The Phosphodiesterase Type 5 Inhibitor Sildenafil Improves DNA Stability and Redox Homeostasis in Systemic Sclerosis Fibroblasts Exposed to Reactive Oxygen Species
Abstract
:1. Introduction
2. Materials and Methods
2.1. Chemicals
2.2. Cell Culture
2.3. Protein Expression Analysis
2.4. Immunofluorescence Microscopy
2.5. 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyl-2H-Tetrazolium Bromide (MTT) Assay
2.6. Cell Viability
2.7. Cell Cycle Analysis
2.8. Measurement of Intracellular ROS Levels
2.9. Thiobarbituric Acid Reactive Substances (TBARS)
2.10. Glutathione Homeostasis
2.11. Enzymatic Activities
2.12. Cytokine Assay
2.13. Statistical Analysis
3. Results
3.1. SSc Fibroblasts Appeared More Sensitive to Oxidative Stress-Induced DNA Double-Strand Breaks (DSBs) in Comparison to the Healthy Control Cells
3.2. Sildenafil Improved the DNA Repair Activation Process in H2O2-Damaged Healthy and SSc Fibroblasts
3.3. Sildenafil Sustained the Cell Viability and Ameliorated the Cell Proliferation in SSc Fibroblasts
3.4. Sildenafil Reduces the ROS Levels in Healthy and SSc Fibroblasts
3.5. Sildenafil Ameliorates an Antioxidant Response in SSc Fibroblasts
3.6. Macrophage Migration Inhibitory Factor Level
3.7. Sildenafil Mitigates the Effect of Oxidative Insult on TBAR Levels
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Antigen | Product Number | Dilution | Manufacturer |
---|---|---|---|
Superoxide dismutase (SOD) 1 | sc-101523 | 1:1000 | Santa Cruz (Santa Cruz, CA, USA) |
Superoxide dismutase (SOD) 2 | sod-110 | 1:1000 | StressGen (San Diego, CA, USA) |
Protein light chain (LC3) I-II | sc-271625 | 1:1000 | Santa Cruz |
Phospho-checkpoint kinase (p-CHK) 2 | 2197 | 1:1000 | Cell Signaling (Leiden, The Netherlands |
Checkpoint kinase (CHK) 2 | sc-5278 | 1:1000 | Santa Cruz |
Caspase (CASP) 3 | sc-56053 | 1:1000 | Santa Cruz |
BCL2 associated X (BAX) | sc-526 | 1:1000 | Santa Cruz |
B-cell lymphoma-2 (BCL-2) | 50E3 | 1:1000 | Cell signaling |
Phospho-extracellular signal-regulated kinases (p-ERK) 1/2 | sc-7383 | 1:1000 | Santa Cruz |
Extracellular signal-regulated kinases (ERK) 2 | sc-154 | 1:1000 | Santa Cruz |
Sequestosome 1 (P62) | sc-48402 | 1:1000 | Santa Cruz |
Catalase (CAT) | sc-271803 | 1:1000 | Santa Cruz |
β-actin | sc-47778 | 1:5000 | Santa Cruz |
γ-histone family member X (γH2AX) | ab26350 | 1:100 | Abcam (Cambridge, UK) |
DNA repair protein RAD51 (RAD51) | ab63801 | 1:1000 | Abcam |
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Di Luigi, L.; Duranti, G.; Antonioni, A.; Sgrò, P.; Ceci, R.; Crescioli, C.; Sabatini, S.; Lenzi, A.; Caporossi, D.; Del Galdo, F.; Dimauro, I.; Antinozzi, C. The Phosphodiesterase Type 5 Inhibitor Sildenafil Improves DNA Stability and Redox Homeostasis in Systemic Sclerosis Fibroblasts Exposed to Reactive Oxygen Species. Antioxidants 2020, 9, 786. https://doi.org/10.3390/antiox9090786
Di Luigi L, Duranti G, Antonioni A, Sgrò P, Ceci R, Crescioli C, Sabatini S, Lenzi A, Caporossi D, Del Galdo F, Dimauro I, Antinozzi C. The Phosphodiesterase Type 5 Inhibitor Sildenafil Improves DNA Stability and Redox Homeostasis in Systemic Sclerosis Fibroblasts Exposed to Reactive Oxygen Species. Antioxidants. 2020; 9(9):786. https://doi.org/10.3390/antiox9090786
Chicago/Turabian StyleDi Luigi, Luigi, Guglielmo Duranti, Ambra Antonioni, Paolo Sgrò, Roberta Ceci, Clara Crescioli, Stefania Sabatini, Andrea Lenzi, Daniela Caporossi, Francesco Del Galdo, Ivan Dimauro, and Cristina Antinozzi. 2020. "The Phosphodiesterase Type 5 Inhibitor Sildenafil Improves DNA Stability and Redox Homeostasis in Systemic Sclerosis Fibroblasts Exposed to Reactive Oxygen Species" Antioxidants 9, no. 9: 786. https://doi.org/10.3390/antiox9090786