Mitochondria Related Pathway Is Essential for Polysaccharides Purified from Sparassis crispa Mediated Neuro-Protection against Glutamate-Induced Toxicity in Differentiated PC12 Cells
<p>The purification of polysaccharides isolated from <span class="html-italic">Sparassis crispa</span>. (<b>A</b>) Scheme for extraction and isolation of polysaccharides from <span class="html-italic">Sparassis crispa</span>; (<b>B</b>) DEAE-52 cellulose anion exchanged the chromatogram of the crude polysaccharides. SCWE I and SCWE II were eluted by double-distilled (D.D.) water and 0.1 mol/L NaCl at a flow rate of 1.0 mL/min, respectively; (<b>C</b>) The crude polysaccharide SCWE I was further purified via Sepharose G-100, and SCWEA was obtained.</p> "> Figure 2
<p>(<b>A</b>) Ultraviolet spectra of SCWEA; (<b>B</b>) Fourier transform infrared spectroscopy spectrum of SCWEA; (<b>C</b>) Congo red test on SCWEA; (<b>D</b>) Periodate oxidation time course; (<b>E</b>) Products of Smith degradation of SCWEA were detected using HPLC.</p> "> Figure 3
<p>The neuro-protective effect of SCWEA against <span class="html-small-caps">l</span>-Glu-induced cell damage in DPC12 cells. Cells were pretreated with SCWEA for 3 h, and then co-incubated with or without 25 mM <span class="html-small-caps">l</span>-Glu for 24 h. Compared with <span class="html-small-caps">l</span>-Glu-treated cells, SCWEA enhanced cell viability (<b>A</b>) and reduced nuclear apoptosis rate (20×; Bar: 100 µm) (<b>B</b>). Data were expressed as a percentage of corresponding control cells and means ± S.D. (<span class="html-italic">n</span> = 6). ### <span class="html-italic">p</span> < 0.001 <span class="html-italic">versus</span> control cells (CTRL); *** <span class="html-italic">p</span> < 0.001 <span class="html-italic">versus</span> <span class="html-small-caps">l</span>-Glu-exposed cells.</p> "> Figure 4
<p>SCWEA suppressed Ca<sup>2+</sup> influx (<b>A</b>) and intracellular reactive oxygen species (ROS) accumulation (<b>B</b>) in <span class="html-small-caps">l</span>-Glu-exposed DPC12 cells (20×; Bar: 100 µm). Cells were pretreated with 4 and 8 µg/mL SCWEA for 3 h, followed by exposure to 25 mM <span class="html-small-caps">l</span>-Glu for another 12 h. The intracellular levels of Ca<sup>2+</sup> and ROS were detected by Fluo-4-AM and DCFH-DA staining, respectively. The experiments were repeated three times.</p> "> Figure 5
<p>The positive regulatory effects of SCWEA on mitochondrial function. DPC12 cells were pretreated with 4 and 8 µg/mL SCWEA for 3 h, followed by exposure to 25 mM <span class="html-small-caps">l</span>-Glu for 12 or 24 h. (<b>A</b>) SCWEA restored <span class="html-small-caps">l</span>-Glu-induced MMP loss. The changes of MMP were determined by JC-1 staining (20×; Bar: 100 µm). Red fluorescence indicates healthy cells with high MMP, whereas green fluorescence indicates apoptotic or unhealthy cells with low MMP. The experiments were repeated three times; (<b>B</b>) SCWEA enhanced the expressions of B-cell lymphoma 2 (Bcl-2) and B-cell lymphoma-extra large (Bcl-xL) in <span class="html-small-caps">l</span>-Glu-exposed DPC12 cells. Quantification data were normalized by GAPDH. Data were expressed as a percentage of corresponding control cells and means ± S.D. (<span class="html-italic">n</span> = 3). ### <span class="html-italic">p</span> < 0.001 <span class="html-italic">versus</span> control cells; * <span class="html-italic">p</span> < 0.05 and *** <span class="html-italic">p</span> < 0.001 <span class="html-italic">versus</span> <span class="html-small-caps">l</span>-Glu-exposed cells.</p> "> Figure 6
