Neuroprotective role of thymoquinone against 1-methyl-4-phenylpyridinium-induced dopaminergic cell death in primary mesencephalic cell culture

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.