3,4-Dihydroxyphenylethanol (DPE or Hydroxytyrosol) Counteracts ERK1/2 and mTOR Activation, Pro-Inflammatory Cytokine Release, Autophagy and Mitophagy Reduction Mediated by Benzo[a]pyrene in Primary Human Colonic Epithelial Cells
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Cultures, Reagents and Treatments
- −
- Cells were grown for 96 h and 6 days, adding Benzo[a]pyrene every other day;
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- Pre-treatments with 1 µM Hydroxytyrosol (H) (MedChemExpress, Cat# HY-N0570, Monmouth Junction, NJ 08852, USA) were performed for 45 min before adding B[a]P;
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- HCoEpC were incubated with 1 µM UC2288 (Calbiochem- Sigma-Aldrich, Cat# 532813, St. Louis, MO, USA), a p21 inhibitor, for 6 days, refreshing medium supplemented with the inhibitor every other day;
- −
- In order to evaluate autophagy, cells treated or not (CT) with Benzo[a]pyrene for 6 days were incubated or not with 20 nM Bafilomycin A1 (MedChemExpress, Cat# HY-100558, Monmouth Junction, NJ 08852, USA) for the last 4 h.
2.2. Cell Viability Assay
2.3. Chemiluminescent Immunometric Assay
2.4. Monocyte Isolation and Macrophage Differentiation
2.5. Immunofluorescence Staining and FACS Analysis
2.6. Western Blotting
2.7. Antibodies
2.7.1. Indirect Immunofluorescence Assay (IFA)
2.7.2. MitoTrackerTM Green-FM Staining
2.8. Statistical Analysis
3. Results
3.1. B[a]P Exerts a Cytotoxic Effect in Primary Colonic Epithelial Cells, Increases the Release of IL-6, VEGF, IL-8, CXCL13, Cathepsin S and Activates Macrophages
3.2. B[a]P Activates ERK1/2 and mTOR in 2D and 3D Culture Models of HCoEpC
3.3. Hydroxytyrosol Slightly Increases Cell Death and Counteracts Cytokine and Chemokine Release as Well as ERK1/2 and mTOR Activation in B[a]P-treated HCoEpC
3.4. Hydroxytyrosol Restores B[a]P-mediated Reduction in Autophagy and Mitophagy in HCoEpC
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Santarelli, R.; Pompili, C.; Gilardini Montani, M.S.; Evangelista, L.; Gonnella, R.; Cirone, M. 3,4-Dihydroxyphenylethanol (DPE or Hydroxytyrosol) Counteracts ERK1/2 and mTOR Activation, Pro-Inflammatory Cytokine Release, Autophagy and Mitophagy Reduction Mediated by Benzo[a]pyrene in Primary Human Colonic Epithelial Cells. Pharmaceutics 2022, 14, 663. https://doi.org/10.3390/pharmaceutics14030663
Santarelli R, Pompili C, Gilardini Montani MS, Evangelista L, Gonnella R, Cirone M. 3,4-Dihydroxyphenylethanol (DPE or Hydroxytyrosol) Counteracts ERK1/2 and mTOR Activation, Pro-Inflammatory Cytokine Release, Autophagy and Mitophagy Reduction Mediated by Benzo[a]pyrene in Primary Human Colonic Epithelial Cells. Pharmaceutics. 2022; 14(3):663. https://doi.org/10.3390/pharmaceutics14030663
Chicago/Turabian StyleSantarelli, Roberta, Chiara Pompili, Maria Saveria Gilardini Montani, Lorenzo Evangelista, Roberta Gonnella, and Mara Cirone. 2022. "3,4-Dihydroxyphenylethanol (DPE or Hydroxytyrosol) Counteracts ERK1/2 and mTOR Activation, Pro-Inflammatory Cytokine Release, Autophagy and Mitophagy Reduction Mediated by Benzo[a]pyrene in Primary Human Colonic Epithelial Cells" Pharmaceutics 14, no. 3: 663. https://doi.org/10.3390/pharmaceutics14030663
APA StyleSantarelli, R., Pompili, C., Gilardini Montani, M. S., Evangelista, L., Gonnella, R., & Cirone, M. (2022). 3,4-Dihydroxyphenylethanol (DPE or Hydroxytyrosol) Counteracts ERK1/2 and mTOR Activation, Pro-Inflammatory Cytokine Release, Autophagy and Mitophagy Reduction Mediated by Benzo[a]pyrene in Primary Human Colonic Epithelial Cells. Pharmaceutics, 14(3), 663. https://doi.org/10.3390/pharmaceutics14030663