Neuroinflammation Based Neurodegenerative In Vitro Model of SH-SY5Y Cells—Differential Effects on Oxidative Stress and Insulin Resistance Relevant to Alzheimer’s Pathology
Abstract
1. Introduction
2. Results
2.1. Effect of Immune Cell-Derived Conditioned Media on Cell Viability
2.2. Effect of Conditioned Media on ROS Production
2.3. Effect of Inflammatory Conditioned Media on Mitochondrial Membrane Potential
2.4. Effect of Inflammatory Conditioned Media on Mitochondrial Mass
2.5. Effect of Inflammatory Conditioned Media on Glucose Uptake
2.6. Effect of Immune Cell-Conditioned Media on Autophagic Activity
2.7. Effect of Immune Cell-Conditioned Media on Insulin Signaling
2.8. Effect of Immune Cell-Conditioned Media on Protein Aggregation
3. Discussion
4. Materials and Methods
4.1. Materials
4.2. Cell Cultures and Treatments
- (1)
- Neutrophil-like differentiation using 1.25% DMSO [46]
- (2)
- Monocyte-like differentiation using 100 nM RA. In both cases, differentiation was initiated on day 0 and continued for five days, with medium replacement and differentiation agent supplementation on day 3. Undifferentiated HL60 cells were also used in the experiments.
4.3. Lactate Dehydrogenase (LDH) Release Cytotoxicity Assay
4.4. Assessment of ROS Levels and Mitochondrial Membrane Potential
4.5. Measurement of Mitochondrial Content
4.6. 2-NBDG Glucose Uptake Assay
4.7. Detection of Autophagic Vacuoles by Acridine Orange
4.8. ELISA for Phospho-GSK-3 Alpha/Beta (S21/S9)
4.9. Thioflavin S Protein Aggregation Staining
4.10. Statistical Evaluation
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
2-NBDG | 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (fluorescent glucose analog) |
AD | Alzheimer’s Disease |
AO | Acridine Orange (fluorescent dye for acidic organelles/autophagy detection) |
APP | Amyloid Precursor Protein |
ATP | Adenosine Triphosphate |
Aβ | Amyloid-Beta Peptide |
C | Control |
DAPI | 4′,6-Diamidino-2-Phenylindole (nuclear stain) |
DCFDA | 2′,7′-Dichlorofluorescin Diacetate (indicator of hydrogen peroxide) |
DMEM/F12 | Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 |
DMSO | Dimethyl Sulfoxide |
ELISA | Enzyme-Linked Immunosorbent Assay |
ER | Endoplasmic Reticulum |
FBS | Fetal Bovine Serum |
GLP-1 | Glucagon-Like Peptide-1 |
GSK-3β | Glycogen Synthase Kinase-3 Beta |
HE | Hydroethidine |
IL-1β | Interleukin-1 Beta |
IL-6 | Interleukin-6 |
iPSC | Induced Pluripotent Stem Cell |
JC-1 | 5,5′,6,6′-Tetrachloro-1,1′,3,3′-Tetraethylbenzimidazolylcarbocyanine Iodide |
LDH | Lactate Dehydrogenase |
LPS | Lipopolysaccharide |
NFT | Neurofibrillary Tangle |
NLRP3 | NOD-, LRR-, and Pyrin Domain-Containing Protein 3 |
NO | Nitric Oxide |
pGSK-3β | Phosphorylated Glycogen Synthase Kinase-3 Beta |
PARP | Poly (ADP-Ribose) Polymerase |
PBS | Phosphate-Buffered Saline |
PI3K | Phosphoinositide 3-Kinase |
RA | Retinoic Acid |
ROS | Reactive Oxygen Species |
SD | Standard Deviation |
SH-SY5Y | Human Neuroblastoma Cell Line |
Thioflavin S | Fluorescent dye for beta-sheet-rich protein aggregates |
TNF-α | Tumor Necrosis Factor-Alpha |
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Group | Description of Treatment |
---|---|
RAW264.7 | Macrophage cell line-produced factors |
RAW264.7 + LPS | Activated macrophage cell line-produced factors |
BV2 | Microglial cell line-produced factors |
BV2 + LPS | Activated microglial cell line-produced factors |
HL60 | Undifferentiated promyeloblast cell line-produced factors |
HL60 + LPS | Activated, undifferentiated promyeloblast cell line-produced factors |
HL60 + DMSO | Neutrophil-like HL60 cell line-produced factors |
HL60 + DMSO + LPS | Activated, neutrophil-like HL60 cell line-produced factors |
HL60 + RA | Monocyte-like HL60 cell line-produced factors |
HL60 + RA + LPS | Activated, monocyte-like HL60 cell line-produced factors |
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Böröczky, C.; Paszternák, A.; Laufer, R.; Tarnóczi, K.; Sikur, N.; Bagaméry, F.; Szökő, É.; Varga, K.; Tábi, T. Neuroinflammation Based Neurodegenerative In Vitro Model of SH-SY5Y Cells—Differential Effects on Oxidative Stress and Insulin Resistance Relevant to Alzheimer’s Pathology. Int. J. Mol. Sci. 2025, 26, 6581. https://doi.org/10.3390/ijms26146581
Böröczky C, Paszternák A, Laufer R, Tarnóczi K, Sikur N, Bagaméry F, Szökő É, Varga K, Tábi T. Neuroinflammation Based Neurodegenerative In Vitro Model of SH-SY5Y Cells—Differential Effects on Oxidative Stress and Insulin Resistance Relevant to Alzheimer’s Pathology. International Journal of Molecular Sciences. 2025; 26(14):6581. https://doi.org/10.3390/ijms26146581
Chicago/Turabian StyleBöröczky, Csenge, Alexandra Paszternák, Rudolf Laufer, Katinka Tarnóczi, Noémi Sikur, Fruzsina Bagaméry, Éva Szökő, Kamilla Varga, and Tamás Tábi. 2025. "Neuroinflammation Based Neurodegenerative In Vitro Model of SH-SY5Y Cells—Differential Effects on Oxidative Stress and Insulin Resistance Relevant to Alzheimer’s Pathology" International Journal of Molecular Sciences 26, no. 14: 6581. https://doi.org/10.3390/ijms26146581
APA StyleBöröczky, C., Paszternák, A., Laufer, R., Tarnóczi, K., Sikur, N., Bagaméry, F., Szökő, É., Varga, K., & Tábi, T. (2025). Neuroinflammation Based Neurodegenerative In Vitro Model of SH-SY5Y Cells—Differential Effects on Oxidative Stress and Insulin Resistance Relevant to Alzheimer’s Pathology. International Journal of Molecular Sciences, 26(14), 6581. https://doi.org/10.3390/ijms26146581