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

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.