RESUMO
OBJECTIVE: To investigate potential mechanisms mediating the neuroprotective effect of thymoquinone (TQ) on dopaminergic neurons. METHODS: This study was conducted in the Chemistry and Biochemistry Institute, University of Veterinary Medicine, Vienna, Austria between June and August 2013. Primary cultures were prepared from embryonic mouse mesencephala (OFI/SPF) at gestation day 14. Four sets of cultures were kept untreated, treated with TQ on the eighth day in vitro (DIV) for 4 days, treated with 1-methyl-4-phenylpyridinium (MPP+) on the tenth DIV for 48 hours and co-treated with thymoquinone and MPP+. On the twelfth DIV, cultures were subjected to immunohistochemistry against tyrosine hydroxylase and fluorescent staining using LysoTracker Deep Red, 5,5`,6,6`-tetrachloro-1,1`,3,3`-tetraethylbenzimidazolylcarbocyanine (JC-1) and 4`,6-diamidino-2-phenylindole stains. RESULTS: The MPP+ decreased the number of dopaminergic neurons by 40%, and increased the release of lactate dehydrogenase (LDH) into the culture medium. The TQ significantly rescued dopaminergic neurons and decreased the release of LDH at the concentrations of 0.1 and 1 uM. The TQ significantly shifted the red fluorescent intensity of the LysoTracker Deep Red, increased the mitochondrial membrane potential as it increased the red:green florescent ratio of JC-1, and decreased MPP+-induced apoptotic cell death. CONCLUSION: The TQ protects dopaminergic neurons in primary mesencephalic culture by enhancing lysosomal degradation that clears damaged mitochondria and inhibits mitochondria-mediated apoptotic cell death.
