γ-Oryzanol from Rice Bran Antagonizes Glutamate-Induced Excitotoxicity in an In Vitro Model of Differentiated HT-22 Cells
<p>Differentiation transformed into a mature neuron and expressed the NMDA receptor in HT-22 cells. (<b>A</b>) HT-22 cell morphology observed before differentiation (left panel) and after 48 h in a differentiation medium (right panel). After 48 h of culture in differentiation medium, HT-22 cells developed a neuron-like morphology, characterized by the presence of extended neurites. Black arrows indicate neurites. Original magnification: 200×; scale bar: 50 μm. (<b>B</b>) Differentiation conditions induce the expression of the NMDA receptor in HT-22 cells after 48 h. NMDA receptor subunit 1 (NMDAR1) was detected as the NMDA receptor. β-actin was used as loading control. The results are shown as the mean ± SD of five independent experiments (<span class="html-italic">n</span> = 5) performed in triplicate. <sup>a</sup> <span class="html-italic">p</span> < 0.05 compared to the undifferentiated group.</p> "> Figure 2
<p>γ-Oryzanol alleviates glutamate-induced neuronal damage. (<b>A</b>) Cell morphology, (<b>B</b>) cell viability, (<b>C</b>) neurite outgrowth, and (<b>D</b>) protein levels and phosphorylation degree of NMDA receptor were evaluated when the differentiated HT-22 cells were treated with 0.1 mmol/L glutamate for 24 h and received γ-oryzanol (Ory) at 0.2 (Ory 0.2), 0.3 (Ory 0.3), and 0.4 (Ory 0.4) mmol/L or memantine (10 µmol/L) for another 24 h. The cell morphology was examined using an Zeiss Axiovert 135 inverted phase-contrast microscope at ×100 magnification. Cell viability was determined with a CCK-8 assay and expressed as a percentage of untreated cells taken as a control group. The total number of neurites per cell was determined by counting the number of neurites directly from the soma using Image J software (version 1.6.0). Protein levels and phosphorylation degree of NMDAR1 are shown via representative immunoblots. β-actin was used as loading control. The ratio between phosphoprotein and total protein of NMDAR1 (p-NMDAR1 and NMDAR1) was calculated. The results are shown as the mean ± SD of five independent experiments (<span class="html-italic">n</span> = 5) performed in triplicate. <sup>a</sup> <span class="html-italic">p</span> < 0.05 and <sup>b</sup> <span class="html-italic">p</span> < 0.01 compared to the untreated control group (control). <sup>c</sup> <span class="html-italic">p</span> < 0.05 and <sup>d</sup> <span class="html-italic">p</span> < 0.01 compared to the data from glutamate-stimulated cells that received vehicle treatment.</p> "> Figure 3
<p>γ-Oryzanol protects differentiated HT-22 cells against glutamate-induced oxidative stress. The differentiated HT-22 cells were treated with 0.1 mmol/L glutamate for 24 h and received γ-oryzanol (0.4 mmol/L) or memantine (10 µmol/L) for another 24 h. (<b>A</b>) The ROS fluorescence intensity is expressed as a percentage of the untreated control cells. (<b>B</b>) The activities of SOD, GSH-Px, CAT, and GSH content were normalized to the corresponding protein concentration for each group. The results are shown as the mean ± SD of five independent experiments (<span class="html-italic">n</span> = 5) performed in triplicate. <sup>a</sup> <span class="html-italic">p</span> < 0.05 and <sup>b</sup> <span class="html-italic">p</span> < 0.01 compared to the untreated control group (control). <sup>c</sup> <span class="html-italic">p</span> < 0.05 and <sup>d</sup> <span class="html-italic">p</span> < 0.01 compared to the data from glutamate-stimulated cells that received vehicle treatment.</p> "> Figure 4
