Polyphenol Metabolite Pyrogallol-O-Sulfate Decreases Microglial Activation and VEGF in Retinal Pigment Epithelium Cells and Diabetic Mouse Retina
Abstract
:1. Introduction
2. Results
2.1. Metabolic Activity of RPE Cells Is Not Affected by Treatment with Pyrogallol-O-Sulfate
2.2. Pre-Treatment of RPE Cells with Pyrogallol-O-Sulfate before Hypoxia Has No Significant Effect on the Expression of Neovascularization-Related Proteins
2.3. Treatment of RPE Cells with Pyrogallol-O-Sulfate after Hypoxia Does Not Compromise the Expression of PEDF, VEGF, Or GLUT1
2.4. Treatment of RPE Cells with Pyrogallol-O-Sulfate for 24 H Decreases the Expression of Pro-Angiogenic VEGF
2.5. A Single Intraocular Injection of Pyrogallol-O-Sulfate Decreases the Expression of Microglial Marker Iba1 in The Retina of a Diabetic Mice
2.6. A Single Subretinal Injection of Pyrogallol-O-Sulfate Decreases the Expression of Pro-Angiogenic VEGF in the RPE of a Diabetic Mouse Model
3. Discussion
4. Materials and Methods
4.1. Cell Culture
4.2. Glucose and Hypoxia
4.3. Treatment with (Poly)Phenol-Derived Metabolites
4.4. Protein Extraction and Quantification
4.4.1. Whole Cell Lysates
4.4.2. Protein Quantification
4.5. MTT Assay
4.6. Immunoblotting
4.7. Animals
4.7.1. Housing
4.7.2. Intraocular Injections of Pyrogallol-O-Sulfate in C57BL/Ins2Akita Mice
4.7.3. Retina and RPE Protein Extraction
4.8. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Primary Antibody | Dilution | Reference | Brand |
---|---|---|---|
VEGF-A | 1:500–1:1000 | ab46154 | Abcam, Cambridge, UK |
PEDF | 1:500–1:1000 | 07-280 | Merck Millipore, Burlington, MA, USA |
Iba1 | 1:500 | SAB2500041 | Sigma-Aldrich, St. Louis, MO, USA |
GFAP | 1:10,000 | ab7260 | Abcam, Cambridge, UK |
GLUT-1 | 1:2000 | ab32551 | Abcam, Cambridge, UK |
β-actin | 1:5000 | A5441 | Sigma-Aldrich, St. Louis, MO, USA |
Goat anti-mouse igG-HRP | 1:5000 | sc-516162 | Santa Cruz Biotechnology, Dallas, TX, USA |
Goat anti-rabbit igG-HRP | 1:5000 | sc-2004 | Santa Cruz Biotechnology, Dallas, TX, USA |
Donkey anti-goat igG-HRP | 1:5000 | sc-2020 | Santa Cruz Biotechnology, Dallas, TX, USA |
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Santos, D.F.; Pais, M.; Santos, C.N.; Silva, G.A. Polyphenol Metabolite Pyrogallol-O-Sulfate Decreases Microglial Activation and VEGF in Retinal Pigment Epithelium Cells and Diabetic Mouse Retina. Int. J. Mol. Sci. 2021, 22, 11402. https://doi.org/10.3390/ijms222111402
Santos DF, Pais M, Santos CN, Silva GA. Polyphenol Metabolite Pyrogallol-O-Sulfate Decreases Microglial Activation and VEGF in Retinal Pigment Epithelium Cells and Diabetic Mouse Retina. International Journal of Molecular Sciences. 2021; 22(21):11402. https://doi.org/10.3390/ijms222111402
Chicago/Turabian StyleSantos, Daniela F., Mariana Pais, Cláudia N. Santos, and Gabriela A. Silva. 2021. "Polyphenol Metabolite Pyrogallol-O-Sulfate Decreases Microglial Activation and VEGF in Retinal Pigment Epithelium Cells and Diabetic Mouse Retina" International Journal of Molecular Sciences 22, no. 21: 11402. https://doi.org/10.3390/ijms222111402