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

Thesis (PhD)--Stellenbosch University, 2017.ENGLISH SUMMARY: Introduction Doxorubicin (DOX) is an anthracycline that has significantly improved the outcome of cancer patients since its discovery. However, its clinical success remains limited due to the dose-dependent cardiotoxic side effects associated with its use. While the mechanisms responsible for this condition are still not well defined, oxidative stress alongside apoptosis, remain the classical but major contributors. Ghrelin, an endogenous brain-gut peptide, most well-known for its effects on appetite and growth hormone release, has been shown to exert anti-apoptotic, antioxidative, anti-inflammatory and anti-fibrotic effects in several models of cardiovascular disease. These cardioprotective effects offered by ghrelin have been indicated to 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 DOXinduced cardiotoxicity. Methods Male 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 per week, the DOX group received 2.5 mg/kg per week, the combination group received both DOX 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 after the last injection, the rats were euthanized, blood was collected and the hearts were subjected to a 40-minute working heart perfusion protocol to obtain functional data. The hearts were weighed and then cut transversely into two sections, where one half was snap frozen for biochemical analysis and the other half was preserved in formalin for histological analysis. Cardiovascular markers of damage and pro-inflammatory cytokines were measured in serum using a luminex assay, and fibrosis and general morphology were assessed using the Masson’s Trichrome and H&E stains, respectively. Cytochrome c expression was evaluated by immunohistochemistry, while oxidative stress was assessed using the TBARS, conjugated diene, ORAC, SOD and glutathione assays. Apoptosis was determined using the Caspase Glo® assay and the expression of pro-survival proteins was measured using Western blot analysis. Results During the eight weeks of treatment, DOX significantly reduced weight gain and food consumption, whereas ghrelin maintained normal body weight and stimulated appetite. As expected, DOX induced significant oxidative stress when compared to the control, as demonstrated 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, as evident 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 fold change, p = 0.0081). The decrease in cell death and oxidative stress in this scenario was associated with a reduction in TNF- and an improvement in cardiac function, which was otherwise worsened in the DOX group due to the decline cardiac output, coronary flow, ratepressure product, left ventricular developed pressure and total work. Even though ghrelin treatment in the presence of DOX did not induce noteworthy changes in the protein expression of ERK1/2 and Akt/PKB, the phosphorylation of STAT3 was improved with ghrelin administration. Discussion and Conclusion The observations presented in this study indicate that while DOX is a known effective chemotherapeutic agent, it produces cardiotoxic effects through the induction of oxidative stress and consequently apoptosis, possibly due to the downregulation of ERK1/2 and Akt/PKB. The co-administration of ghrelin with DOX significantly decreased the detrimental effects associated with DOX treatment alone. The effects of ghrelin were not only beneficial at organ level, but also at the organism level, as a result of improved general well-being of the experimental animals and through the maintenance of cardiac function, a decline in myocardial TNF- production and the stimulation of the STAT3 signaling pathway. The fact that ghrelin alone did not exert any detrimental effects makes this peptide an appealing cardioprotective agent and may therefore have the potential to improve the high morbidity and mortality rates of cancer survivors. While ghrelin in this context may possess anticardiotoxic effects, further research is required to determine the effects of ghrelin on cancer cell proliferation.AFRIKAANS OPSOMMING: Inleiding Doksorubisien (DOX) is ‘n antrasiklien wat die uitkoms vir kankerpasiënte betekenisvol verbeter het sedert die ontdekking daarvan. Die kliniese sukses is steeds beperk as gevolg van die dosis-afhangklike kardiotoksiese newe-effekte wat verband hou met die gebruik daarvan. Terwyl die meganismes wat