhang Y, Wang W, Yu G, Guan H, Zhang L, Li C. Acetylated Chitosan Oligosaccharides Act as Antagonists against Glutamate-Induced PC12 Cell Death via Bcl-2/Bax Signal Pathway. Marine Drugs. 2015; 13(3):1267-1289. https://doi.org/10.3390/md13031267
Chicago/Turabian StyleHao, Cui, Lixia Gao, Yiran Zhang, Wei Wang, Guangli Yu, Huashi Guan, Lijuan Zhang, and Chunxia Li. 2015. "Acetylated Chitosan Oligosaccharides Act as Antagonists against Glutamate-Induced PC12 Cell Death via Bcl-2/Bax Signal Pathway" Marine Drugs 13, no. 3: 1267-1289. https://doi.org/10.3390/md13031267