Mecanismos redox de adaptação celular e suplementação antioxidante como estratégia terapêutica na anemia falciforme: um ensaio pré-clínico.

Abstract

Redox homeostasis loss is associated with the pathophysiological mechanisms of sickle cell anemia (SCA). Thus, understanding the redox mechanisms of cellular adaptation enables the identification of new targets and/or targeted interventions, such as therapeutic alternatives with antioxidant properties. Therefore, this study evaluated the expression profile of redox signaling pathways in hematopoietic progenitor cells from humanized mice for SCA, as well as their adaptive responses under the effects of supplementation with melatonin (MEL) and ergothioneine (ERT). Four groups were evaluated, generated at CEMIB/UNICAMP over 60 days: the control group, characterized by transgenic HbSA+Fetal (SAF) mice; homozygous untreated SCA (SS) mice; and SCA mice treated with MEL (SS+MEL; orally, 10 mg/kg) or ERT (SS+ERT; orally, 35 mg/kg). After euthanasia (CEUA 5955-1/2022), RNA was extracted from bone marrow progenitor cells for RT-qPCR analysis, in pseudo quintuplicates, targeting key redox biology markers: ATF4, NRF2, FOXO3, MST1, YWHAZ, TRX, TXNIP, SLC22A4 (ETT), eIF2α, CAT, SOD1, GPX1, PRDX1, and the endogenous control ACTB. Fold change levels were calculated using the 2^-ΔΔCT formula. Statistical analyses were performed using GLM in a one-way ANOVA format, followed by Bonferroni's test. Significant disease effects (SF vs. SS) observed included the induction of NRF2 (~9x), FOXO3 (~3x), MST1 (~7x), ETT (~8x), CAT (~4x), and GPX1 (~8x); and inhibition of TRX1 (~2.5x), SOD1 (~1.4x), and PRDX1 (~3x). These findings validate the robustness of the model and underscore the critical role of central redox homeostasis elements in SCA pathophysiology. For MEL supplementation (SS vs. SS+MEL), the observed effects included induction of YWHAZ (~2x), TRX (~3x), SOD1 (~2x), and PRDX1 (~3x), alongside inhibition of NRF2 (~147x), FOXO3 (~9x), MST1 (~9x), ETT (~7x), CAT (~26x), and GPX1 (~111x). Overall, in the SS+MEL group, transcript levels were restored to patterns observed in the SAF group, suggesting a direct antioxidant effect of MEL, mitigating redox pathway activation. With ERT supplementation (SS vs. SS+ERT), the observed effects included induction of ATF4 (~3x), YWHAZ (~4x), TRX1 (~3x), TXNIP (~6x), ETT (~2x), CAT (~2x), and SOD1 (~2x), along with inhibition of NRF2 (~4x), FOXO3 (~79x), and GPX1 (~2x). The inhibitory pattern suggests a direct antioxidant action of ERT, mitigating disease effects, while the inductions appear to involve indirect actions, reinforcing its critical and little-understood role during hematopoiesis, as ATF4 and TXNIP are proposed as new regulators of erythroid proliferation and differentiation. In conclusion, the evaluated mechanisms play a critical role in the redox homeostasis of progenitor cells in the tested model. Additionally, ERT treatment appears to have a broad-spectrum modulatory action associated with erythropoiesis, while MEL treatment has a cytoprotective effect, mitigating the activation of redox signaling pathways exacerbated by the disease.