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

Abstract

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 lymphoma-extra 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.

Toll-Like Receptor 4 Activation Prevents Rat Cardiac Fibroblast Death Induced by Simulated Ischemia/Reperfusion

“…It is wellknown that LPS administration increases the expression of inflammatory cytokines (such as IL-1β, IL-6, and TNF-α) in the host through the TLR4/NFkB pathway but can also induce the PI3K/Akt pathway, which has been reported as an endogenous negative feedback regulator and/or compensatory mechanism of TLR4/NFkB-mediated proinflammatory responses, thus preventing an excessive innate immune response (25,26,33,34). In this sense, in our previous findings, we showed that I/R triggers the activation of p38 and JNK signaling pathways (11), which correspond to proapoptotic signals. In addition, a recent study showed that miR-20a has a protective action on cardiomyocytes subjected to sI/R by inactivating the p38 MAPK/JNK pathway and promoting cell viability (35).…”

“…It is well-known that simulated ischemia/reperfusion (sI/R) can produce deleterious effects on CF viability; however, strategies to prevent sI/R injury have been poorly evaluated. This is an important topic since CFs participates in wound healing processes; therefore, viability protection is of utmost importance for their protection (8)(9)(10)(11).…”

“…At the cardiac cellular level, it is well-known that sI/R triggers CF necrosis and apoptosis ( 10 ), and we recently showed that the use of antioxidants prevents CF death ( 11 ). Therefore, the search of alternative therapies to decrease CF death and, consequently, to improve the wound healing process has become relevant; however, until now, it is unknown whether TLR4 activation can produce a cytoprotective effect in CFs exposed to sI/R conditions.…”

Targeting of CAT and VCAM1 as Novel Therapeutic Targets for DMD Cardiomyopathy

“…All concentrations of the active compounds were derived from previous published studies and DMD hiPSC-CMs were treated for 5 days prior to functional assay. Specifically: Quercetin, Naringenin, Kaempferol, and Hesperidin were used at 10 μM ( Gutiérrez-Venegas et al, 2014 ; Denaro et al, 2021 ); Diosgenin at 200 ng/mL ( Zhao et al, 2018 ); D -lactic acid at 10 μM ( Pohanka, 2020 ); Vitamin C at 10 μM ( Parra-Flores et al, 2019 ); Oxalic acid (10 μM) ( Liu et al, 2018 ); and Allicin at 10 μM ( Xiang et al, 2020 ).…”

Modulators of Oxidative Stress: Chemical and Pharmacological Aspects

“…A huge generation of ROS at the beginning of reperfusion initiates lipid peroxidation and protein/nucleic acid oxidation, consequently activating pro-apoptotic pathways associated with p38-MAPK and JNK proteins [ 5 ]. In their manuscript, Parra-Flores et al [ 6 ] showed for the first time synergistic cytoprotective effect of ascorbic acid, deferoxamine, and N -acetylcysteine in simulated IR in CF. When added at low concentrations (10 μM) each at the onset of simulated reperfusion, the three antioxidants synergistically reduced intracellular ROS production and increased cell viability, induced phosphorylation of pro-survival kinases ERK1/2 (extracellular-signal-regulated kinases 1/2) and PKB (protein kinase B)/Akt, and decreased phosphorylation of the pro-apoptotic kinases p38-MAPK (proteins p38-mitogen-activated protein kinase) and JNK (c-Jun-N-terminal kinase).…”