Protective effects of saffron and trans-crocetin on glutamate mediated excitotoxicity in rat neuroblastoma cells

Neuronal dysfunctions or even cell death are often accompanied by an exceeding release of glutamate. Excessive glutamate level induces unregulated stimulation of NMDA receptors. In previous studies we found an antagonistic effect of hydro-ethanolic saffron extract (CSE) and trans-crocetin, a carotenoid from saffron, on NMDA receptors (Berger et al., Neuroscience, 180; 238–247, 2011). In this study we evaluated theprotective effects of CSE and trans-crocetin on glutamate mediated excitotoxictiy on rat B104 neuroblastoma cells using cell-based cytotoxicity tests and a fluorescent stain with 4',6-diamidino-2-phenylindole (DAPI-staining). Glutamate applied for 24h decreased concentration dependently (0.1–20 mM) cell viability (MTT-test) and increased LDH activity (LDH-test). The number of annexin-V and 7-AAD positive cells was also augmented and DAPI staining showed an enhanced number of pycnotic nuclei. The glutamate effect was partially inhibited by kynurenic acid (10 mM), an antagonist on excitatory amino acid receptors. After preincubation with CSE or trans-crocetin for 2h followed by 24h co-incubation with 10 mM glutamate a reduced excitotoxicity could be found. CSE 500µg/ml significantly increased cell viability from 27±4% to 63±5% and decreased LDH activity from 207±5% to 125±18%). DAPI-staining showed no differences between CSE treated and control cells. Trans-crocetin 50µM fully abolished the glutamate effects on cell viability, LDH activity, and DAPI-staining. We conclude that the neuroprotective effects of saffron and trans-crocetin which was previously demonstrated on cortical neurons are partially mediated by attenuation of glutamate mediated excitotoxicity