<p>γ-Oryzanol suppressed glutamate-induced intracellular calcium overload and CaMKII activation. The differentiated HT-22 cells were treated with 0.1 mmol/L glutamate for 24 h and received γ-oryzanol (0.4 µmol/L) or memantine (10 µmol/L) for another 24 h. (<b>A</b>) The fluorescence intensities for intracellular Ca<sup>2+</sup> was measured via live cell imaging. Original magnification: 200×; scale bar: 50 μm. Represents bar diagram of relative fluorescence intensity. (<b>B</b>) Representative immunoblots depicting the protein levels and phosphorylation degree of CaMKII, β-actin was used as loading control. The ratio of p-CaMKII to total CaMKII was calculated. The results are shown as the mean ± SD of five independent experiments (<span class="html-italic">n</span> = 5) performed in triplicate. <sup>a</sup> <span class="html-italic">p</span> < 0.05 and <sup>b</sup> <span class="html-italic">p</span> < 0.01 compared to the untreated control group (control). <sup>c</sup> <span class="html-italic">p</span> < 0.05 and <sup>d</sup> <span class="html-italic">p</span> < 0.01 compared to the data from glutamate-stimulated cells that received vehicle treatment.</p> "> Figure 5
<p>γ-Oryzanol prevents glutamate-induced mitochondrial dysfunction. The differentiated HT-22 cells were treated with 0.1 mmol/L glutamate for 24 h and received γ-oryzanol (0.4 µmol/L) or memantine (10 µmol/L) for another 24 h. (<b>A</b>) The mitochondrial transmembrane potential has been measured with the JC-1 fluorescence probe. (<b>B</b>) The bioluminescent detection of ADP and ATP levels was used to measure the ADP/ATP ratio in cells with a commercial assay kit. (<b>C</b>) The oxidation of NADH was measured to determine the activity of mitochondrial complex I. The results are shown as the mean ± SD of five independent experiments (<span class="html-italic">n</span> = 5) performed in triplicate. <sup>a</sup> <span class="html-italic">p</span> < 0.05 and <sup>b</sup> <span class="html-italic">p</span> < 0.01 compared to the untreated control group (control). <sup>c</sup> <span class="html-italic">p</span> < 0.05 and <sup>d</sup> <span class="html-italic">p</span> < 0.01 compared to the data from glutamate-stimulated cells that received vehicle treatment.</p> "> Figure 6
<p>γ-Oryzanol downregulates ASK1/JNK signaling pathway induced by glutamate. The differentiated HT-22 cells were treated with 0.1 mmol/L glutamate for 24 h and received γ-oryzanol (0.4 µmol/L) or memantine (10 µmol/L) for another 24 h. Protein expression and extent of phosphorylation on (<b>A</b>) ASK1, (<b>B</b>) MKK4, (<b>C</b>) MKK7, (<b>D</b>) JNK, (<b>E</b>) c-Jun, and (<b>F</b>) c-Fos were analyzed by Western blot. The degree of phosphorylation was calculated as a ratio of the total protein. β-actin was used as loading control. The results are shown as the mean ± SD of five independent experiments (<span class="html-italic">n</span> = 5) performed in triplicate. <sup>a</sup> <span class="html-italic">p</span> < 0.05 and <sup>b</sup> <span class="html-italic">p</span> < 0.01 compared to the untreated control group (control). <sup>c</sup> <span class="html-italic">p</span> < 0.05 and <sup>d</sup> <span class="html-italic">p</span> < 0.01 compared to the data from glutamate-stimulated cells that received vehicle treatment.</p> "> Figure 7
<p>γ-Oryzanol declined cytochrome c-initiated caspase cascade activation induced by glutamate. The differentiated HT-22 cells were treated with 0.1 mmol/L glutamate for 24 h and received γ-oryzanol (0.4 µmol/L) or memantine (10 µmol/L) for another 24 h. (<b>A</b>) The concentrations of cytochrome c in both mitochondrial and cytosolic fractions were determined using an immunoassay. (<b>B</b>) Protein expression of Apaf-1, procaspase-9, cleaved caspase-9, procaspase-3, cleaved caspase-3, proPARP, and cleaved PARP were analyzed by Western blot. Β-actin was used as loading control. The results are shown as the mean ± SD of five independent experiments (<span class="html-italic">n</span> = 5) performed in triplicate. <sup>a</sup> <span class="html-italic">p</span> < 0.05 and <sup>b</sup> <span class="html-italic">p</span> < 0.01 compared to the untreated control group (control). <sup>c</sup> <span class="html-italic">p</span> < 0.05 and <sup>d</sup> <span class="html-italic">p</span> < 0.01 compared to the data from glutamate-stimulated cells that received vehicle treatment.</p> "> Figure 8
