Trimethylamine N-Oxide as a Mediator Linking Peripheral to Central Inflammation: An In Vitro Study
Abstract
:1. Introduction
2. Results
2.1. TMAO Favored Differentiation of Adipocytes from Preadipocytes (3T3-L1)
2.2. TMAO Increased Expression of Pro-Inflammatory Cytokines in Macrophages (RAW 264.7)
2.3. TMAO Decreased Expression of Tight Junction Proteins in hCMECs
2.4. TMAO Increased Expression of Pro-Inflammatory Cytokines in Primary Neuronal Cultures
2.5. TMAO Induces a Pro-Inflammatory State in Primary Microglial Cultures and Promotes Phagocytosis
3. Discussion
4. Materials and Methods
4.1. In Vitro Cell Lines
4.1.1. Adipocytes (Cell Line 3T3-L1)
4.1.2. Macrophages (Cell Line RAW 264.7)
4.1.3. Blood–Brain Barrier Cells (Cell Line hCMEC/D3)
4.2. Primary Neuronal Cultures
4.3. Primary Microglial Cultures
4.4. Differentiation Assay
4.5. Cell Metabolic Activity Assay (MTT)
4.6. Immunochemistry
4.7. Phagocytosis Assay
4.8. Western Blot
4.9. RNA Extraction and qPCR
4.10. Statistical Analysis
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Name | Dilution | Reference |
---|---|---|
IBA-1 | 1:1000 | Abcam (ab5076) |
CD16/CD32 | 1:100 | BD Biosciences (553142) |
Alexa fluor 546 donkey anti-goat | 1:500 | Invitrogen |
Alexa fluor 488 donkey anti-rat | 1:500 | Invitrogen |
Alexa fluor 488 donkey anti-goat | 1:500 | Invitrogen |
Name | Dilution | Reference |
---|---|---|
Ocluddin | (1:1000) | 40-4700, Thermo Fisher, MA, USA |
ZO-1/TJP1 | (1:1000) | 40-2200, Invitrogen, MA, USA |
p-eNOS at Thr 495 | (1:1000) | 9574S, Cell signaling, MA, USA |
Total eNOS | (1:1000) | 9572, Cell signaling, MA, USA |
p-SAPK/JNK (Thr 183/Tyr 185) | (1:1000) | 9251, Cell signaling, MA, USA |
Total SAPK/JNK | (1:1000) | 9252S, Cell signaling, MA, USA |
GFAP | (1:1000) | 3670-S, Cell signaling, MA, USA |
CD11b | (1:1000) | NB110-89474, Novus biologicals, Centennial, CO, USA |
β-actin | (1:5000) | A1978, Sigma, St. Louis, MO, USA |
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Janeiro, M.H.; Solas, M.; Orbe, J.; Rodríguez, J.A.; Sanchez de Muniain, L.; Escalada, P.; Yip, P.K.; Ramirez, M.J. Trimethylamine N-Oxide as a Mediator Linking Peripheral to Central Inflammation: An In Vitro Study. Int. J. Mol. Sci. 2023, 24, 17557. https://doi.org/10.3390/ijms242417557
Janeiro MH, Solas M, Orbe J, Rodríguez JA, Sanchez de Muniain L, Escalada P, Yip PK, Ramirez MJ. Trimethylamine N-Oxide as a Mediator Linking Peripheral to Central Inflammation: An In Vitro Study. International Journal of Molecular Sciences. 2023; 24(24):17557. https://doi.org/10.3390/ijms242417557
Chicago/Turabian StyleJaneiro, Manuel H., Maite Solas, Josune Orbe, Jose A. Rodríguez, Leyre Sanchez de Muniain, Paula Escalada, Ping K. Yip, and Maria J. Ramirez. 2023. "Trimethylamine N-Oxide as a Mediator Linking Peripheral to Central Inflammation: An In Vitro Study" International Journal of Molecular Sciences 24, no. 24: 17557. https://doi.org/10.3390/ijms242417557