Antioxidant and neuroprotective effects of Hexarelin in Neuro-2A cells

In several neurodegenerative diseases, including amyotrophic lateral sclerosis, alterations of redox homeostasis within the cell lead to oxidative stress, abnormalities in mitochondrial morphology and biochemistry, damage to DNA, RNA, proteins and lipids, and ultimately to apoptosis. Intracellular hydrogen peroxide (H2O2), generated through oxidative stress, activates the apoptotic pathway by the phosphorylation of the pro-apoptotic regulator Bax and the downregulation of the anti-apoptotic protein Bcl-2, with the subsequent activation of caspases – 3 and – 7. Consequently, phosphoinositide 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) are modulated, supporting cell survival or inducing cell death. Hexarelin is a well-known growth hormone secretagogue endowed with growth hormone-releasing effects and neuroprotective activities, such as prevention of status epilepticus and promotion of neurogenesis. Moreover, hexarelin reduced the activation of caspase – 3 and caspase – 7 caused by brain hypoxia-ischemia procedures in neonatal rats, and exerted protective effects against β-amyloid cytotoxicity in vitro. In this study, we explored the protective effects of hexarelin against oxidative stress and its anti-apoptotic mechanism to attenuate H2O2-induced neurotoxicity in Neuro-2A cells, a mouse neuroblastoma cell line. Neuro-2A cells were incubated with increasing concentration of H2O2 for 24 h. MTT assay was used to measure changes in the viability of cells, and 100 µM H2O2 was selected as the lower concentration that significantly induced a reduction of cells survival. Further measurements by real-time PCR analysis showed that 24 hours treatment with 100 µM H2O2 induced significant changes in caspases – 3 and – 7, Bax and Bcl-2 mRNA levels, inducing cell apoptosis. Hexarelin blunted H2O2 effects both increasing cell viability and by opposing changes in caspases – 3 and – 7, Bax and Bcl-2 mRNA levels. Western blot analysis confirmed that hexarelin activate the Akt, p38 and ERK1/2 pathways. In conclusion, our results suggest that hexarelin exerts neuroprotective activities against H2O2-induced toxicity in Neuro-2A cells. Further experiments are needed to identify the precise molecular mechanisms underlying this neuroprotective effects and to clarify whether other GHS moieties exert the same neuroprotective effects.