An investigation into the potential cardioprotective effects of ghrelin in a rat model of chronic Doxorubicin-induced cardiotoxicity

[摘要] ENGLISH SUMMARY: IntroductionDoxorubicin (DOX) is an anthracycline that has significantly improved the outcome ofcancer patients since its discovery. However, its clinical success remains limited due to thedose-dependent cardiotoxic side effects associated with its use. While the mechanismsresponsible for this condition are still not well defined, oxidative stress alongside apoptosis,remain the classical but major contributors. Ghrelin, an endogenous brain-gut peptide, mostwell-known for its effects on appetite and growth hormone release, has been shown to exertanti-apoptotic, antioxidative, anti-inflammatory and anti-fibrotic effects in several models ofcardiovascular disease. These cardioprotective effects offered by ghrelin have been indicatedto occur through the activation of the pro-survival proteins, ERK1/2, Akt/PKB and STAT3.This study therefore investigated the protective effects of ghrelin in a chronic model of DOXinducedcardiotoxicity.MethodsMale Sprague-Dawley rats were acclimatized and divided into five experimental groups.While the control group remained untreated, the ghrelin group received 300 μg/kg ghrelin perweek, the DOX group received 2.5 mg/kg per week, the combination group received bothDOX and ghrelin at the previously mentioned doses and the vehicle group received saline.All injections were performed via intraperitoneal injection for eight weeks. One week afterthe last injection, the rats were euthanized, blood was collected and the hearts were subjectedto a 40-minute working heart perfusion protocol to obtain functional data. The hearts wereweighed and then cut transversely into two sections, where one half was snap frozen forbiochemical analysis and the other half was preserved in formalin for histological analysis.Cardiovascular markers of damage and pro-inflammatory cytokines were measured in serumusing a luminex assay, and fibrosis and general morphology were assessed using theMasson's Trichrome and H&E stains, respectively. Cytochrome c expression was evaluatedby immunohistochemistry, while oxidative stress was assessed using the TBARS, conjugateddiene, ORAC, SOD and glutathione assays. Apoptosis was determined using the CaspaseGlo® assay and the expression of pro-survival proteins was measured using Western blotanalysis. ResultsDuring the eight weeks of treatment, DOX significantly reduced weight gain and foodconsumption, whereas ghrelin maintained normal body weight and stimulated appetite. Asexpected, DOX induced significant oxidative stress when compared to the control, asdemonstrated by the formation of conjugated dienes (1.64 ± 0.11 vs 0.55 ± 0.12 μmol/gram, p= 0.0003) and a reduction in the GSH:GSSG ratio (2.10 ± 0.47 vs 9.66 ± 1.08 arbitrary units,p< 0.05), whereas ghrelin attenuated these effects. Apoptotic cell death was also induced, asevident by an increase in cytochrome c staining, caspase activity (67877 ± 15686 vs 13421 ±1871 relative light units, p< 0.0001) and PARP cleavage (2.11 ± 0.24 vs 1.00 ± 0.09 foldchange, p = 0.0081). The decrease in cell death and oxidative stress in this scenario wasassociated with a reduction in TNF-and an improvement in cardiac function, which wasotherwise worsened in the DOX group due to the decline cardiac output, coronary flow, ratepressureproduct, left ventricular developed pressure and total work. Even though ghrelintreatment in the presence of DOX did not induce noteworthy changes in the proteinexpression of ERK1/2 and Akt/PKB, the phosphorylation of STAT3 was improved withghrelin administration.Discussion and ConclusionThe observations presented in this study indicate that while DOX is a known effectivechemotherapeutic agent, it produces cardiotoxic effects through the induction of oxidativestress and consequently apoptosis, possibly due to the downregulation of ERK1/2 andAkt/PKB. The co-administration of ghrelin with DOX significantly decreased the detrimentaleffects associated with DOX treatment alone. The effects of ghrelin were not only beneficialat organ level, but also at the organism level, as a result of improved general well-being ofthe experimental animals and through the maintenance of cardiac function, a decline inmyocardial TNF-production and the stimulation of the STAT3 signaling pathway. The factthat ghrelin alone did not exert any detrimental effects makes this peptide an appealingcardioprotective agent and may therefore have the potential to improve the high morbidityand mortality rates of cancer survivors. While ghrelin in this context may possess anticardiotoxiceffects, further research is required to determine the effects of ghrelin on cancercell proliferation.

[发布机构] Stellenbosch University