Esculetin inhibits N-methyl-D-aspartate neurotoxicity via glutathione preservation in primary cortical cultures

Abstract

Recently, loss of endogenous glutathione during N-methyl-D-aspartate (NMDA) receptor-mediated excitotoxic injury, and the resultant overproduction of reactive oxygen species (ROS) through an arachidonic acid cascade process in brain, have been implicated in neuronal damage in various neurodegenerative diseases. Glutathione depletion induced by L-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of glutathione synthesis, is known to cause arachidonic acid-mediated excitotoxicity in primary mixed cortical cultures. The aim of this study was to investigate whether esculetin (6,7-dihydroxycoumarin), an inhibitor of lipoxygenase, protects against neurotoxicity induced by NMDA or BSO. We observed that neurotoxicity induced by NMDA but not kainic acid was attenuated by esculetin. At the same concentration (100 µM), esculetin attenuated the (45)Ca(2+) uptake elevation induced by NMDA. Free radical-mediated neuronal injury induced by H(2)O(2) and xanthine/xanthine oxidase was concentration-dependently blocked by esculetin. Esculetin (1-30 µM) dose-dependently inhibited BSO-induced neuronal injury. In addition, arachidonate-induced neurotoxicity was completely blocked by esculetin. BSO also reduced glutathione peroxidase (GPx) activity, but did not change glutathione reductase (GR) activity 24 h after treatment. Esculetin dose-dependently increased GR activity, but did not alter GPx activity. These findings suggest that esculetin can contribute to the rescue of neuronal cells from NMDA neurotoxicity and that this protective effect occurs partly through NMDA receptor modulation and the sparing of glutathione depletion.

Keywords: Esculetin; L-buthionine-(S,R)-sulfoximine; N-methyl-D-aspartate; cortical cultures; lipoxygenase.

Figure 1

Morphological evidence of neuroprotection by esculetin against N-methyl-D-aspartate (NMDA) neurotoxicity. Phase-contrast photomicrographs of sister cultures of neurons cultured with NMDA (150 µM for 5 min) alone (A) or in the presence of 100 mM esculetin (B). Neurons were stained with trypan blue. Scale bar =50 µm.

Figure 2

Esculetin attenuates NMDA-induced ⁴⁵Ca²⁺ uptake. Sister cultures were exposed to 100 µM NMDA for 5 min in the presence of extracellular ⁴⁵Ca²⁺: immediately following exposure, the cells were washed and lysed and intracellular ⁴⁵Ca²⁺ measured. Bars represents mean neuronal uptake after NMDA exposure in the presence of MK-801 (10 µM) or esculetin (10 or 100 µM). ^*,^**Significantly different from control (^*P<0.05, ^**P<0.01).

Figure 3

Esculetin concentration-dependently inhibits BSO-induced neurotoxicity (A). Phase-contrast photomicrographs of sister cultures of neurons cultured with vehicle (B), BSO (1 mM for 24 h) alone (C) or in the presence of 30 µM esculetin (D). ^*Significantly different from control (P<0.05). Scale bar=50 µm.

Figure 4

Esculetin, but not cycloheximide (CHX) or vitamin E (Vit-E), significantly attenuates arachidonic acid (AA)-induced neurotoxicity in mixed cortical cultures. ^*Significantly different from control (P<0.05).

Figure 5

Esculetin does not change glutathione peroxidase (GP) activity (A) but increases glutathione reductase (GRx) activity (B) in BSO neurotoxicity. ^*,^**Significantly different from normal (^*P<0.05, ^**P<0.01). ^#,^##Significantly different from BSO control (^#P<0.05, ^##P<0.01).