Abstract
BackgroundExposure to ethanol during early development triggers severe neuronal death by activating multiple stress pathways and causes neurological disorders, such as fetal alcohol effects or fetal alcohol syndrome. This study investigated the effect of ethanol on intracellular events that predispose developing neurons for apoptosis via calcium-mediated signaling. Although the underlying molecular mechanisms of ethanol neurotoxicity are not completely determined, mitochondrial dysfunction, altered calcium homeostasis and apoptosis-related proteins have been implicated in ethanol neurotoxicity. The present study was designed to evaluate the neuroprotective mechanisms of metformin (Met) and thymoquinone (TQ) during ethanol toxicity in rat prenatal cortical neurons at gestational day (GD) 17.5.ResultsWe found that Met and TQ, separately and synergistically, increased cell viability after ethanol (100 mM) exposure for 12 hours and attenuated the elevation of cytosolic free calcium [Ca2+]c. Furthermore, Met and TQ maintained normal physiological mitochondrial transmembrane potential (ΔψM), which is typically lowered by ethanol exposure. Increased cytosolic free [Ca2+]c and lowered mitochondrial transmembrane potential after ethanol exposure significantly decreased the expression of a key anti-apoptotic protein (Bcl-2), increased expression of Bax, and stimulated the release of cytochrome-c from mitochondria. Met and TQ treatment inhibited the apoptotic cascade by increasing Bcl-2 expression. These compounds also repressed the activation of caspase-9 and caspase-3 and reduced the cleavage of PARP-1. Morphological conformation of cell death was assessed by TUNEL, Fluoro-Jade-B, and PI staining. These staining methods demonstrated more cell death after ethanol treatment, while Met, TQ or Met plus TQ prevented ethanol-induced apoptotic cell death.ConclusionThese findings suggested that Met and TQ are strong protective agents against ethanol-induced neuronal apoptosis in primary rat cortical neurons. The collective data demonstrated that Met and TQ have the potential to ameliorate ethanol neurotoxicity and revealed a possible protective target mechanism for the damaging effects of ethanol during early brain development.
Highlights
Exposure to ethanol during early development triggers severe neuronal death by activating multiple stress pathways and causes neurological disorders, such as fetal alcohol effects or fetal alcohol syndrome
Effect of TQ and Met on ethanol-induced cell death in primary rat cortical neurons Cortical neurons were treated with ethanol (100 mM), TQ (10, 15, 25 and 35 μM) and Met (10 mM) in different combinations for 12 h, and cell viability was measured by 3-[4,5-dimethylthiazol-2]-2,5 diphenyltetrazolium bromide (MTT) assay
We observed that the intracellular Ca2+ concentration was significantly elevated in cells exposed to ethanol (100 mM) for 12 h compared to control, Figure 2 Effect of Ethanol on elevation of cytosolic free [Ca2+]c in primary cortical neurons
Summary
Exposure to ethanol during early development triggers severe neuronal death by activating multiple stress pathways and causes neurological disorders, such as fetal alcohol effects or fetal alcohol syndrome. This study investigated the effect of ethanol on intracellular events that predispose developing neurons for apoptosis via calcium-mediated signaling. Mitochondria play an important role in apoptosis under a variety of pro-apoptotic conditions, such as oxidative stress and cytochrome-c release. The latter is a key event in the activation of caspase-3, a pivotal downstream step in apoptosis initiation [10,11]. Cellular or sustained Ca2+ overload is an important mechanism that contributes to ethanol-induced programmed cell death through the activation of post-mitochondrial events involving caspase activation [13,14]
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