Inibition of H2O2-induced neurotoxicity in mouse neuroblastoma Neuro2A cells by microvescicles released from hexarelin-stimulated microglial cells

Background: In neurodegenerative diseases, hydrogen peroxide (H2O2) and the production of reactive oxygen species (ROS) have been implicated in the activation of well-known inflammatory signalling pathways. Microglial cells can assume two opposite phenotypes: M2 anti-inflammatory phenotype, in the early stages of neurodegenerative diseases, and the M1 pro-inflammatory subtype that become prevalentwith the diseases progression. Recently, different studies have demonstrated that microglia communicate with neuronal and non neuronal cells by releasing microvescicles (MVs) that contribute to disease progression. Targeting microglia M2 polarization represent an interesting therapeutic approach to inhibit local neurodegeneration, for example inducing the release of insulin growth factor-1 (IGF-1), a beneficial factor against inflammation. Our previous studies have demonstrated that hexarelin, a well-known growth hormone secretagogues (GHS), is endowed with antioxidant, anti-inflammatory and neuroprotective activities, promotes neurogenesis and inhibits cytotoxic effects of β-amyloid in N9 cells, but can also stimulate the release of IGF-1. In this study, we explored the ability of hexarelin to modulated the phenotype of N9 cells and how the induction of M2 microglia phenotype can oppose H2O2-induced neurotoxicity in mouse neuroblastoma Neuro2A cells. Methods: N9 cells were incubated with hexarelin 10-6 M for 24 h. Cells were used to quantify mRNA levels of iNOS and Arg1 for the evaluation of M1/M2 polarization by Real Time PCR, while medium was collected for subsequent experiments. N9 collected media were processed by two centrifugations at 1000xg for 10 and 20 minutes, respectively. The supernatant was ultracentrifugated at 110’000xg for 70 minutes and the pellet resuspended. Before use, MVs were quantified by NanoSight instrument. Neuro2A cells were treated with H2O2 100 µM for 24 h. At the end of the treatment, H2O2 was removed and substituted with N9 conditioned media, purified MVs, or N9 conditioned media deprived of MVs for 24 h. mRNA levels of Bax, BCL-2, caspase-3 and caspase-7 were measured by Real Time PCR. Results: Hexarelin-treated N9 cells showed a prevalence of M2 anti-inflammatory phenotype, with the down-regulation of iNOS and the up-regulation of Arg1 mRNA levels. H2O2 100 µM induced the activation of apoptosis pathway: the mRNA level of pro-apoptotic Bax significantly increased, as well as the levels of caspase-3 and caspase-7, while BCL-2 mRNA levels were reduced. N9 collected media and MVs extracted inhibited the apoptosis activation: Bax, caspase-3 and caspase-7 mRNA levels were reduced while anti-apoptotic BCL-2 mRNA levels were increased. Conclusion: Our results suggest that hexarelin modulated the phenotype of N9 microglial cells, inducing a polarization toward the M2 anti-inflammatory phenotype. Medium derived by hexarelin-treated N9 cells and MVs reduced the toxic effect of H2O2. Further experiments are needed to clarify the composition of MVs underlying the neuroprotective effects.