Inhibitory regulation of glutamate release from rat cortical nerve terminals by thymoquinone

Thymoquinone (TQ), a natural compound derived from the oil of Nigella sativa seeds, has demonstrated neuroprotective properties. This study investigated the effects of TQ on glutamate release from rat cortical synaptosomes and explored the underlying mechanisms. TQ inhibited 4-aminopyridine (4-AP)-evoked glutamate release in a concentration-dependent manner, with an estimated IC₅₀ of 8.1 μM. This inhibitory effect was absent under Ca^2 + -free conditions and in the presence of bafilomycin A1, an inhibitor of vesicular glutamate transporter, indicating a Ca²⁺-dependent exocytotic mechanism. Consistently, TQ significantly reduced 4-AP-induced uptake of the synaptotagmin 1 luminal domain antibody (syt1-L ab), confirming suppression of synaptic vesicle exocytosis. Moreover, TQ attenuated the 4-AP-induced elevation of intraterminal Ca²⁺ without affecting synaptosomal membrane potential. Notably, the inhibitory effect of TQ on glutamate release was abolished by blockade of P/Q-type Ca²⁺ channels or inhibition of protein kinase C (PKC). Western blot analysis further revealed that TQ reduced 4-AP-induced phosphorylation of PKC, SNAP-25, and Munc18–1 in synaptosomes. Collectively, these findings suggest that TQ inhibits glutamate exocytosis from cortical synaptosomes by reducing Ca²⁺ influx through P/Q-type Ca²⁺ channels and subsequently downregulating the PKC/SNAP-25/Munc18–1 signaling cascade.