verantwoordelik is vir hierdie toestand steeds nie bekend is nie, is oksidatiewe stres, tesame met apoptose, die klassieke bydraers. Ghrelien,’n endogene brein-derm peptied wat wel bekend is vir sy effekte op aptyt en vrystelling van groeihormoon, vertoon anti-apoptotiese, antioksidatiewe, anti-inflammatoriese en antifibrotiese effekte in verskeie kardiovaskulêre siekte modelle. Hierdie kardio-beskermende effekte van ghrelien is te wyte aan die aktivering van die pro-oorlewingsproteïene, ERK1/2, Akt/PKB en STAT3. Hierdie studie het die beskermende effekte van ghrelien in ‘n chroniese model van DOX-geïnduseerde kardiotoksisiteit ondersoek. Metodes Manlike Sprague-Dawley rotte is geaklimatiseer en in vyf eksperimentele groepe verdeel. Terwyl die kontrole groep onbehandel is, het die ghrelien groep weekliks 300 μg/kg ghrelien, die DOX groep 2.5 mg/kg per week, die kombinasie groep beide DOX en ghrelien by dieselfde vorige dossisse, en die draer groep ‘n soutoplossing ontvang. Alle toedienings is via intraperitoneale inspuiting vir agt weke gedoen. Een week voor die laaste inspuiting, is die rotte doodgemaak, bloed is versamel, en die harte is blootgestel aan ‘n 40-minute werkende hartperfusie protokol om funksionele data te versamel. Die harte is geweeg en transversaal in twee dele gedissekteer, waar die een helfte geklampvries is vir biochemiese analises, en die ander helfte is in formalien gepreserveer vir histologiese analises. Kardiovaskulêre merkers, vir skade en pro-inflammatoriese sitokiene is in serum bepaal deur ‘n Luminex platvorm, terwyl fibrose en algemene morfologiese ondersoeke deur Masson se Trichrome en H&E kleuringstegniek onderskeidelik van gebruik te maak. Sitochroom-c uitdrukking is deur middel van immune-histochemie bepaal, en oksidatiewe stres deur van TBARS, gekonjugeerde diëne, ORAC, SOD en glutatioon toetse. Apoptose is deur middel die Caspase Glo® getoets, en uitdrukking van die pro-oorlewingsproteïene is bepaal deur Westerse kladtegniek. Resultate Gedurende die agt weke behandeling het DOX toename in gewig. asook voedselinname betekenigsvol verlaag, terwyl ghrelien normale liggaamsgewig en aptyt gestimuleer. Soos verwag, het DOX oksidatiewe stres betekenisvol geïnduseer vergeleke met die kontrole groep soos waargeneem deur die vorming van gekonjugeerde diëne (1.64 ± 0.11 vs 0.55 ± 0.12 μmol/gram, p = 0.0003), en ‘n verlaging in die GSH:GSSG verhouding (2.10 ± 0.47 vs 9.66 ± 1.08 AU, p< 0.05), terwyl ghrelien hierdie effekte verlaag het. Apoptotiese seldood is ook geïnduseer soos waargeneem deur die toename in sitochroom-c kleuring, kaspase aktiwiteit (67877 ± 15686 vs 13421 ± 1871 RLU, p< 0.0001) en PARP klowing (2.11 ± 0.24 vs 1.00 ± 0.09 voudige verandering, p = 0.0081). Die verlaging in seldood en oksidatiewe stres in hierdie scenario is met ‘n verlaging in TNF- vorming en verbeterde kardiale funksionering geassosiëer, wat nie in die DOX groep waargeneem is nie weens verlaagde kardiale omset. koronêre vloei, tempo-druk produk, linker ventrikulêre-ontwikkelde druk en totale arbeid. Hoewel ghrelien behandeling in die teenwoordigheid van DOX behandeling geen waarneembare veranderinge in die proteïenuitdrukking van ERK1/2 en Akt/PKB vertoon het nie, het ghrelien toediening wel die fosforilering van STAT3 verbeter. Bespreking en gevolgtrekking Die waarnemings wat in hierdie studie rapporteur word wys daarop dat terwyl DOX ‘n welbekende effektiewe chemoterapeutiese middel is, veroorsaak dit kardiotoksiese effekte deur die induksie van oksidatiewe stres, en gevolglik apoptose, moontlik deur die afregulering van ERK1/2 en Akt/PKB. Die toediening van ghrelien met DOX verlaag die skadelike effekte betekenisvol vergeleke met enkel behandeling van DOX. Die effek van ghrelien is beide voordelig op orgaanvlak sowel organisme vlak, weens verbeterde algemene welstand van die eksperimentele diere en deur die handhawing van kardiale funksie, ‘n afname in miokardiale TNF- vorming, en die stimulering van die STAT3 seinweg. Die feit dat ghrelien as ‘n enkel middel geen skadelike effekte vertoon het nie, maak van hierdie peptied ‘n gepaste kardiobeskermende middel en kan dus daarom die potensiaal hê om die hoë morbiditeit en mortaliteit van kanker pasiente te verbeter. Alhoewel ghrelien in hierdie konteks anti-kardiotoksiese effekte vertoon het, daar word voorgestel dat die effek van ghrelien op kankerselle eers ondersoek word alvorens enige behandelingsvoorstelle toegepas kan word