Hexarelin Modulation of MAPK and PI3K/Akt Pathways in Neuro-2A Cells Inhibits Hydrogen Peroxide—Induced Apoptotic Toxicity

Abstract

Hexarelin, a synthetic hexapeptide, exerts cyto-protective effects at the mitochondrial level in cardiac and skeletal muscles, both in vitro and in vivo, may also have important neuroprotective bioactivities. This study examined the inhibitory effects of hexarelin on hydrogen peroxide (H2O2)-induced apoptosis in Neuro-2A cells. Neuro-2A cells were treated for 24 h with various concentrations of H2O2 or with the combination of H2O2 and hexarelin following which cell viability and nitrite (NO2−) release were measured. Cell morphology was also documented throughout and changes arising were quantified using Image J skeleton and fractal analysis procedures. Apoptotic responses were evaluated by Real-Time PCR (caspase-3, caspase-7, Bax, and Bcl-2 mRNA levels) and Western Blot (cleaved caspase-3, cleaved caspase-7, MAPK, and Akt). Our results indicate that hexarelin effectively antagonized H2O2-induced damage to Neuro-2A cells thereby (i) improving cell viability, (ii) reducing NO2− release and (iii) restoring normal morphologies. Hexarelin treatment also reduced mRNA levels of caspase-3 and its activation, and modulated mRNA levels of the BCL-2 family. Moreover, hexarelin inhibited MAPKs phosphorylation and increased p-Akt protein expression. In conclusion, our results demonstrate neuroprotective and anti-apoptotic effects of hexarelin, suggesting that new analogues could be developed for their neuroprotective effects.

Protective Effects of Hexarelin and JMV2894 in a Human Neuroblastoma Cell Line Expressing the SOD1-G93A Mutated Protein

“…First, we quantified the number of SH-SY5Y SOD1 G93A cells in a fixed area by the use of a specific macro for ImageJ software [ 28 ]. The number of cells in each field was used for normalizing the data of skeleton analysis, which, in turn, was used to quantify endpoints and process length [ 12 , 29 , 30 ], since the loss of ramifications is a typical characteristic of morphological cytoskeletal changes in apoptosis. Briefly, the Analyze Skeleton Plugin was applied to skeleton images obtained using ImageJ plugin protocols of original photomicrographs, as described in Material and Methods.…”

“…We quantified, by fractal analysis, the following parameters: Fractal dimension (D), an index of cell complexity pattern, that is used to identify cellular forms, ranging from simple rounded to complex branched [ 12 , 30 , 31 ]. Lacunarity, a property of the soma, based on the heterogeneity or translational and rotational invariance in a shape; lower lacunarity values indicate a loss of shape heterogeneity [ 12 , 30 , 31 ]. Maximum Span Across the Convex Hull (MSACH), which is the maximum distance between two points across the convex hull.…”

“…Fractal dimension (D), an index of cell complexity pattern, that is used to identify cellular forms, ranging from simple rounded to complex branched [ 12 , 30 , 31 ].…”

“…Growth hormone secretagogues (GHSs) are a large family of synthetic and natural compounds endowed with the following: (i) endocrine activities, as demonstrated by their ability to stimulate the release of growth hormone (GH) and insulin-like growth factor 1 (IGF-1); (ii) extra-endocrine activities, including the stimulation of food intake, anticonvulsant and anti-inflammatory effects, and protection of muscle tissue in different pathological conditions [ 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 ].…”

“…Among the GHS, hexarelin is a synthetic hexapeptide that exerts cytoprotective effects, both in vivo and in vitro, by acting at the mitochondrial level in cardiac and skeletal muscle, and with important neuroprotective and anti-apoptotic activities [ 9 , 11 , 12 , 18 ]. It has been shown that hexarelin modulates the activation of mitogen-activated protein kinases (MAPKs) and phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) [ 19 , 20 ], and, thereby, could modulate intracellular Ca 2+ concentrations [ 13 ].…”