NeuropharmacologyGenistein inhibits glutamate-induced apoptotic processes in primary neuronal cell cultures: An involvement of aryl hydrocarbon receptor and estrogen receptor/glycogen synthase kinase-3β intracellular signaling pathway
Section snippets
Primary hippocampal, neocortical, and cerebellar cell cultures
Hippocampal and neocortical tissues for primary cultures were prepared from Swiss mouse embryos (Institute of Pharmacology, Polish Academy of Sciences, Krakow, Poland) at 15–17 days of gestation and were cultivated essentially as described (Brewer 1995, Junghans and Kappler 1999). Pregnant females were anesthetized with CO2 vapor, killed by cervical dislocation, and subjected to cesarean section in order to remove fetal brains. In case of cerebellar cultures, tissue originated from 7-day-old
Impact of glutamate on caspase-3 activity and LDH-release
In hippocampal cultures exposed to 1 mM glutamate, the activity of caspase-3 was growing in a time-dependent manner. It rose by 48% at 3 h, and it was still enhanced by 58–71% over the control level until the end of experiment (Fig. 1, panel a). Likewise the glutamate-induced caspase-3 activity, LDH-release was also increasing with time of glutamate treatment. Thus, it was elevated to 167%, 184%, and 236% of control value, at 3, 6, and 24 h, respectively (Fig. 1, panel b).
Influence of genistein on glutamate-induced caspase-3 activity and LDH-release
In hippocampal
Discussion
The present data have shown that the stimulation of glutamate receptors evokes pro-apoptotic effects in the mouse hippocampal, neocortical and cerebellar neurons. We observed a time-dependent activation of caspase-3, which was found within several hours after the treatment and was followed by a substantial LDH-release in cell cultures on 7 days in vitro (DIV). These biochemical data were confirmed at the cellular level with Hoechst 33342 and calcein AM staining. There is a strong linkage
Conclusion
In summary, the present data demonstrated strong effects of genistein at low micromolar concentrations in various brain tissues against glutamate-induced apoptosis. This study has also provided evidence for the involvement of aryl hydrocarbon receptor and estrogen receptor/GSK3β intracellular signaling pathway in anti-apoptotic action of genistein, which may have implication for the treatment or the prevention of neurodegenerative disorders.
Acknowledgment
We wish to thank Dr. Marzena Mackowiak for her expertise in identification of apoptotically dying cells and the excellent assistance in use of MetaMorph software. The authors gratefully acknowledge financial support from the Polish Ministry of Education and Science, grant No. 2P05A 123 30.
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