Synergistic Protection by Isoquercitrin and Quercetin against Glutamate-Induced Oxidative Cell Death in HT22 Cells via Activating Nrf2 and HO-1 Signaling Pathway: Neuroprotective Principles and Mechanisms of Dendropanax morbifera Leaves
Abstract
:1. Introduction
2. Materials and Methods
2.1. Chemicals and Reagents
2.2. Plant Material
2.3. Extraction, Isolation, and Characterization of Compounds
2.4. Antioxidant Activity Assays
2.4.1. DPPH Radical Scavenging Assay
2.4.2. Superoxide Radical Scavenging Assay
2.4.3. Reducing Power Assay
2.5. Cell Culture and Treatments
2.6. Cell Viability Assay
2.7. PI/Annexin V Apoptosis Analysis
2.8. Measurement of Intracellular and Mitochondrial ROS Generation
2.9. Measurement of Intracellular Calcium Levels
2.10. Measurement of Mitochondrial Membrane Potential
2.11. Measurement of Mitochondrial Membrane Permeabilization
2.12. Subcellular Fractionation
2.13. Western Blotting
2.14. Molecular Docking Simulation
2.15. Quantitative HPLC-UV Assay and Method Validation
2.16. Analysis of Synergistic Effect
2.17. Statistical Analysis
3. Results
3.1. Extraction, Isolation, and Structual Characterization of Antioxidant Compounds from DML
3.2. Antioxidant Activities of Isolated Compounds
3.3. Isoquercitrin and Quercetin Inhibit Glu-Induced Apoptotic Cell Death in HT22 Cells
3.4. Isoquercitrin and Quercetin Attenuate Glu-Induced Intracellular/Mitochondrial ROS Generation and Ca2+ Dysregulation
3.5. Isoquercitrin and Quercetin Restore Glu-Induced Mitochondrial Dysfunction
3.6. Isoquercitrin and Quercetin Inhibit Glu-Induced Nuclear Translocation of AIF
3.7. Isoquercitrin and Quercetin Activate Nrf2/HO-1 Signaling Pathway
3.8. Molecular Docking Simulation of Isoquercitrin and Quercetin to Keap1-BTB Domain
3.9. Identification of Active Principles in DMLE Using HPLC Assay and Combinatory Treatment
3.10. Combination of Isoquercitrin and Quercetin Synergistically Inhibited Glu-Induced Cell Death
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Park, H.-J.; Kim, H.-N.; Kim, C.Y.; Seo, M.-D.; Baek, S.-H. Synergistic Protection by Isoquercitrin and Quercetin against Glutamate-Induced Oxidative Cell Death in HT22 Cells via Activating Nrf2 and HO-1 Signaling Pathway: Neuroprotective Principles and Mechanisms of Dendropanax morbifera Leaves. Antioxidants 2021, 10, 554. https://doi.org/10.3390/antiox10040554
Park H-J, Kim H-N, Kim CY, Seo M-D, Baek S-H. Synergistic Protection by Isoquercitrin and Quercetin against Glutamate-Induced Oxidative Cell Death in HT22 Cells via Activating Nrf2 and HO-1 Signaling Pathway: Neuroprotective Principles and Mechanisms of Dendropanax morbifera Leaves. Antioxidants. 2021; 10(4):554. https://doi.org/10.3390/antiox10040554
Chicago/Turabian StylePark, Hye-Jin, Ha-Neul Kim, Chul Young Kim, Min-Duk Seo, and Seung-Hoon Baek. 2021. "Synergistic Protection by Isoquercitrin and Quercetin against Glutamate-Induced Oxidative Cell Death in HT22 Cells via Activating Nrf2 and HO-1 Signaling Pathway: Neuroprotective Principles and Mechanisms of Dendropanax morbifera Leaves" Antioxidants 10, no. 4: 554. https://doi.org/10.3390/antiox10040554