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The Association of Ascorbic Acid, Deferoxamine and N-Acetylcysteine Improves Cardiac Fibroblast Viability and Cellular Function Associated with Tissue Repair Damaged by Simulated Ischemia/Reperfusion

1
Laboratorio de Farmacología Molecular, Departamento de Química Farmacológica y Toxicológica, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Santos Dumont 964, Independencia, Santiago 8380492, Chile
2
Advanced Center for Chronic Diseases (ACCDiS), Facultad de Ciencias Químicas y Farmacéuticas and Facultad de Medicina, Universidad de Chile, Santos Dumont 964, Independencia, Santiago 8380492, Chile
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Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Independencia 1027, Independencia, Santiago 8380453, Chile
4
Cardiology Division, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390-8573, USA
*
Author to whom correspondence should be addressed.
Antioxidants 2019, 8(12), 614; https://doi.org/10.3390/antiox8120614
Received: 12 October 2019 / Revised: 18 November 2019 / Accepted: 26 November 2019 / Published: 3 December 2019
(This article belongs to the Special Issue Modulators of Oxidative Stress: Chemical and Pharmacological Aspects)
Acute myocardial infarction is one of the leading causes of death worldwide and thus, an extensively studied disease. Nonetheless, the effects of ischemia/reperfusion injury elicited by oxidative stress on cardiac fibroblast function associated with tissue repair are not completely understood. Ascorbic acid, deferoxamine, and N-acetylcysteine (A/D/N) are antioxidants with known cardioprotective effects, but the potential beneficial effects of combining these antioxidants in the tissue repair properties of cardiac fibroblasts remain unknown. Thus, the aim of this study was to evaluate whether the pharmacological association of these antioxidants, at low concentrations, could confer protection to cardiac fibroblasts against simulated ischemia/reperfusion injury. To test this, neonatal rat cardiac fibroblasts were subjected to simulated ischemia/reperfusion in the presence or absence of A/D/N treatment added at the beginning of simulated reperfusion. Cell viability was assessed using trypan blue staining, and intracellular reactive oxygen species (ROS) production was assessed using a 2′,7′-dichlorofluorescin diacetate probe. Cell death was measured by flow cytometry using propidium iodide. Cell signaling mechanisms, differentiation into myofibroblasts and pro-collagen I production were determined by Western blot, whereas migration was evaluated using the wound healing assay. Our results show that A/D/N association using a low concentration of each antioxidant increased cardiac fibroblast viability, but that their separate administration did not provide protection. In addition, A/D/N association attenuated oxidative stress triggered by simulated ischemia/reperfusion, induced phosphorylation of pro-survival extracellular-signal-regulated kinases 1/2 (ERK1/2) and PKB (protein kinase B)/Akt, and decreased phosphorylation of the pro-apoptotic proteins p38- mitogen-activated protein kinase (p38-MAPK) and c-Jun-N-terminal kinase (JNK). Moreover, treatment with A/D/N also reduced reperfusion-induced apoptosis, evidenced by a decrease in the sub-G1 population, lower fragmentation of pro-caspases 9 and 3, as well as increased B-cell lymphomaextra large protein (Bcl-xL)/Bcl-2-associated X protein (Bax) ratio. Furthermore, simulated ischemia/reperfusion abolished serum-induced migration, TGF-β1 (transforming growth factor beta 1)-mediated cardiac fibroblast-to-cardiac myofibroblast differentiation, and angiotensin II-induced pro-collagen I synthesis, but these effects were prevented by treatment with A/D/N. In conclusion, this is the first study where a pharmacological combination of A/D/N, at low concentrations, protected cardiac fibroblast viability and function after simulated ischemia/reperfusion, and thereby represents a novel therapeutic approach for cardioprotection.
Keywords: ascorbic acid; deferoxamine; N-acetylcysteine; ischemia/reperfusion; cardiac fibroblasts; reactive oxygen species ascorbic acid; deferoxamine; N-acetylcysteine; ischemia/reperfusion; cardiac fibroblasts; reactive oxygen species
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Parra-Flores, P.; Riquelme, J.A.; Valenzuela-Bustamante, P.; Leiva-Navarrete, S.; Vivar, R.; Cayupi-Vivanco, J.; Castro, E.; Espinoza-Pérez, C.; Ruz-Cortés, F.; Pedrozo, Z.; Lavandero, S.; Rodrigo, R.; Diaz-Araya, G. The Association of Ascorbic Acid, Deferoxamine and N-Acetylcysteine Improves Cardiac Fibroblast Viability and Cellular Function Associated with Tissue Repair Damaged by Simulated Ischemia/Reperfusion. Antioxidants 2019, 8, 614.

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