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Hexarelin exerts neuroprotective and antioxidant effects against hydrogen peroxide-induced toxicity through the modulation of MAPK and PI3K/Akt patways in Neuro-2A cells

Hexarelin, a synthetic hexapeptide, protects cardiac and skeletal muscles by inhibiting apoptosis, both in vitro and in vivo. Moreover, evidence suggests that hexarelin could have important neuroprotective bioactivity. Oxidative stress and the generation of free radicals has been implicated in the etiologies of several neurodegenerative diseases, including amyotrophic lateral sclerosis, Parkinson’s disease, Alzheimer’s disease, Huntington’s disease and multiple sclerosis. In addition to direct oxidative stress, exogenous hydrogen peroxide (H2O2) can penetrate biological membranes and enhance the formation of other reactive oxygen species. The aim of this study was to examine the inhibitory influence of hexarelin on H2O2-induced apoptosis in Neuro-2A cells, a mouse neuroblastoma cell line. Our results indicate that H2O2 reduced the viability of Neuro-2A cells in a dose-related fashion. Furthermore, H2O2 induced significant changes in the morphology of Neuro-2A cells, reflected in the formation of apoptotic cell bodies, and an increase of nitric oxide (NO) production. Hexarelin effectively antagonized H2O2 oxidative damage to Neuro-2A cells as indicated by improved cell viability, normal morphology and reduced nitrite (NO2−) release. Hexarelin treatment of Neuro-2A cells also reduced mRNA levels of caspases−3 and −7 and those of the pro-apoptotic molecule Bax; by contrast, hexarelin treatment increased anti-apoptotic Bcl-2 mRNA levels. Hexarelin also reduced MAPKs phosphorylation induced by H2O2 and concurrently increased p-Akt protein expression. In conclusion, our results identify several neuroprotective and anti-apoptotic effects of hexarelin. These properties suggest that further investigation of hexarelin as a neuroprotective agent in an investigational and therapeutic context are merited.


Introduction
Growth hormone secretagogues (GHS) are a class of synthetic oligopeptides and non-peptidyl 37 molecules with endocrine and extra-endocrine properties. GHS are preferentially recognized and 38 bound by the ghrelin receptor, known as growth hormone secretagogue receptor type-1a (GHS-R1a) 39 [1]. The GHS-R1a is predominantly found in the hypothalamus and pituitary gland where it mediates 40 the release of growth hormone (GH). The GHS-R1a is also implicated in the regulation of 41 gastrointestinal motility, as well as energy and glucose homeostasis [2]. 42 In addition to its endocrine effects, GHS also target peripheral tissues; to illustrate, GHS improve 43 muscle function in several pathological conditions by inhibiting the apoptosis pathway, reducing NO 44 release and counteracting inflammation [3,4]. 45 Among the GHS, hexarelin (a synthetic hexapeptide), binds not only to GHS-R1a but also to the 46 CD36 receptor and manifests varied beneficial effects [5][6][7] in diseases associated with muscle 47 wasting, chronic heart failure [8], excitotoxicity, neurological disorders, epilepsy and diabetes [9]. 48 Despite the emerging biological importance of hexarelin, its signalling mechanisms have been only 49 partially elucidated. Some studies have demonstrated that hexarelin modulates activation of different 50 intracellular pathways, like mitogen-activated protein kinases (MAPKs) and phosphoinositide 3-51 kinase (PI3K)/protein kinase B (Akt) [10,11], and, thereby, could indirectly influence intracellular 52 calcium (Ca 2+ ) concentrations [12]. Furthermore, hexarelin protects cells in vitro from apoptosis by 53 inhibiting nitric oxide (NO) synthesis and reactive oxygen species (ROS) release, modulating 54 caspases activity as well as the expression of proteins belonging to the BCL-2 family [6,[12][13][14][15][16]. 55 Oxidative stress is involved in the progression of many neuronal disorders, such as Alzheimer's 56 disease, Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis, as well as in 57 cancer, diabetes and aging [17][18][19]. 58 Hydrogen peroxide (  MTT was added to each well and incubated at 37°C for 3 h. Then, the culture medium was removed 93 and a 200 µl aliquot of acidified isopropanol was added in order to dissolve the formazan crystals.

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Absorbance was read at 570 nm using a multilabel spectrophotometer VICTOR 3 (Perkin Elmer, 95 Waltham, MA, USA). Cell viability of control cells was set to 100% and the relative absorbance of 96 experimental groups were converted to relative percentages (relative absorbance of experimental 97 group/relative absorbance of control) x 100= % of viable cells.   In order to monitor the apoptosis pathway, Neuro-2A cells were plated in 24-well culture plates at a 115 density of 2 × 10 5 cells/well and incubated and treated at 37°C for 24 h according to specific protocols.

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Following treatment, Neuro-2A cells were washed with PBS and total RNA was extracted using  independently replicated at least three times. Student's t-test was used for comparisons between two 154 groups. One-way ANOVA followed by Tukey's t-test was used for multiple comparisons. A p-value 155 of less than 0.05 was considered significant.  at higher H 2 O 2 concentrations (Fig 2A). These changes were obviously attenuated with hexarelin co-180 treatment ( Fig 2B).    (Figs 4C and 4D). Notably, the presence of 1 M hexarelin in cells co-incubated with 213 100 µM H 2 O 2 caused a non-significant reduction in caspase-7 mRNA (Fig 4D), but, significantly 214 reduced caspase-3 mRNA (p<0.001, Fig 4C).

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In addition to the activation of caspases, mitochondria also play a crucial role in the process of cell

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Exposure to 100 µM H 2 O 2 significantly increased the p-ERK/t-ERK ratio (p<0.05), by comparison, 243 1 M hexarelin did not affect ERK protein levels, both compared to the control group. Notably, co-244 incubation with hexarelin and H 2 O 2 decreased p-ERK protein levels (Fig 6A). 245 Western blot analysis revealed that hexarelin treatment of Neuro-2A cells exposed to H 2 O 2 246 significantly decreased the level of p-p38 when compared to the H 2 O 2 -treated group (p<0.05).

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Furthermore, levels of p-Akt, associated with cell survival after oxidative stress [24], were not 250 changed by hexarelin treatment alone compared to the control but were reduced by H 2 O 2 treatment 251 (p<0.05, Fig 6C). Notably, p-Akt levels in cells treated with hexarelin and H 2 O 2 were significantly 252 (p<0.01) greater than those in cells treated with H 2 O 2 alone.

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There are numerous inducers of oxidative stress that are able to cause cytotoxicity in in vitro models.

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In our study, we used H 2 O 2 as it is an established method for measurement of potential