Efeito cardioprotetor do complexo de inclusão D-Limoneno Hidroxipropil-β-Ciclodextrina na Cardiotoxicidade induzida por Doxorrubicina em modelo animal

Abstract

Introduction: Cardiotoxicity (cdtx) induced by Doxorubicin (Doxo) is a common problem in the treatment of cancer, which is the main limiting factor for its use in the clinic as it promotes mechanical and electrocardiographic cardiac changes; Still, there is no specific, widely accepted treatment for Doxo-induced cdtx. Natural products have been successfully tested in the treatment of cardiovascular diseases, more specifically, the monoterpene D-limonene (DL) was successfully tested as a cardioprotective agent in animal models; however, it is volatile and has low solubility in water, which limits its clinical use. Thus, the DL complex with hydroxypropyl-β-cyclodextrin (HPβCD) could improve its properties. The general objective of this work was to investigate, through experimental testing, the cardioprotective effect of the D-limonene-hydroxypropyl-β-cyclodextrin (HβDL) inclusion complex on Doxo-induced cdtx. Methods: The animals were divided into groups (n=5) and treated intraperitoneally with either DL (10 mg/kg), or HβDL (10, 30 and 100 mg/kg), or HPβCD (100 mg/kg ), or with vehicle (0.9% NaCl/0.1% DMSO); cdtx was induced with Doxo (20 mg/kg). To assess cardiac injury, CK- MB and LDH were measured in the blood serum of all groups. For other assessments such as histological analysis, electrocardiographic parameters, susceptibility to arrhythmias in vivo and in vitro, action potential (AP), L-type calcium current (ICa,L), production of cytosolic and mitochondrial reactive oxygen species, mitochondrial mass , calcium transient, contractility, activity of antioxidant enzymes, lipoperoxidation, expression of Ca2+/calmodulin-dependent kinase II (CaMKII), Bax, BCl2 and Caspase 3, HβDL 10 mg/kg was used. Molecular docking was also performed. For statistical analysis, one-way/two-way ANOVA was used, as appropriate, or the chi-square test, followed by the Bonferroni post hoc test, with significance being considered when P < 0.05. Results: Cardiac injury biomarkers CK-MB and LDH were elevated in the Doxo group compared to control, and were reversed by HβDL at all doses. Histological changes of cellular infiltrate and vacuoles presented in the Doxo group were prevented by HβDL. HβDL led to body weight gain similar to the control group. Electrocardiographic parameters of QTc, QRS and BPM were increased in the Doxo group, which was prevented in the HβDL group; in vivo arrhythmia was 50% higher in the Doxo group than in the HβDL group. AP arrhythmia, duration and mean standard deviation of AP, transient amplitude and calcium waves were increased in the Doxo group, which was prevented by HβDL. ICa,L was increased in the Doxo group, which was also prevented by HβDL. Sarcomeric shortening and time to relaxation by 50% were reduced in the Doxo group, and HβDL restored both parameters. HβDL prevented the phenotype of increased expression of phosphorylated and oxidized CaMKII by Doxo administration, as well as the increase in cytoplasmic and mitochondrial reactive species and mitochondrial mass. The activities of mitochondrial glutathione peroxidase and superoxide dismutase (SOD), which were decreased in the Doxo group, were restored in the HβDL group. There was no difference in catalase and cytoplasmic SOD activities. HβDL prevented the increase in pro- apoptotic proteins Bax and Caspase 3, and lipoperaxidation, induced by Doxo. There was no change in Bcl-2 expression. Molecular docking showed affinity between DL and the two ligands evaluated – CaMKII and myosin light chain kinase. Conclusion: HβDL 10 mg/kg prevented the increase in biomarkers of cardiac injury and structural changes in cardiac tissue, improved the electrocardiographic profile, decreased the percentage of arrhythmia in vivo and in vitro, reduced calcium waves, prevented changes in BP and blood pressure dynamics. contraction/relaxation of the cardiomyocyte, prevented the increase in ICa,L, inhibited CaMKII activation, improved redox profile and suppressed apoptotic pathways. The results validate the study design and show a promising potential cardioprotective effect of HβDL.