Cell signaling pathways in the mechanisms of neuroprotection afforded by bergamot essential oil against NMDA-induced cell death in vitro

Background and purpose: The effects of bergamot essential oil (BEO; Citrus bergamia, Risso) on excitotoxic neuronal damagewas investigated in vitro.Experimental approach: The study was performed in human SH-SY5Y neuroblastoma cells exposed to N-methyl-D-aspartate(NMDA). Cell viability was measured by dye exclusion. Reactive oxygen species (ROS) and caspase-3 activity were measuredfluorimetrically. Calpain I activity and the activation (phosphorylation) of Akt and glycogen synthase kinase-3b (GSK-3b) wereassayed by Western blotting.Key results: NMDA induced concentration-dependent, receptor-mediated, death of SH-SY5Y cells, ranging from 11 to 25%(0.25–5mM). Cell death induced by 1mM NMDA (21%) was preceded by a significant accumulation of intracellular ROS andby a rapid activation of the calcium-activated protease calpain I. In addition, NMDA caused a rapid deactivation of Akt kinaseand this preceded the detrimental activation of the downstream kinase, GSK-3b. BEO (0.0005–0.01%) concentrationdependently reduced death of SH-SY5Y cells caused by 1mM NMDA. In addition to preventing ROS accumulation andactivation of calpain, BEO (0.01%) counteracted the deactivation of Akt and the consequent activation of GSK-3b, induced byNMDA. Results obtained by using specific fractions of BEO, suggested that monoterpene hydrocarbons were responsible forneuroprotection afforded by BEO against NMDA-induced cell death.Conclusions and Implications: Our data demonstrate that BEO reduces neuronal damage caused in vitro by excitotoxicstimuli and that this neuroprotection was associated with prevention of injury-induced engagement of critical death pathways.