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 damage was 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 measured fluorimetrically. Calpain I activity and the activation (phosphorylation) of Akt and glycogen synthase kinase‐3 β (GSK‐3 β ) were assayed by Western blotting. Key results: NMDA induced concentration‐dependent, receptor‐mediated, death of SH‐SY5Y cells, ranging from 11 to 25% (0.25–5 m M ). Cell death induced by 1 m M NMDA (21%) was preceded by a significant accumulation of intracellular ROS and by a rapid activation of the calcium‐activated protease calpain I. In addition, NMDA caused a rapid deactivation of Akt kinase and this preceded the detrimental activation of the downstream kinase, GSK‐3 β . BEO (0.0005–0.01%) concentration dependently reduced death of SH‐SY5Y cells caused by 1 m M NMDA. In addition to preventing ROS accumulation and activation of calpain, BEO (0.01%) counteracted the deactivation of Akt and the consequent activation of GSK‐3 β , induced by NMDA. Results obtained by using specific fractions of BEO, suggested that monoterpene hydrocarbons were responsible for neuroprotection afforded by BEO against NMDA‐induced cell death. Conclusions and Implications: Our data demonstrate that BEO reduces neuronal damage caused in vitro by excitotoxic stimuli and that this neuroprotection was associated with prevention of injury‐induced engagement of critical death pathways. British Journal of Pharmacology (2007) 151 , 518–529; doi: 10.1038/sj.bjp.0707237