Acylated and unacylated ghrelin inhibit doxorubicin-induced apoptosis in skeletal muscle

Abstract

Aim: Doxorubicin, a potent chemotherapeutic drug, has been demonstrated previously as an inducer of apoptosis in muscle cells. Extensive induction of apoptosis may cause excessive loss of muscle cells and subsequent functional decline in skeletal muscle. This study examined the effects of acylated ghrelin, a potential agent for treating cancer cachexia, on inhibiting apoptotic signalling in doxorubicin-treated skeletal muscle. Unacylated ghrelin, a form of ghrelin that does not bind to GHSR-1a, is also employed in this study to examine the GHSR-1a signalling dependency of the effects of ghrelin.

Methods: Adult C57BL/6 mice were randomly assigned to saline control (CON), doxorubicin (DOX), doxorubicin with treatment of acylated ghrelin (DOX+Acylated Ghrelin) and doxorubicin with treatment of unacylated ghrelin (DOX+Unacylated Ghrelin). Mice in all groups that involved DOX were intraperitoneally injected with 15 mg of doxorubicin per kg body weight, whereas mice in CON group received saline as placebo. Gastrocnemius muscle tissues were harvested after the experimental period for analysis.

Results: The elevation of apoptotic DNA fragmentation and number of TUNEL-positive nuclei were accompanied with the upregulation of Bax in muscle after exposure to doxorubicin, but all these changes were neither seen in the muscle treated with acylated ghrelin nor unacylated ghrelin after doxorubicin exposure. Protein abundances of autophagic markers including LC3 II-to-LC3 I ratio, Atg12-5 complex, Atg5 and Beclin-1 were not altered by doxorubicin but were upregulated by the treatment of either acylated or unacyated ghrelin. Histological analysis revealed that the amount of centronucleated myofibres was elevated in doxorubicin-treated muscle while muscle of others groups showed normal histology.

Conclusions: Collectively, our data demonstrated that acylated ghrelin administration suppresses the doxorubicin-induced activation of apoptosis and enhances the cellular signalling of autophagy. The treatment of unacylated ghrelin has similar effects as acylated ghrelin on apoptotic and autophagic signalling, suggesting that the effects of ghrelin are probably mediated through a signalling pathway that is independent of GHSR-1a. These findings were consistent with the hypothesis that acylated ghrelin inhibits doxorubicin-induced upregulation of apoptosis in skeletal muscle while treatment of unacylated ghrelin can achieve similar effects as the treatment of acylated ghrelin. The inhibition of apoptosis and enhancement of autophagy induced by acylated and unacylated ghrelin might exert myoprotective effects on doxorubicin-induced toxicity in skeletal muscle.

Keywords: acylated ghrelin; adriamycin; muscle damage; skeletal myofibre.