Investigating the effects of chronic doxorubicin and ghrelin treatment on hepatic tissue
Thesis (MSc)--Stellenbosch University, 2018.
ENGLISH ABSTRACT: Introduction: Doxorubicin (DOX) is an effective and widely used chemotherapeutic agent. However, its toxicity on healthy tissue limits its usage. DOX is known to induce damage mainly via oxidative stress which stimulates oxidative damage and apoptosis either intrinsically or extrinsically. While literature has primarily focused on the side effects of DOX on the myocardium, very little attention has been paid to the potential toxic effects of DOX on peripheral organs such as the liver which metabolises drugs and various toxins. Ghrelin is a hormone naturally found in the body which has been found to possess some beneficial properties that could protect the liver against DOX toxicity. Up until now, the therapeutic role of ghrelin in a chronic model of DOX toxicity has not been evaluated. Therefore, this study aimed to evaluate the effects of DOX on the liver and whether the use of ghrelin in this context will confer protection. Methods: Four week old male Sprague-Dawley rats were randomly divided into four experimental groups: vehicle (saline), DOX (2.5 mg/kg/week), ghrelin (300 μg/kg/wk) and DOX+Ghrelin (both treatment regimens). Treatment was administered through intraperitoneal (i.p) injections for eight weeks where body weight and food consumption was monitored. One week after the last injection, the rats were sacrificed and the liver was harvested for histological and molecular analysis. Blood, which was separated into serum and plasma was also collected to evaluate liver function. Histological staining included Haematoxylin and Eosin (H&E), Masson’s Trichome, Reticulin and Oil red O, while molecular experiments included oxidative stress analysis, apoptotic and autophagic activity as well as endoplasmic reticulum (ER stress). Results: Terminal body weights were recorded per week and it was evident by the fourth week that the DOX treated animals (228.30 ± 7.09 g, p < 0.001) were not gaining as much weight as the vehicle treated animals (285.50 ± 19.24 g). This lack of weight gain was maintained until the end of the treatment protocol and coincided with reduced food consumption. Ghrelin significantly increased food consumption in the DOX+ghrelin group (159.20 g ± 9.43 g, p < 0.05) versus the DOX group (128.50 g ± 7.27), however this effect was not reflected in the body weight measurements of this group. Interestingly, DOX induced the fusion of liver lobes and this was prevented in the presence of ghrelin. While a normal rat liver and those that were treated with ghrelin alone displayed seven lobes, DOX treated animals presented with an average of four lobes and the combination group, an average of six lobes. This fusion did not affect survival, liver function or oxidative stress parameters. Although autophagic activity was maintained in all treatment groups, DOX+ghrelin prevented caspase-3 cleavage and permitted binding immunoglobin protein (BiP) and activating transcription factor 4 (ATF4) protein expression. Discussion & Conclusion: This study demonstrated that the liver is a robust organ that can withstand a great deal of stress and toxicity as weekly administration of DOX did not induce severe liver damage. Due to this organs regenerative capabilities, it is highly likely that DOX induced early damage of which the liver was able to recover from. While liver morphology was altered by DOX, structural parameters measured and function tests remained unchanged. No adverse oxidative damage was observed, nor was there any variation in the anti-oxidant status of this tissue. Molecularly, ghrelin reduced apoptosis and elevated autophagy. Therefore, while ghrelin’s protective effects were mainly observed as a result of its orexigenic properties, its effect against DOX on the liver requires further investigation.
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