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Purpose: To investigate the mechanism of the anti-epileptic effect of Ganoderma lucidum polysaccharides (GLP), the changes of intracellular calcium and CaMK II α expression in a model of epileptic neurons were investigated., Method: Primary hippocampal neurons were divided into: 1) Control group, neurons were cultured with Neurobasal medium, for 3 hours; 2) Model group I: neurons were incubated with Mg(2+) free medium for 3 hours; 3) Model group II: neurons were incubated with Mg(2+) free medium for 3 hours then cultured with the normal medium for a further 3 hours; 4) GLP group I: neurons were incubated with Mg(2+) free medium containing GLP (0.375 mg/ml) for 3 hours; 5) GLP group II: neurons were incubated with Mg(2+) free medium for 3 hours then cultured with a normal culture medium containing GLP for a further 3 hours. The CaMK II α protein expression was assessed by Western-blot. Ca(2+) turnover in neurons was assessed using Fluo-3/AM which was added into the replacement medium and Ca(2+) turnover was observed under a laser scanning confocal microscope., Results: The CaMK II α expression in the model groups was less than in the control groups, however, in the GLP groups, it was higher than that observed in the model group. Ca(2+) fluorescence intensity in GLP group I was significantly lower than that in model group I after 30 seconds, while in GLP group II, it was reduced significantly compared to model group II after 5 minutes., Conclusion: GLP may inhibit calcium overload and promote CaMK II α expression to protect epileptic neurons.

Epilepsy can cause cerebral transient dysfunctions. Ganoderma lucidum spores (GLS), a traditional Chinese medicinal herb, has shown some antiepileptic effects in our previous studies. This was the first study of the effects of GLS on cultured primary hippocampal neurons, treated with Mg(2+) free medium. This in vitro model of epileptiform discharge hippocampal neurons allowed us to investigate the anti-epileptic effects and mechanism of GLS activity. Primary hippocampal neurons from <1 day old rats were cultured and their morphologies observed under fluorescence microscope. Neurons were confirmed by immunofluorescent staining of neuron specific enolase (NSE). Sterile method for GLS generation was investigated and serial dilutions of GLS were used to test the maximum non-toxic concentration of GLS on hippocampal neurons. The optimized concentration of GLS of 0.122 mg/ml was identified and used for subsequent analysis. Using the in vitro model, hippocampal neurons were divided into 4 groups for subsequent treatment i) control, ii) model (incubated with Mg(2+) free medium for 3 hours), iii) GLS group I (incubated with Mg(2+) free medium containing GLS for 3 hours and replaced with normal medium and incubated for 6 hours) and iv) GLS group II (neurons incubated with Mg(2+) free medium for 3 hours then replaced with a normal medium containing GLS for 6 hours). Neurotrophin-4 and N-Cadherin protein expression were detected using Western blot. The results showed that the number of normal hippocampal neurons increased and the morphologies of hippocampal neurons were well preserved after GLS treatment. Furthermore, the expression of neurotrophin-4 was significantly increased while the expression of N-Cadherin was decreased in the GLS treated group compared with the model group. This data indicates that GLS may protect hippocampal neurons by promoting neurotrophin-4 expression and inhibiting N-Cadherin expression.