<p>γ-Oryzanol reversed Bcl-2 protein downregulation and DNA fragmentation induced by glutamate. The differentiated HT-22 cells were treated with 0.1 mmol/L glutamate for 24 h and received γ-oryzanol (0.4 µmol/L) or memantine (10 µmol/L) for another 24 h. (<b>A</b>) The expression of Bcl-2 and Bax protein were measured by Western blot. β-actin was used as loading control. The ratio of relative intensities in Bcl-2 to Bax (Bcl-2/Bax) was reported. (<b>B</b>) The extent of apoptosis was quantified using the ELISA kit to detect DNA fragments associated with cytoplasmic histones. The results are shown as the mean ± SD of five independent experiments (<span class="html-italic">n</span> = 5) performed in triplicate. <sup>a</sup> <span class="html-italic">p</span> < 0.05 and <sup>b</sup> <span class="html-italic">p</span> < 0.01 compared to the untreated control group (control). <sup>c</sup> <span class="html-italic">p</span> < 0.05 and <sup>d</sup> <span class="html-italic">p</span> < 0.01 compared to the data from glutamate-stimulated cells that received vehicle treatment.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Culture
2.2. Procedure of Glutamate-Induced Excitotoxicity and Treatment
2.3. Assement of Cell Viability
2.4. Measurement of Intracellular Reactive Oxygen Species (ROS)
2.5. Measurement of Antioxidant Enzymes Activity and Intracellular Glutathione Level
2.6. Measurement of Intracellular Calcium
2.7. Assessment of Mitochondrial Transmembrane Potential
2.8. Quantification of ADP and ATP Concentrations
2.9. Quantification of Released Cytochrome C
2.10. Measurement of Mitochondrial Complex I Activity
2.11. Quantification of Apoptotic DNA Fragmentation
2.12. Western Blot Analysis
2.13. Statistical Analysis
3. Results
3.1. Differentiation Induces Morphological Changes and an Increase in NMDA Receptor Expression in HT-22 Cells
3.2. γ-Oryzanol Alleviates Glutamate-Induced Neuronal Insults
3.3. γ-Oryzanol Alleviates Glutamate-Induced Oxidative Stress
3.4. γ-Oryzanol Suppressed Glutamate-Induced Intracellular Calcium Overload and CaMKII Activation
3.5. γ-Oryzanol Attenuates Glutamate-Induced Mitochondrial Dysfunction
3.6. γ-Oryzanol Downregulates ASK1/JNK Signaling Pathway Induced by Glutamate
3.7. γ-Oryzanol Declined Cytochrome c-Initiated Caspase Cascade Activation Induced by Glutamate
3.8. γ-Oryzanol Reversed Bcl-2 Protein Downregulation and DNA Fragmentation Induced by Glutamate
4. Discussion
5. Conclusions
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Chen, L.-C.; Lai, M.-C.; Hong, T.-Y.; Liu, I.-M. γ-Oryzanol from Rice Bran Antagonizes Glutamate-Induced Excitotoxicity in an In Vitro Model of Differentiated HT-22 Cells. Nutrients 2024, 16, 1237. https://doi.org/10.3390/nu16081237
Chen L-C, Lai M-C, Hong T-Y, Liu I-M. γ-Oryzanol from Rice Bran Antagonizes Glutamate-Induced Excitotoxicity in an In Vitro Model of Differentiated HT-22 Cells. Nutrients. 2024; 16(8):1237. https://doi.org/10.3390/nu16081237
Chicago/Turabian StyleChen, Li-Chai, Mei-Chou Lai, Tang-Yao Hong, and I-Min Liu. 2024. "γ-Oryzanol from Rice Bran Antagonizes Glutamate-Induced Excitotoxicity in an In Vitro Model of Differentiated HT-22 Cells" Nutrients 16, no. 8: 1237. https://doi.org/10.3390/nu16081237