<p>AKT/GSK3β pathway contributes SCWEA-mediated neuro-protective effect. DPC12 cells were pre-treated with 4 and 8 µg/mL SCWEA for 3 h, followed by exposure to 25 mM glutamate for 24 h. (<b>A</b>) The activation of AKT and GSK-3β was detected via Western blot. Quantification data were normalized by corresponding T-AKT and T-GSK3β, respectively; (<b>B</b>) Protective effects of SCWEA against <span class="html-small-caps">l</span>-Glu on cell viability were abolished by LY294002 pre-treatment. DPC12 cells were pre-treated with 10 µM LY294002 for 30 min, followed by treatment with 4 µg/mL SCWEA for 3 h and exposure to 25 mM <span class="html-small-caps">l</span>-Glu for 24 h. Data were expressed as a percentage of corresponding control cells and means ± S.D. (<span class="html-italic">n</span> = 6). ### <span class="html-italic">p</span> < 0.001 <span class="html-italic">versus</span> control cells; *** <span class="html-italic">p</span> < 0.001 <span class="html-italic">versus</span> <span class="html-small-caps">l</span>-Glu-exposed cells; ^^^ <span class="html-italic">p</span> < 0.001 <span class="html-italic">versus</span> SCWEA and <span class="html-small-caps">l</span>-Glu co-treated cells.</p> "> Figure 7
<p>The regulatory effects of SCWEA on the morphology of PC12 cells. PC12 cells were exposed to 4 and 8 µg/mL SCWEA for 24 h and the morphologies were detected via microphotograph. 10×, scale bar: 50 µm.</p> "> Figure 8
<p>Effects of various concentrations of <span class="html-small-caps">l</span>-Glu on the viability of DPC12 cells. DPC12 cells were exposed to 10–40 mM of <span class="html-small-caps">l</span>-Glu for 24 h. Data were expressed as a percentage of corresponding control cells and means ± S.D. (<span class="html-italic">n</span> = 6). ** <span class="html-italic">p</span> < 0.01, *** <span class="html-italic">p</span> < 0.001 <span class="html-italic">versus</span> control cells.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Purification and Characterization of Sparassis crispa Polysaccharides
Molecular weight 1 | Mr (kDa) | molar ratio, mol % |
75 | 96.7 | |
Particle size 2 | Z-Average (r.nm) 3 | PdI 4 |
44.45 | 0.306 |
2.2. The Effect of SCWEA against l-Glu Induced Cell Damage in DPC12 Cells
2.3. The Effects of SCWEA on Intracellular Calcium Concentration and Reactive Oxygen Species (ROS) Levels
2.4. The Effects of SCWEA on Mitochondrial Function
2.5. Protein Kinase B (AKT) Contributing to SCWEA-Mediated Neuro-Protective Effect
3. Discussion
4. Materials and Methods
4.1. Submerge Fermentation of Sparassis crispa and Crude Extract Preparation
4.2. Polysaccharide Purification and Characterization
4.2.1. Purification of Sparassis crispa Polysaccharides
4.2.2. Molecular Weight and Particle Size Measurements
4.2.3. Ultraviolet (UV) Spectra Measurement
4.2.4. Fourier Transform Infrared Spectroscopy (FTIR) Determination
4.2.5. Congo Red Test
4.2.6. Monosaccharides Analysis
4.2.7. Periodate Oxidation-Smith Degradation Reaction of Polysaccharides
4.3. Cell Culture
4.4. Cell Viability Analysis
4.5. Cellular Morphology Analysis
4.6. Measurement of ROS
4.7. Measurement of Intracellular Calcium Concentration ([Ca2+]i)
4.8. Mitochondrial Membrane Potential (MMP) Analysis
4.9. Western Blot
4.10. Statistical Analysis
Acknowledgments
Author Contributions
Conflicts of Interest
Appendix
References
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Hu, S.; Wang, D.; Zhang, J.; Du, M.; Cheng, Y.; Liu, Y.; Zhang, N.; Wang, D.; Wu, Y. Mitochondria Related Pathway Is Essential for Polysaccharides Purified from Sparassis crispa Mediated Neuro-Protection against Glutamate-Induced Toxicity in Differentiated PC12 Cells. Int. J. Mol. Sci. 2016, 17, 133. https://doi.org/10.3390/ijms17020133
Hu S, Wang D, Zhang J, Du M, Cheng Y, Liu Y, Zhang N, Wang D, Wu Y. Mitochondria Related Pathway Is Essential for Polysaccharides Purified from Sparassis crispa Mediated Neuro-Protection against Glutamate-Induced Toxicity in Differentiated PC12 Cells. International Journal of Molecular Sciences. 2016; 17(2):133. https://doi.org/10.3390/ijms17020133
Chicago/Turabian StyleHu, Shuang, Di Wang, Junrong Zhang, Mengyan Du, Yingkun Cheng, Yan Liu, Ning Zhang, Di Wang, and Yi Wu. 2016. "Mitochondria Related Pathway Is Essential for Polysaccharides Purified from Sparassis crispa Mediated Neuro-Protection against Glutamate-Induced Toxicity in Differentiated PC12 Cells" International Journal of Molecular Sciences 17, no. 2: 133. https://doi.org/10.3390/ijms17020133